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Posts Tagged ‘Progenitor cell’


Pathway Specific Targeting in Anticancer Therapies

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

7.7 Pathway specific targeting in anticancer therapies

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

 

7.7.1 Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Thangavelua, CQ Pana, …, BC Lowa, and J. Sivaramana
Proc Nat Acad Sci 2012; 109(20):7705–7710
http://dx.doi.org:/10.1073/pnas.1116573109

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA’s active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

The Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (12). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (23). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (25).

Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly, direct suppression of miR23a and miR23b (9) or activation of TGF-β (10) enhances KGA expression. Similarly, Rho GTPase that controls cytoskeleton and cell division also up-regulates KGA expression in an NF-κB–dependent manner (11). In addition, KGA is a substrate for the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1, linking glutaminolysis to cell cycle progression (12). In comparison, function and regulation of LGA is not well studied, although it was recently shown to be linked to p53 pathway (1314). Although intense efforts are being made to develop a specific KGA inhibitor such as BPTES [bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide] (15), its mechanism of inhibition and selectivity is not yet understood. Equally important is to understand how KGA function is regulated in normal and cancer cells so that a better treatment strategy can be considered.

The previous crystal structures of microbial (Mglu) and Escherichia coli glutaminases show a conserved catalytic domain of KGA (1617). However, detailed structural information and regulation are not available for human glutaminases especially the KGA, and this has hindered our strategies to develop inhibitors. Here we report the crystal structure of the catalytic domain of human apo KGA and its complexes with substrate (L-glutamine), product (L-glutamate), BPTES, and its derived inhibitors. Further, Raf-Mek-Erk module is identified as the regulator of KGA activity. Although BPTES is not recognized in the active site, its binding confers a drastic conformational change of a key loop (Glu312-Pro329), which is essential in stabilizing the catalytic pocket. Significantly, EGF activates KGA activity, which can be abolished by the kinase-dead, dominant negative mutants of Mek2 (Mek2-K101A) or its upstream activator Raf-1 (Raf-1-K375M), which are the kinase components of the growth-promoting Raf-Mek2-Erk signaling node. Furthermore, coexpression of phosphatase PP2A and treatment with Mek-specific inhibitor or alkaline phosphatase all abolished enhanced KGA activity inside the cells and in vitro, indicating that stimulation of KGA is phosphorylation dependent. Our results therefore provide mechanistic insights into KGA inhibition by BPTES and its regulation by EGF-mediated Raf-Mek-Erk module in cell growth and possibly cancer manifestation.

Structures of cKGA and Its Complexes with L-Glutamine and L-Glutamate.
The human KGA consists of 669 amino acids. We refer to Ile221-Leu533 as the catalytic domain of KGA (cKGA) (Fig. 1A). The crystal structures of the apo cKGA and in complex with L-glutamine or L-glutamate were determined (Table S1). The structure of cKGA has two domains with the active site located at the interface. Domain I comprises (Ile221-Pro281 and Cys424 -Leu533) of a five-stranded anti-parallel β-sheet (β2↓β1↑β5↓β4↑β3↓) surrounded by six α-helices and several loops. The domain II (Phe282-Thr423) mainly consists of seven α-helices. L-Glutamine/L-glutamate is bound in the active site cleft (Fig. 1B and Fig. S1B). Overall the active site is highly basic, and the bound ligand makes several hydrogen-bonding contacts to Gln285, Ser286, Asn335, Glu381, Asn388, Tyr414, Tyr466, and Val484 (Fig. 1C and Fig. S1C), and these residues are highly conserved among KGA homologs (Fig. S1D). Notably, the putative serine-lysine catalytic dyad (286-SCVK-289), corresponding to the SXXK motif of class D β-lactamase (17), is located in close proximity to the bound ligand. In the apo structure, two water molecules were located in the active site, one of them being displaced by glutamine in the substrate complex. The substrate side chain is within hydrogen-bonding distance (2.9 Å) to the active site Ser286. Other key residues involved in catalysis, such as Lys289, Tyr414, and Tyr466, are in the vicinity of the active site. Lys289 is within hydrogen-bonding distance to Ser286 (3.1 Å) and acts as a general base for the nucleophilic attack by accepting the proton from Ser286. Tyr466, which is close to Ser286 and in hydrogen-bonding contact (3.2 Å) with glutamine, is involved in proton transfer during catalysis. Moreover, the carbonyl oxygen of the glutamine is hydrogen-bonded with the main chain amino groups of Ser286 and Val484, forming the oxyanion hole. Thus, we propose that in addition to the putative catalytic dyad (Ser286 XX Lys289), Tyr466 could play an important role in the catalysis (Fig. 1Cand Fig. S2).

structure of the cKGA-L-glutamine complex

structure of the cKGA-L-glutamine complex

http://www.pnas.org/content/109/20/7705/F1.medium.gif

Fig. 1.  Schematic view and structure of the cKGA-L-glutamine complex. (A) Human KGA domains and signature motifs (refer to Fig. S1A for details). (B) Structure of the of cKGA and bound substrate (L-glutamine) is shown as a cyan stick. (C) Fourier 2Fo-Fc electron density map (contoured at 1 σ) for L-glutamine, that makes hydrogen bonds with active site residues are shown.

Allosteric Binding Pocket for BPTES. The chemical structure of BPTES has an internal symmetry, with two exactly equivalent parts including a thiadiazole, amide, and a phenyl group (Fig. S3A), and it equally interacts with each monomer. The thiadiazole group and the aliphatic linker are well buried in a hydrophobic cluster that consists of Leu321, Phe322, Leu323, and Tyr394 from both monomers, which forms the allosteric pocket (Fig. 2 B–E). The side chain of Phe322 is found at the bottom of the allosteric pocket. The phenyl-acetamido moiety of BPTES is partially exposed on the loop (Asn324-Glu325), where it interacts with Phe318, Asn324, and the aliphatic part of the Glu325 side chain. On the basis of our observations we synthesized a series of BPTES-derived inhibitors (compounds2–5) (Fig. S3 AF and SI Results) and solved their cocrystal structure of compounds 2–4. Similar to BPTES, compounds 24 all resides within the hydrophobic cluster of the allosteric pocket (Fig. S3 CF).

Fig. 2. Structure of cKGA: BPTES complex and the allosteric binding mode of BPTES.

Allosteric Binding of BPTES Triggers Major Conformational Change in the Key Loop Near the Active Site.  The overall structure of these inhibitor complexes superimposes well with apo cKGA. However, a major conformational change at the Glu312 to Pro329 loop was observed in the BPTES complex (Fig. 2F). The most conformational changes of the backbone atoms that moved away from the active site region are found at the center of the loop (Leu316-Lys320). The backbone of the residues Phe318 and Asn319 is moved ≈9 Å and ≈7 Å, respectively, compared with the apo structure, whereas the side chain of these residues moved ≈14 Å and ≈12 Å, respectively. This loop rearrangement in turn brings Phe318 closer to the phenyl group of the inhibitor and forms the inhibitor binding pocket, whereas in the apo structure the same loop region (Leu316-Lys320) was found to be adjacent to the active site and forms a closed conformation of the active site.

Binding of BPTES Stabilizes the Inactive Tetramers of cKGA.  To understand the role of oligomerization in KGA function, dimers and tetramers of cKGA were generated using the symmetry-related monomers (Fig. 2 A–E and Fig. S4 D and E). The dimer interface in the cKGA: BPTES complex is formed by residues from the helix Asp386-Lys398 of both monomers and involves hydrogen bonding, salt bridges, and hydrophobic interactions (Phe389, Ala390, Tyr393, and Tyr394), besides two sulfate ions located in the interface (Fig. 2E). The dimers are further stabilized by binding of BPTES, where it binds to loop residues (Glu312-Pro329) and Tyr394 from both monomers (Fig. 2 D and E). Similarly, residues from Lys311-Asn319 loop and Arg454, His461, Gln471, and Asn529-Leu533 are involved in the interface with neighboring monomers to form the tetramer in the BPTES complex.

BPTES Induces Allosteric Conformational Changes That Destabilize Catalytic Function of KGA

Fig. 3A shows that 293T cells overexpressing KGA produced higher level of glutamate compared with the vector control cells. Most significantly, all of these mutants, except Phe322Ala, greatly diminished the KGA activity.

Fig. 3. Mutations at allosteric loop and BPTES binding pocket abrogate KGA activity and BPTES sensitivity.

Raf-Mek-Erk Signaling Module Regulates KGA Activity. Because KGA supports cell growth and proliferation, we first validated that treatment of cells with BPTES indeed inhibits KGA activity and cell proliferation (Fig. S5 A–D and SI Results). Next, as cells respond to various physiological stimuli to regulate their metabolism, with many of the metabolic enzymes being the primary targets of modulation (18), we examined whether KGA activity can be regulated by physiological stimuli, in particular EGF, which is important for cell growth and proliferation. Cells overexpressing KGA were made quiescent and then stimulated with EGF for various time points. Fig. 4A shows that the basal KGA activity remained unchanged 30 min after EGF stimulation, but the activity was substantially enhanced after 1 h and then gradually returned to the basal level after 4 h. Because EGF activates the Raf-Mek-Erk signaling module (19), treatment of cells with Mek-specific inhibitor U0126 could block the enhanced KGA activity with parallel inhibition of Erk phosphorylation (Fig. 4A). Interestingly, such Mek-induced KGA activity is specific to EGF and lysophosphatidic acid (LPA) but not with other growth factors, such as PDGF, TGF-β, and basic FGF (bFGF), despite activation of Mek-Erk by bFGF (Fig. S6A).

The results show that KGA could interact equally well with the wild-type or mutant forms of Raf-1 and Mek2 (Fig. 4C). Importantly, endogenous Raf-1 or Erk1/2, including the phosphorylated Erk1/2 (Fig. 4 C and D), could be detected in the KGA complex. Taken together, these results indicate that the activity of KGA is directly regulated by Raf-Mek-Erk downstream of EGF receptor. To further show that Mek2-enhanced KGA activity requires both the kinase activity of Mek2 and the core residues for KGA catalysis, wild-type or triple mutant (Leu321Ala/Phe322Ala/Leu323Ala) of KGA was coexpressed with dominant negative Mek2-KA or the constitutive active Mek2-SD and their KGA activities measured. The result shows that the presence of Mek2-KA blocks KGA activity, whereas the triple mutant still remains inert even in the presence of the constitutively active Mek2 (Fig. 4E), and despite Mek2 binding to the KGA triple mutant (Fig. S7B). Consequently, expressing triple mutant did not support cell proliferation as well as the wild-type control (Fig. S7C).

Fig. 4. EGFR-Raf-Mek-Erk signaling stimulates KGA activity.

When cells expressing both KGA and Mek2-K101A were treated with subthreshold levels of BPTES, there was a synergistic reduction in cell proliferation (Fig. S6C and SI Results). Lastly, to determine whether regulation of KGA by Raf-Mek-Erk depends on its phosphorylation status, cells were transfected with KGA with or without the protein phosphatase PP2A and assayed for the KGA activity. PP2A is a ubiquitous and conserved serine/threonine phosphatase with broad substrate specificity. The results indicate that KGA activity was reduced down to the basal level in the presence of PP2A (Fig. 5A). Coimmunoprecipitation study also revealed that KGA interacts with PP2A (Fig. 5B), suggesting a negative feedback regulation by this protein phosphatase. Furthermore, treatment of immunoprecipitated and purified KGA with calf-intestine alkaline phosphatase (CIAP) almost completely abolished the KGA activity in vitro (Fig. S6D). Taken together, these results indicate that KGA activity is regulated by Raf-Mek2, and KGA activation by EGF could be part of the EGF-stimulated Raf-Mek-Erk signaling program in controlling cell growth and proliferation (Fig. 5C).

KGA activity is regulated by phosphorylation

KGA activity is regulated by phosphorylation

http://www.pnas.org/content/109/20/7705/F5.medium.gif

Fig. 5. KGA activity is regulated by phosphorylation. (C) Schematic model depicting the synergistic cross-talk between KGA-mediated glutaminolysis and EGF-activated Raf-Mek-Erk signaling. Exogenous glutamine can be transported across the membrane and converted to glutamate by glutaminase (KGA), thus feeding the metabolite to the ATP-producing tricarboxylic acid (TCA) cycle. This process can be stimulated by EGF receptor-mediated Raf-Mek-Erk signaling via their phosphorylation-dependent pathway, as evidenced by the inhibition of KGA activity by the kinase-dead and dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-specific inhibitor U0126. Consequently, inhibiting KGA with BPTES and blocking Raf-Mek pathway with Mek2-K101A provide a synergistic inhibition on cell proliferation.

Small-molecule inhibitors that target glutaminase activity in cancer cells are under development. Earlier efforts targeting glutaminase using glutamine analogs have been unsuccessful owing to their toxicities (2). BPTES has attracted much attention as a selective, nontoxic inhibitor of KGA (15), and preclinical testing of BPTES toward human cancers has just begun (20). BPTES selectively suppresses the growth of glioma cells (21) and inhibits the growth of lymphoma tumor growth in animal model studies (22). Wang et al. (11) reported a small molecule that targets glutaminase activity and oncogenic transformation. Despite extensive studies, nothing is known about the structural and molecular basis for KGA inhibitory mechanisms and how their function is regulated during normal and cancer cell metabolism. Such limited information impedes our effort in producing better generations of inhibitors for better treatment regimens.

Comparison of the complex structures with apo cKGA structure, which has well-defined electron density for the key loop, we provide the atomic view of an allosteric binding pocket for BPTES and elucidate the inhibitory mechanism of KGA by BPTES. The key residues of the loop (Glu312-Pro329) undergo major conformational changes upon binding of BPTES. In addition, structure-based mutagenesis studies suggest that this loop is essential for stabilizing the active site. Therefore, by binding in an allosteric pocket, BPTES inhibits the enzymatic activity of KGA through (i) triggering a major conformational change on the key residues that would normally be involved in stabilizing the active sites and regulating its enzymatic activity; and (ii) forming a stable inactive tetrameric KGA form. Our findings are further supported by two very recent reports on KGA isoform (GAC) (2324), although these studies lack full details owing to limitation of their electron density maps. BPTES is specific to KGA but not to LGA (15). Sequence comparison of KGA with LGA (Fig. S8A) reveals two unique residues on KGA, Phe318 and Phe322, which upon mutation to LGA counterparts, become resistant to BPTES. Thus, our study provides the molecular basis of BPTES specificity.

7.7.2 Sonic hedgehog (Shh) signaling promotes tumorigenicity and stemness via activation of epithelial-to-mesenchymal transition (EMT) in bladder cancer.

Islam SS, Mokhtari RB, Noman AS, …, van der Kwast T, Yeger H, Farhat WA.
Molec Carcinogenesis mar 2015; 54(5). http://dx.doi.org:/10.1002/mc.22300

shh sonic hedgehog signaling pathway nri2151-f1

shh sonic hedgehog signaling pathway nri2151-f1

Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis

Sonic_hedgehog_pathway

Sonic_hedgehog_pathway

7.7.3 Differential activation of NF-κB signaling is associated with platinum and taxane resistance in MyD88 deficient epithelial ovarian cancer cells

Gaikwad SM, Thakur B, Sakpal A, Singh RK, Ray P.
Int J Biochem Cell Biol. 2015 Apr; 61:90-102
http://dx.doi.org:/10.1016/j.biocel.2015.02.001

Development of chemoresistance is a major impediment to successful treatment of patients suffering from epithelial ovarian carcinoma (EOC). Among various molecular factors, presence of MyD88, a component of TLR-4/MyD88 mediated NF-κB signaling in EOC tumors is reported to cause intrinsic paclitaxel resistance and poor survival. However, 50-60% of EOC patients do not express MyD88 and one-third of these patients finally relapses and dies due to disease burden. The status and role of NF-κB signaling in this chemoresistant MyD88(negative) population has not been investigated so far. Using isogenic cellular matrices of cisplatin, paclitaxel and platinum-taxol resistant MyD88(negative) A2780 ovarian cancer cells expressing a NF-κB reporter sensor, we showed that enhanced NF-κB activity was required for cisplatin but not for paclitaxel resistance. Immunofluorescence and gel mobility shift assay demonstrated enhanced nuclear localization of NF-κB and subsequent binding to NF-κB response element in cisplatin resistant cells. The enhanced NF-κB activity was measurable from in vivo tumor xenografts by dual bioluminescence imaging. In contrast, paclitaxel and the platinum-taxol resistant cells showed down regulation in NF-κB activity. Intriguingly, silencing of MyD88 in cisplatin resistant and MyD88(positive) TOV21G and SKOV3 cells showed enhanced NF-κB activity after cisplatin but not after paclitaxel or platinum-taxol treatments. Our data thus suggest that NF-κB signaling is important for maintenance of cisplatin resistance but not for taxol or platinum-taxol resistance in absence of an active TLR-4/MyD88 receptor mediated cell survival pathway in epithelial ovarian carcinoma.

7.7.4 Activation of apoptosis by caspase-3-dependent specific RelB cleavage in anticancer agent-treated cancer cells

Kuboki MIto ASimizu SUmezawa K.
Biochem Biophys Res Commun. 2015 Jan 16; 456(3):810-4
http://dx.doi.org:/10.1016/j.bbrc.2014.12.024

Activation of caspase 3 and caspase-dependent apoptosis  nrmicro2071-f1

Activation of caspase 3 and caspase-dependent apoptosis nrmicro2071-f1

Highlights

  • We have prepared RelB mutants that are resistant to caspase 3-induced scission.
  • Vinblastine induced caspase 3-dependent site-specific RelB cleavage in cancer cells.
  • Cancer cells expressing cleavage-resistant RelB showed less sensitivity to vinblastine.
  • Caspase 3-induced RelB cleavage may provide positive feedback mechanism in apoptosis.

DTCM-glutarimide (DTCM-G) is a newly found anti-inflammatory agent. In the course of experiments with lymphoma cells, we found that DTCM-G induced specific RelB cleavage. Anticancer agent vinblastine also induced the specific RelB cleavage in human fibrosarcoma HT1080 cells. The site-directed mutagenesis analysis revealed that the Asp205 site in RelB was specifically cleaved possibly by caspase-3 in vinblastine-treated HT1080 cells. Moreover, the cells stably overexpressing RelB Asp205Ala were resistant to vinblastine-induced apoptosis. Thus, the specific Asp205 cleavage of RelB by caspase-3 would be involved in the apoptosis induction by anticancer agents, which would provide the positive feedback mechanism.

apoptotic-caspases-control-microglia-activation-cdd2011107f3

apoptotic-caspases-control-microglia-activation-cdd2011107f3

 

 

7.7.5 Identification of Liver Cancer Progenitors Whose Malignant Progression Depends on Autocrine IL-6 Signaling

He GDhar DNakagawa HFont-Burgada JOgata HJiang Y, et al.
Cell. 2013 Oct 10; 155(2):384-96
http://dx.doi.org/10.1016%2Fj.cell.2013.09.031

Il-6 signaling in cancer cells

Il-6 signaling in cancer cells

Hepatocellular carcinoma (HCC) is a slowly developing malignancy postulated to evolve from pre-malignant lesions in chronically damaged livers. However, it was never established that premalignant lesions actually contain tumor progenitors that give rise to cancer. Here, we describe isolation and characterization of HCC progenitor cells (HcPCs) from different mouse HCC models. Unlike fully malignant HCC, HcPCs give rise to cancer only when introduced into a liver undergoing chronic damage and compensatory proliferation. Although HcPCs exhibit a similar transcriptomic profile to bipotential hepatobiliary progenitors, the latter do not give rise to tumors. Cells resembling HcPCs reside within dysplastic lesions that appear several months before HCC nodules. Unlike early hepatocarcinogenesis, which depends on paracrine IL-6 production by inflammatory cells, due to upregulation of LIN28 expression, HcPCs had acquired autocrine IL-6 signaling that stimulates their in vivo growth and malignant progression. This may be a general mechanism that drives other IL-6-producing malignancies.

Clonal evolution and selective pressure may cause some descendants of the initial progenitor to cross the bridge of no return and form a premalignant lesion. Cancer genome sequencing indicates that most cancers require at least five genetic changes to evolve (Wood et al., 2007). It has been difficult to isolate and propagate cancer progenitors prior to detection of tumor masses. Further, it is not clear whether cancer progenitors are the precursors for the  cancer stem cells (CSCs)isolated from cancers. An answer to these critical questions depends on identification and isolation of cancer progenitors, which may also enable definition of molecular markers and signaling pathways suitable for early detection and treatment.

Hepatocellular carcinoma (HCC), the end product of chronic liver diseases, requires several decades to evolve (El-Serag, 2011). It is the third most deadly and fifth most common cancer worldwide, and in the United States its incidence has doubled in the past two decades. Furthermore, 8% of the world’s population are chronically infected with hepatitis B or C viruses (HBV and HCV) and are at a high risk of new HCC development (El-Serag, 2011). Up to 5% of HCV patients will develop HCC in their lifetime, and the yearly HCC incidence in patients with cirrhosis is 3%–5%. These tumors may arise from premalignant lesions, ranging from dysplastic foci to dysplastic hepatocyte nodules that are often seen in damaged and cirrhotic livers and are more proliferative than the surrounding parenchyma (Hytiroglou et al., 2007). There is no effective treatment for HCC and, upon diagnosis, most patients with advanced disease have a remaining lifespan of 4–6 months. Premalignant lesions, called foci of altered hepatocytes (FAH), were described in chemically induced HCC models (Pitot, 1990), but it was questioned whether these lesions harbor tumor progenitors or result from compensatory proliferation (Sell and Leffert, 2008). The aim of this study was to determine whether HCC progenitor cells (HcPCs) exist and if so, to isolate these cells and identify some of the signaling networks that are involved in their maintenance and progression.

We now describe HcPC isolation from mice treated with the procarcinogen diethyl nitrosamine (DEN), which induces poorly differentiated HCC nodules within 8 to 9 months (Verna et al., 1996). The use of a chemical carcinogen is justified because the finding of up to 121 mutations per HCC genome suggests that carcinogens may be responsible for human HCC induction (Guichard et al., 2012). Furthermore, 20%–30% of HCC, especially in HBV-infected individuals, evolve in noncirrhotic livers (El-Serag, 2011). Nonetheless, we also isolated HcPCs fromTak1Δhep mice, which develop spontaneous HCC as a result of progressive liver damage, inflammation, and fibrosis caused by ablation of TAK1 (Inokuchi et al., 2010). Although the etiology of each model is distinct, both contain HcPCs that express marker genes and signaling pathways previously identified in human HCC stem cells (Marquardt and Thorgeirsson, 2010) long before visible tumors are detected. Furthermore, DEN-induced premalignant lesions and HcPCs exhibit autocrine IL-6 production that is critical for tumorigenic progression. Circulating IL-6 is a risk indicator in several human pathologies and is strongly correlated with adverse prognosis in HCC and cholangiocarcinoma (Porta et al., 2008Soresi et al., 2006). IL-6 produced by in-vitro-induced CSCs was suggested to be important for their maintenance (Iliopoulos et al., 2009). Little is known about the source of IL-6 in HCC.

DEN-Induced Collagenase-Resistant Aggregates of HCC Progenitors

A single intraperitoneal (i.p.) injection of DEN into 15-day-old BL/6 mice induces HCC nodules first detected 8 to 9 months later. However, hepatocytes prepared from macroscopically normal livers 3 months after DEN administration already contain cells that progress to HCC when transplanted into the permissive liver environment of MUP-uPA mice (He et al., 2010), which express urokinase plasminogen activator (uPA) from a mouse liver-specific major urinary protein (MUP) promoter and undergo chronic liver damage and compensatory proliferation (Rhim et al., 1994). HCC markers such as α fetoprotein (AFP), glypican 3 (Gpc3), and Ly6D, whose expression in mouse liver cancer was reported (Meyer et al., 2003), were upregulated in aggregates from DEN-treated livers, but not in nonaggregated hepatocytes or aggregates from control livers (Figure S1A). Using 70 μm and 40 μm sieves, we separated aggregated from nonaggregated hepatocytes (Figure 1A) and tested their tumorigenic potential by transplantation into MUP-uPA mice (Figure 1B). To facilitate transplantation, the aggregates were mechanically dispersed and suspended in Dulbecco’s modified Eagle’s medium (DMEM). Five months after intrasplenic (i.s.) injection of 104 viable cells, mice receiving cells from aggregates developed about 18 liver tumors per mouse, whereas mice receiving nonaggregated hepatocytes developed less than 1 tumor each (Figure 1B). The tumors exhibited typical trabecular HCC morphology and contained cells that abundantly express AFP (Figure S1B).

Only liver tumors were formed by the transplanted cells. Other organs, including the spleen into which the cells were injected, remained tumor free (Figure 1B), suggesting that HcPCs progress to cancer only in the proper microenvironment. Indeed, no tumors appeared after HcPC transplantation into normal BL/6 mice. But, if BL/6 mice were first treated with retrorsine (a chemical that permanently inhibits hepatocyte proliferation [Laconi et al., 1998]), intrasplenically transplanted with HcPC-containing aggregates, and challenged with CCl4 to induce liver injury and compensatory proliferation (Guo et al., 2002), HCCs readily appeared (Figure 1C). CCl4 omission prevented tumor development. Notably, MUP-uPA or CCl4-treated livers are fragile, rendering direct intrahepatic transplantation difficult. CCl4-induced liver damage, especially within a male liver, generates a microenvironment that drives HcPC proliferation and malignant progression. To examine this point, we transplanted GFP-labeled HcPC-containing aggregates into retrorsine-treated BL/6 mice and examined their ability to proliferate with or without subsequent CCl4 treatment. Indeed, the GFP+ cells formed clusters that grew in size only in CCl4-treated host livers (Figure S1E). Omission of CC14 prevented their expansion.

Because CD44 is expressed by HCC stem cells (Yang et al., 2008Zhu et al., 2010), we dispersed the aggregates and separated CD44+ from CD44 cells and transplanted both into MUP-uPA mice. Whereas as few as 103 CD44+ cells gave rise to HCCs in 100% of recipients, no tumors were detected after transplantation of CD44 cells (Figure 1E). Remarkably, 50% of recipients developed at least one HCC after receiving as few as 102 CD44+ cells.

HcPC-Containing Aggregates in Tak1Δhep Mice

We applied the same HcPC isolation protocol to Tak1Δhep mice, which develop HCC of different etiology from DEN-induced HCC. Importantly, Tak1Δhep mice develop HCC as a consequence of chronic liver injury and fibrosis without carcinogen or toxicant exposure (Inokuchi et al., 2010). Indeed, whole-tumor exome sequencing revealed that DEN-induced HCC contained about 24 mutations per 106 bases (Mb) sequenced, with B-RafV637E being the most recurrent, whereas 1.4 mutations per Mb were detected inTak1Δhep HCC’s exome (Table S1). By contrast, Tak1Δhep HCC exhibited gene copy number changes. HCC developed in 75% of MUP-uPA mice that received dispersed Tak1Δhep aggregates, but no tumors appeared in mice receiving nonaggregated Tak1Δhep or totalTak1f/f hepatocytes (Figure 2B). bile duct ligation (BDL) or feeding with 3,5-dicarbethoxy-1,4-dihydrocollidine (DDC), treatments that cause cholestatic liver injuries and oval cell expansion (Dorrell et al., 2011), did increase the number of small hepatocytic cell aggregates (Figure S2A). Nonetheless, no tumors were observed 5 months after injection of such aggregates into MUP-uPA mice (Figure S2B). Thus, not all hepatocytic aggregates contain HcPCs, and HcPCs only appear under tumorigenic conditions.

The HcPC Transcriptome Is Similar to that of HCC and Oval Cells

To determine the relationship between DEN-induced HcPCs, normal hepatocytes, and fully transformed HCC cells, we analyzed the transcriptomes of aggregated and nonaggregated hepatocytes from male littermates 5 months after DEN administration, HCC epithelial cells from DEN-induced tumors, and normal hepatocytes from age- and gender-matched littermate controls. Clustering analysis distinguished the HCC samples from other samples and revealed that the aggregated hepatocyte samples did not cluster with each other but rather with nonaggregated hepatocytes derived from the same mouse (Figure S3A). 57% (583/1,020) of genes differentially expressed in aggregated relative to nonaggregated hepatocytes are also differentially expressed in HCC relative to normal hepatocytes (Figure 3B, top), a value that is highly significant (p < 7.13 × 10−243). More specifically, 85% (494/583) of these genes are overexpressed in both HCC and HcPC-containing aggregates (Figure 3B, bottom table). Thus, hepatocyte aggregates isolated 5 months after DEN injection contain cells that are related in their gene expression profile to HCC cells isolated from fully developed tumor nodules.

Figure 3 Aggregated Hepatocytes Exhibit an Altered Transcriptome Similar to that of HCC Cells

We examined which biological processes or cellular compartments were significantly overrepresented in the induced or repressed genes in both pairwise comparisons (Gene Ontology Analysis). As expected, processes and compartments that were enriched in aggregated hepatocytes relative to nonaggregated hepatocytes were almost identical to those that were enriched in HCC relative to normal hepatocytes (Figure 3C). Several human HCC markers, including AFP, Gpc3 and H19, were upregulated in aggregated hepatocytes (Figures 3D and 3E). Aggregated hepatocytes also expressed more Tetraspanin 8 (Tspan8), a cell-surface glycoprotein that complexes with integrins and is overexpressed in human carcinomas (Zöller, 2009). Another cell-surface molecule highly expressed in aggregated cells is Ly6D (Figures 3D and 3E). Immunofluorescence (IF) analysis revealed that Ly6D was undetectable in normal liver but was elevated in FAH and ubiquitously expressed in most HCC cells (Figure S3C). A fluorescent-labeled Ly6D antibody injected into HCC-bearing mice specifically stained tumor nodules (Figure S3D). Other cell-surface molecules that were upregulated in aggregated cells included syndecan 3 (Sdc3), integrin α 9 (Itga9), claudin 5 (Cldn5), and cadherin 5 (Cdh5) (Figure 3D). Aggregated hepatocytes also exhibited elevated expression of extracellular matrix proteins (TIF3 and Reln1) and a serine protease inhibitor (Spink3). Elevated expression of such proteins may explain aggregate formation. Aggregated hepatocytes also expressed progenitor cell markers, including the epithelial cell adhesion molecule (EpCAM) (Figure 3E) and Dlk1 (Figure 3D). We compared the HcPC and HCC (Figure 3A) to the transcriptome of DDC-induced oval cells (Shin et al., 2011). This analysis revealed a striking similarity between the HCC, HcPC, and the oval cell transcriptomes (Figure S3B). Despite these similarities, some genes that were upregulated in HcPC-containing aggregates and HCC were not upregulated in oval cells. Such genes may account for the tumorigenic properties of HcPC and HCC.

Figure 4  DEN-Induced HcPC Aggregates Express Pathways and Markers Characteristic of HCC and Hepatobiliary Stem Cells

We examined the aggregates for signaling pathways and transcription factors involved in hepatocarcinogenesis. Many aggregated cells were positive for phosphorylated c-Jun and STAT3 (Figure 4A), transcription factors involved in DEN-induced hepatocarcinogenesis (Eferl et al., 2003He et al., 2010). Sox9, a transcription factor that marks hepatobiliary progenitors (Dorrell et al., 2011), was also expressed by many of the aggregated cells, which were also positive for phosphorylated c-Met (Figure 4A), a receptor tyrosine kinase that is activated by hepatocyte growth factor (HGF) and is essential for liver development (Bladt et al., 1995) and hepatocarcinogenesis (Wang et al., 2001). Few of the nonaggregated hepatocytes exhibited activation of these signaling pathways. Despite different etiology, HcPC-containing aggregates from Tak1Δhep mice exhibit upregulation of many of the same markers and pathways that are upregulated in DEN-induced HcPC-containing aggregates. Flow cytometry confirmed enrichment of CD44+ cells as well as CD44+/CD90+ and CD44+/EpCAM+ double-positive cells in the HcPC-containing aggregates from either DEN-treated or Tak1Δhep livers (Figure S4B).

HcPC-Containing Aggregates Originate from Premalignant Dysplastic Lesions

FAH are dysplastic lesions occurring in rodent livers exposed to hepatic carcinogens (Su et al., 1990). Similar lesions are present in premalignant human livers (Su et al., 1997). Yet, it is still debated whether FAH correspond to premalignant lesions or are a reaction to liver injury that does not lead to cancer (Sell and Leffert, 2008). In DEN-treated males, FAH were detected as early as 3 months after DEN administration (Figure 5A), concomitant with the time at which HcPC-containing aggregates were detected. In females, FAH development was delayed. FAH contained cells positive for the same progenitor cell markers and activated signaling pathways present in HcPC-containing aggregates, including AFP, CD44, and EpCAM (Figure 5C). FAH also contained cells positive for activated STAT3, c-Jun, and PCNA (Figure 5C).

HcPCs Exhibit Autocrine IL-6 Expression Necessary for HCC Progression

In situ hybridization (ISH) and immunohistochemistry (IHC) revealed that DEN-induced FAH contained IL-6-expressing cells (Figures 6A, 6B, and S5), and freshly isolated DEN-induced aggregates contained more IL-6 messenger RNA (mRNA) than nonaggregated hepatocytes (Figure 6C). We examined several factors that control IL-6 expression and found that LIN28A and B were significantly upregulated in HcPCs and HCC (Figures 6D and 6E). LIN28-expressing cells were also detected within FAH (Figure 6F). As reported (Iliopoulos et al., 2009), knockdown of LIN28B in cultured HcPC or HCC cell lines decreased IL-6 expression (Figure 6G). LIN28 exerts its effects through downregulation of the microRNA (miRNA) Let-7 (Iliopoulos et al., 2009).

Figure 6  Liver Premalignant Lesions and HcPCs Exhibit Elevated IL-6 and LIN28 Expression

Figure 7  HCC Growth Depends on Autocrine IL-6 Production

The isolation and characterization of cells that can give rise to HCC only after transplantation into an appropriate host liver undergoing chronic injury demonstrates that cancer arises from progenitor cells that are yet to become fully malignant. Importantly, unlike fully malignant HCC cells, the HcPCs we isolated cannot form s.c. tumors or even liver tumors when introduced into a nondamaged liver. Liver damage induced by uPA expression or CCl4 treatment provides HcPCs with the proper cytokine and growth factor milieu needed for their proliferation. Although HcPCs produce IL-6, they may also depend on other cytokines such as TNF, which is produced by macrophages that are recruited to the damaged liver. In addition, uPA expression and CCl4 treatment may enhance HcPC growth and progression through their fibrogenic effect on hepatic stellate cells. Although HCC and other cancers have been suspected to arise from premalignant/dysplastic lesions (Hruban et al., 2007Hytiroglou et al., 2007), a direct demonstration that such lesions progress into malignant tumors has been lacking. Based on expression of common markers—EpCAM, CD44, AFP, activated STAT3, and IL-6—that are not expressed in normal hepatocytes, we postulate that HcPCs originate from FAH or dysplastic foci, which are first observed in male mice within 3 months of DEN exposure.

7.7.6 Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth

Lin R1Tao RGao XLi TZhou XGuan KLXiong YLei QY.
Mol Cell. 2013 Aug 22; 51(4):506-18
http://dx.doi.org:/10.1016/j.molcel.2013.07.002

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

Fatty acid synthesis occurs at low rates in most nondividing cells of normal tissues that primarily uptake lipids from circulation. In contrast, increased lipogenesis, especially de novo lipid synthesis, is a key characteristic of cancer cells. Many studies have demonstrated that in cancer cells, fatty acids are preferred to be derived from de novo synthesis instead of extracellular lipid supply (Medes et al., 1953Menendez and Lupu, 2007;Ookhtens et al., 1984Sabine et al., 1967). Fatty acids are key building blocks for membrane biogenesis, and glucose serves as a major carbon source for de novo fatty acid synthesis (Kuhajda, 2000McAndrew, 1986;Swinnen et al., 2006). In rapidly proliferating cells, citrate generated by the tricarboxylic acid (TCA) cycle, either from glucose by glycolysis or glutamine by anaplerosis, is preferentially exported from mitochondria to cytosol and then cleaved by ATP citrate lyase (ACLY) (Icard et al., 2012) to produce cytosolic acetyl coenzyme A (acetyl-CoA), which is the building block for de novo lipid synthesis. As such, ACLY couples energy metabolism with fatty acids synthesis and plays a critical role in supporting cell growth. The function of ACLY in cell growth is supported by the observation that inhibition of ACLY by chemical inhibitors or RNAi dramatically suppresses tumor cell proliferation and induces differentiation in vitro and in vivo (Bauer et al., 2005Hatzivassiliou et al., 2005). In addition, ACLY activity may link metabolic status to histone acetylation by providing acetyl-CoA and, therefore, gene expression (Wellen et al., 2009).

While ACLY is transcriptionally regulated by sterol regulatory element-binding protein 1 (SREBP-1) (Kim et al., 2010), ACLY activity is regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Berwick et al., 2002Migita et al., 2008Pierce et al., 1982). Akt can directly phosphorylate and activate ACLY (Bauer et al., 2005Berwick et al., 2002Migita et al., 2008Potapova et al., 2000). Covalent lysine acetylation has recently been found to play a broad and critical role in the regulation of multiple metabolic enzymes (Choudhary et al., 2009Zhao et al., 2010). In this study, we demonstrate that ACLY protein is acetylated on multiple lysine residues in response to high glucose. Acetylation of ACLY blocks its ubiquitinylation and degradation, thus leading to ACLY accumulation and increased fatty acid synthesis. Our observations reveal a crosstalk between protein acetylation and ubiquitylation in the regulation of fatty acid synthesis and cell growth.

Acetylation of ACLY at Lysines 540, 546, and 554

Recent mass spectrometry-based proteomic analyses have potentially identified a large number of acetylated proteins, including ACLY (Figure S1A available online; Choudhary et al., 2009Zhao et al., 2010). We detected the acetylation level of ectopically expressed ACLY followed by western blot using pan-specific anti-acetylated lysine antibody. ACLY was indeed acetylated, and its acetylation was increased by nearly 3-fold after treatment with nicotinamide (NAM), an inhibitor of the SIRT family deacetylases, and trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC) class I and class II (Figure 1A). Experiments with endogenous ACLY also showed that TSA and NAM treatment enhanced ACLY acetylation (Figure 1B).

Figure 1  ACLY Is Acetylated at Lysines 540, 546, and 554

Ten putative acetylation sites were identified by mass spectrometry analyses (Table S1). We singly mutated each lysine to either a glutamine (Q) or an arginine (R) and found that no single mutation resulted in a significant reduction of ACLY acetylation (data not shown), indicating that ACLY may be acetylated at multiple lysine residues. Three lysine residues, K540, K546, and K554, received high scores in the acetylation proteomic screen and are evolutionarily conserved from C. elegans to mammals (Figure S1A). We generated triple Q and R mutants of K540, K546, and K554 (3KQ and 3KR) and found that both 3KQ and 3KR mutations resulted in a significant (~60%) decrease in ACLY acetylation (Figure 1C), indicating that 3K are the major acetylation sites of ACLY.  Further, we found that the acetylation of endogenous ACLY is clearly increased after treatment of cells with NAM and TSA (Figure 1D). These results demonstrate that ACLY is acetylated at K540, K546, and K554.

Glucose Promotes ACLY Acetylation to Stabilize ACLY

In mammalian cells, glucose is the main carbon source for de novo lipid synthesis. We found that ACLY levels increased with increasing glucose concentration, which also correlated with increased ACLY 3K acetylation (Figure 1E). Furthermore, to confirm whether the glucose level affects ACLY protein stability in vivo, we intraperitoneally injected glucose in BALB/c mice and found that high glucose resulted in a significant increase of ACLY protein levels (Figure 1F).

To determine whether ACLY acetylation affects its protein levels, we treated HeLa and Chang liver cells with NAM and TSA and found an increase in ACLY protein levels (Figure S1G, upper panel). ACLY mRNA levels were not significantly changed by the treatment of NAM and TSA (Figure S1G, lower panel), indicating that this upregulation of ACLY is mostly achieved at the posttranscriptional level. Indeed, ACLY protein was also accumulated in cells treated with the proteasome inhibitor MG132, indicating that ACLY stability could be regulated by the ubiquitin-proteasome pathway (Figure 1G). Blocking deacetylase activity stabilized ACLY (Figure S1H). The stabilization of ACLY induced by high glucose was associated with an increase of ACLY acetylation at K540, K546, and K554. Together, these data support a notion that high glucose induces both ACLY acetylation and protein stabilization and prompted us to ask whether acetylation directly regulates ACLY stability. We then generated ACLYWT, ACLY3KQ, and ACLY3KRstable cells after knocking down the endogenous ACLY. We found that the ACLY3KR or ACLY3KQmutant was more stable than the ACLYWT (Figures 1I and S1I). Collectively, our results suggest that glucose induces acetylation at K540, 546, and 554 to stabilize ACLY.

Acetylation Stabilizes ACLY by Inhibiting Ubiquitylation

To determine the mechanism underlying the acetylation and ACLY protein stability, we first examined ACLY ubiquitylation and found that it was actively ubiquitylated (Figure 2A). Previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). In order to identify the ubiquitylation sites, we tested the ubiquitylation levels of double mutants 540R–546R and 546–554R (Figure S2A). We found that the ubiquitylation of the 540R-546R and 546R-554R mutants is partially decreased, while mutation of K540, K546, and K554 (3KR), which changes all three putative acetylation lysine residues of ACLY to arginine residues, dramatically reduced the ACLY ubiquitylation level (Figures 2B and S2A), indicating that 3K lysines might also be the ubiquitylation target residues. Moreover, inhibition of deacetylases by NAM and TSA decreased ubiquitylation of WT but not 3KQ or 3KR mutant ACLY (Figure 2C). These results implicate an antagonizing role of the acetylation towards the ubiquitylation of ACLY at these three lysine residues.

Figure 2  Acetylation Protects ACLY from Proteasome Degradation by Inhibiting Ubiquitylation

We found that ACLY acetylation was only detected in the nonubiquitylated, but not the ubiquitylated (high-molecular-weight), ACLY species. This result indicates that ACLY acetylation and ubiquitylation are mutually exclusive and is consistent with the model that K540, K546, and K554 are the sites of both ubiquitylation and acetylation. Therefore, acetylation of these lysines would block ubiquitylation.

We also found that glucose upregulates ACLY acetylation at 3K and decreases its ubiquitylation (Figure S2B). High glucose (25 mM) effectively decreased ACLY ubiquitylation, while inhibition of deacetylases clearly diminished its ubiquitylation (Figure 2E). We conclude that acetylation and ubiquitylation occur mutually exclusively at K540, K546, and K554 and that high-glucose-induced acetylation at these three sites blocks ACLY ubiquitylation and degradation.

UBR4 Targets ACLY for Degradation

UBR4 was identified as a putative ACLY-interacting protein by affinity purification coupled with mass spectrometry analysis (data not shown). To address if UBR4 is a potential ACLY E3 ligase, we determined the interaction between ACLY and UBR4 and found that ACLY interacted with the E3 ligase domain of UBR4; this interaction was enhanced by MG132 treatment (Figure 3A). UBR4 knockdown in A549 cells resulted in an increase of endogenous ACLY protein level (Figure 3C). Moreover, UBR4 knockdown significantly stabilized ACLY (Figure 3D) and decreased ACLY ubiquitylation (Figure 3E). Taken together, these results indicate that UBR4 is an ACLY E3 ligase that responds to glucose regulation.

Figure 3  UBR4 Is the E3 Ligase of ACLY

PCAF Acetylates ACLY

PCAF knockdown significantly reduced acetylation of 3K, indicating that PCAF is a potential 3K acetyltransferase in vivo (Figure 4C, upper panel). Furthermore, PCAF knockdown decreased the steady-state level of endogenous ACLY, but not ACLY mRNA (Figure 4C, middle and lower panels). Moreover, we found that PCAF knockdown destabilized ACLY (Figure 4D). In addition, overexpression of PCAF decreases ACLY ubiquitylation (Figure 4E), while PCAF inhibition increases the interaction between UBR4 E3 ligase domain and wild-type ACLY, but not 3KR (Figure 4F). Together, our results indicate that PCAF increases ACLY protein level, possibly via acetylating ACLY at 3K.

Figure 4  PCAF Is the Acetylase of ACLY

SIRT2 Deacetylates ACLY

Figure 5  SIRT2 Decreases ACLY Acetylation and Increases Its Protein Levels In Vivo

Acetylation of ACLY Promotes Cell Proliferation and De Novo Lipid Synthesis

The protein levels of ACLY 3KQ and 3KR were accumulated to a level higher than the wild-type cells upon extended culture in low-glucose medium (Figure S6A, right panel), indicating a growth advantage conferred by ACLY stabilization resulting from the disruption of both acetylation and ubiquitylation at K540, K546, and K554. Cellular acetyl-CoA assay showed that cells expressing 3KQ or 3KR mutant ACLY produce more acetyl-CoA than cells expressing the wild-type ACLY under low glucose (Figures 6B and S6B), further supporting the conclusion that 3KQ or 3KR mutation stabilizes ACLY.

Figure 6  Acetylation of ACLY at 3K Promotes Lipogenesis and Tumor Cell Proliferation

ACLY is a key enzyme in de novo lipid synthesis. Silencing ACLY inhibited the proliferation of multiple cancer cell lines, and this inhibition can be partially rescued by adding extra fatty acids or cholesterol into the culture media (Zaidi et al., 2012). This prompted us to measure extracellular lipid incorporation in A549 cells after knockdown and ectopic expression of ACLY. We found that when cultured in low glucose (2.5 mM), cells expressing wild-type ACLY uptake significantly more phospholipids compared to cells expressing 3KQ or 3KR mutant ACLY (Figures 6C, 6D, and S6D). When cultured in the presence of high glucose (25 mM), however, cells expressing either the wild-type, 3KQ, or 3KR mutant ACLY all have reduced, but similar, uptake of extracellular phospholipids (Figures 6C, 6D, and S6D). The above results are consistent with a model that acetylation of ACLY induced by high glucose increases its stability and stimulates de novo lipid synthesis.

3K Acetylation of ACLY Is Increased in Lung Cancer

ACLY is reported to be upregulated in human lung cancer (Migita et al., 2008). Many small chemicals targeting ACLY have been designed for cancer treatment (Zu et al., 2012). The finding that 3KQ or 3KR mutant increased the ability of ACLY to support A549 lung cancer cell proliferation prompted us to examine 3K acetylation in human lung cancers. We collected a total of 54 pairs of primary human lung cancer samples with adjacent normal lung tissues and performed immunoblotting for ACLY protein levels. This analysis revealed that, when compared to the matched normal lung tissues, 29 pairs showed a significant increase of total ACLY protein using b-actin as a loading control (Figures 7A and S7A). The tumor sample analyses demonstrate that ACLY protein levels are elevated in lung cancers, and 3K acetylation positively correlates with the elevated ACLY protein. These data also indicate that ACLY with 3K acetylation may be potential biomarker for lung cancer diagnosis.

Figure 7
  Acetylation of ACLY at 3K Is Upregulated in Human Lung Carcinoma

Dysregulation of cellular metabolism is a hallmark of cancer (Hanahan and Weinberg, 2011Vander Heiden et al., 2009). Besides elevated glycolysis, increased lipogenesis, especially de novo lipid synthesis, also plays an important role in tumor growth. Because most carbon sources for fatty acid synthesis are from glucose in mammalian cells (Wellen et al., 2009), the channeling of carbon into de novo lipid synthesis as building blocks for tumor cell growth is primarily linked to acetyl-CoA production by ACLY. Moreover, the ACLY-catalyzed reaction consumes ATP. Therefore, as the key cellular energy and carbon source, one may expect a role for glucose in ACLY regulation. In the present study, we have uncovered a mechanism of ACLY regulation by glucose that increases ACLY protein level to meet the enhanced demand of lipogenesis in growing cells, such as tumor cells (Figure 7C). Glucose increases ACLY protein levels by stimulating its acetylation.

Upregulation of ACLY is common in many cancers (Kuhajda, 2000Milgraum et al., 1997Swinnen et al., 2004Yahagi et al., 2005). This is in part due to the transcriptional activation by SREBP-1 resulting from the activation of the PI3K/AKT pathway in cancers (Kim et al., 2010Nadler et al., 2001Wang and Dey, 2006). In this study, we report a mechanism of ACLY regulation at the posttranscriptional level. We propose that acetylation modulated by glucose status plays a crucial role in coordinating the intracellular level of ACLY, hence fatty acid synthesis, and glucose availability. When glucose is sufficient, lipogenesis is enhanced. This can be achieved, at least in part, by the glucose-induced stabilization of ACLY. High glucose increases ACLY acetylation, which inhibits its ubiquitylation and degradation, leading to the accumulation of ACLY and enhanced lipogenesis. In contrast, when glucose is limited, ACLY is not acetylated and thus can be ubiquitylated, leading to ACLY degradation and reduced lipogenesis. Moreover, our data indicate that acetylation and ubiquitylation in ACLY may compete with each other by targeting the same lysine residues at K540, K546, and K554. Consistently, previous proteomic analyses have identified K546 in ACLY as a ubiquitylation site (Wagner et al., 2011). Similar models of different modifications on the same lysine residues have been reported in the regulation of other proteins (Grönroos et al., 2002Li et al., 20022012). We propose that acetylation and ubiquitylation have opposing effects in the regulation of ACLY by competitively modifying the same lysine residues. The acetylation-mimetic 3KQ and the acetylation-deficient 3KR mutants behaved indistinguishably in most biochemical and functional assays, mainly due to the fact that these mutations disrupt lysine ubiquitylation that primarily occurs on these three residues.

ACLY is increased in lung cancer tissues compared to adjacent tissues. Consistently, ACLY acetylation at 3K is also significantly increased in lung cancer tissues. These observations not only confirm ACLY acetylation in vivo, but also suggest that ACLY 3K acetylation may play a role in lung cancer development. Our study reveals a mechanism of ACLY regulation in response to glucose signals.

 

7.7.7 Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Nomura DK1Long JZNiessen SHoover HSNg SWCravatt BF.
Cell. 2010 Jan 8; 140(1):49-61
http://dx.doi.org/10.1016.2Fj.cell.2009.11.027

Highlights

  • Monoacylglycerol lipase (MAGL) is elevated in aggressive human cancer cells
  • Loss of MAGL lowers fatty acid levels in cancer cells and impairs pathogenicity
  • MAGL controls a signaling network enriched in protumorigenic lipids
  • A high-fat diet can restore the growth of tumors lacking MAGL in vivo
monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

monoacylglycerol-lipase-magl-is-highly-expressed-in-aggressive-human-cancer-cells-and-primary-tumors

http://www.cell.com/cms/attachment/1082768/7977146/fx1.jpg

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity—phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals.

We show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in non-aggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity — phenotypes that are reversed by an MAGL inhibitor. Interestingly, impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of pro-tumorigenic signals.

The conversion of cells from a normal to cancerous state is accompanied by reprogramming of metabolic pathways (Deberardinis et al., 2008Jones and Thompson, 2009Kroemer and Pouyssegur, 2008), including those that regulate glycolysis (Christofk et al., 2008Gatenby and Gillies, 2004), glutamine-dependent anaplerosis (DeBerardinis et al., 2008DeBerardinis et al., 2007Wise et al., 2008), and the production of lipids (DeBerardinis et al., 2008Menendez and Lupu, 2007). Despite a growing appreciation that dysregulated metabolism is a defining feature of cancer, it remains unclear, in many instances, how such biochemical changes occur and whether they play crucial roles in disease progression and malignancy.

Among dysregulated metabolic pathways, heightened de novo lipid biosynthesis, or the development a “lipogenic” phenotype (Menendez and Lupu, 2007), has been posited to play a major role in cancer. For instance, elevated levels of fatty acid synthase (FAS), the enzyme responsible for fatty acid biosynthesis from acetate and malonyl CoA, are correlated with poor prognosis in breast cancer patients, and inhibition of FAS results in decreased cell proliferation, loss of cell viability, and decreased tumor growth in vivo (Kuhajda et al., 2000Menendez and Lupu, 2007Zhou et al., 2007). FAS may support cancer growth, at least in part, by providing metabolic substrates for energy production (via fatty acid oxidation) (Buzzai et al., 2005Buzzai et al., 2007Liu, 2006). Many other features of lipid biochemistry, however, are also critical for supporting the malignancy of cancer cells, including:

Prominent examples of lipid messengers that contribute to cancer include:

Here, we use functional proteomic methods to discover a lipolytic enzyme, monoacylglycerol lipase (MAGL), that is highly elevated in aggressive cancer cells from multiple tissues of origin. We show that MAGL, through hydrolysis of monoacylglycerols (MAGs), controls free fatty acid (FFA) levels in cancer cells. The resulting MAGL-FFA pathway feeds into a diverse lipid network enriched in pro-tumorigenic signaling molecules and promotes migration, survival, and in vivo tumor growth. Aggressive cancer cells thus pair lipogenesis with high lipolytic activity to generate an array of pro-tumorigenic signals that support their malignant behavior.

Activity-Based Proteomic Analysis of Hydrolytic Enzymes in Human Cancer Cells

To identify enzyme activities that contribute to cancer pathogenesis, we conducted a functional proteomic analysis of a panel of aggressive and non-aggressive human cancer cell lines from multiple tumors of origin, including melanoma [aggressive (C8161, MUM2B), non-aggressive (MUM2C)], ovarian [aggressive (SKOV3), non-aggressive (OVCAR3)], and breast [aggressive (231MFP), non-aggressive (MCF7)] cancer. Aggressive cancer lines were confirmed to display much greater in vitro migration and in vivo tumor-growth rates compared to their non-aggressive counterparts (Figure S1), as previously shown (Jessani et al., 2004;Jessani et al., 2002Seftor et al., 2002Welch et al., 1991). Proteomes from these cancer lines were screened by activity-based protein profiling (ABPP) using serine hydrolase-directed fluorophosphonate (FP) activity-based probes (Jessani et al., 2002Patricelli et al., 2001). Serine hydrolases are one of the largest and most diverse enzyme classes in the human proteome (representing ~ 1–1.5% of all human proteins) and play important roles in many biochemical processes of potential relevance to cancer, such as proteolysis (McMahon and Kwaan, 2008Puustinen et al., 2009), signal transduction (Puustinen et al., 2009), and lipid metabolism (Menendez and Lupu, 2007Zechner et al., 2005). The goal of this study was to identify hydrolytic enzyme activities that were consistently altered in aggressive versus non-aggressive cancer lines, working under the hypothesis that these conserved enzymatic changes would have a high probability of contributing to the pathogenic state of cancer cells.

Among the more than 50 serine hydrolases detected in this analysis (Tables S13), two enzymes, KIAA1363 and MAGL, were found to be consistently elevated in aggressive cancer cells relative to their non-aggressive counterparts, as judged by spectral counting (Jessani et al., 2005Liu et al., 2004). We confirmed elevations in KIAA1363 and MAGL in aggressive cancer cells by gel-based ABPP, where proteomes are treated with a rhodamine-tagged FP probe and resolved by 1D-SDS-PAGE and in-gel fluorescence scanning (Figure 1A). In both cases, two forms of each enzyme were detected (Figure 1A), due to differential glycoslyation for KIAA1363 (Jessani et al., 2002), and possibly alternative splicing for MAGL (Karlsson et al., 2001). We have previously shown that KIAA1363 plays a role in regulating ether lipid signaling pathways in aggressive cancer cells (Chiang et al., 2006). On the other hand, very little was known about the function of MAGL in cancer.

Figure 1  MAGL is elevated in aggressive cancer cells, where the enzyme regulates monoacylgycerol (MAG) and free fatty acid (FFA) levels

The heightened activity of MAGL in aggressive cancer cells was confirmed using the substrate C20:4 MAG (Figure 1B). Since several enzymes have been shown to display MAG hydrolytic activity (Blankman et al., 2007), we confirmed the contribution that MAGL makes to this process in cancer cells using the potent and selective MAGL inhibitor JZL184 (Long et al., 2009a).

MAGL Regulates Free Fatty Acid Levels in Aggressive Cancer Cells

MAGL is perhaps best recognized for its role in degrading the endogenous cannabinoid 2-arachidonoylglycerol (2-AG, C20:4 MAG), as well as other MAGs, in brain and peripheral tissues (Dinh et al., 2002Long et al., 2009aLong et al., 2009bNomura et al., 2008). Consistent with this established function, blockade of MAGL by JZL184 (1 μM, 4 hr) produced significant elevations in the levels of several MAGs, including 2-AG, in each of the aggressive cancer cell lines (Figure 1C and Figure S2). Interestingly, however, MAGL inhibition also caused significant reductions in the levels of FFAs in aggressive cancer cells (Figure 1D and Figure S2). This surprising finding contrasts with the function of MAGL in normal tissues, where the enzyme does not, in general, control the levels of FFAs (Long et al., 2009aLong et al., 2009b;Nomura et al., 2008).

Metabolic labeling studies using the non-natural C17:0-MAG confirmed that MAGs are converted to LPC and LPE by aggressive cancer cells, and that this metabolic transformation is significantly enhanced by treatment with JZL184 (Figure S1). Finally, JZL184 treatment did not affect the levels of MAGs and FFAs in non-aggressive cancer lines (Figure 1C, D), consistent with the negligible expression of MAGL in these cells (Figure 1A, B).

We next stably knocked down MAGL expression by RNA interference technology using two independent shRNA probes (shMAGL1, shMAGL2), both of which reduced MAGL activity by 70–80% in aggressive cancer lines (Figure 2A, D and Figure S2). Other serine hydrolase activities were unaffected by shMAGL probes (Figure 2A, D and Figures S2), confirming the specificity of these reagents. Both shMAGL probes caused significant elevations in MAGs and corresponding reductions in FFAs in aggressive melanoma (Figure 2B, C), ovarian (Figure 2E, F), and breast cancer cells (Figure S2).

Figure 2  Stable shRNA-mediated knockdown of MAGL lowers FFA levels in aggressive cancer cells.

Together, these data demonstrate that both acute (pharmacological) and stable (shRNA) blockade of MAGL cause elevations in MAGs and reductions in FFAs in aggressive cancer cells. These intriguing findings indicate that MAGL is the principal regulator of FFA levels in aggressive cancer cells. Finally, we confirmed that MAGL activity (Figure 3A, B) and FFA levels (Figure 3C) are also elevated in high-grade primary human ovarian tumors compared to benign or low-grade tumors. Thus, heightened expression of the MAGL-FFA pathway is a prominent feature of both aggressive human cancer cell lines and primary tumors.

Figure 3  High-grade primary human ovarian tumors possess elevated MAGL activity and FFAs compared to benign tumors.

Disruption of MAGL Expression and Activity Impairs Cancer Pathogenicity

shMAGL cancer lines were next examined for alterations in pathogenicity using a set of in vitro and in vivo assays. shMAGL-melanoma (C8161), ovarian (SKOV3), and breast (231MFP) cancer cells exhibited significantly reduced in vitro migration (Figure 4A, F and Figure S2), invasion (Figure 4B, G and Figure S2), and cell survival under serum-starvation conditions (Figure 4C, H and Figure S2). Acute pharmacological blockade of MAGL by JZL184 also decreased cancer cell migration (Figure S2), but not survival, possibly indicating that maximal impairments in cancer aggressiveness require sustained inhibition of MAGL.

Figure 4  shRNA-mediated knockdown and pharmacological inhibition of MAGL impair cancer aggressiveness.

MAGL Overexpression Increases FFAs and the Aggressiveness of Cancer Cells

Stable MAGL-overexpressing (MAGL-OE) and control [expressing an empty vector or a catalytically inactive version of MAGL, where the serine nucleophile was mutated to alanine (S122A)] variants of MUM2C and OVCAR3 cells were generated by retroviral infection and evaluated for their respective MAGL activities by ABPP and C20:4 MAG substrate assays. Both assays confirmed that MAGL-OE cells possess greater than 10-fold elevations in MAGL activity compared to control cells (Figure 5A and Figure S4). MAGL-OE cells also showed significant reductions in MAGs (Figure 5B andFigure S4) and elevated FFAs (Figure 5C and Figure S4). This altered metabolic profile was accompanied by increased migration (Figure 5D and Figure S4), invasion (Figure 5E and Figure S4), and survival (Figure S4) in MAGL-OE cells. None of these effects were observed in cancer cells expressing the S122A MAGL mutant, indicating that they require MAGL activity.  MAGL-OE MUM2C cells also showed enhanced tumor growth in vivo compared to control cells (Figure 5F). Notably, the increased tumor growth rate of MAGL-OE MUM2C cells nearly matched that of aggressive C8161 cells (Figure S4). These data indicate that the ectopic expression of MAGL in non-aggressive cancer cells is sufficient to elevate their FFA levels and promote pathogenicity both in vitro and in vivo.

Figure 5 Ectopic expression of MAGL elevates FFA levels and enhances the in vitro and in vivo pathogenicity of MUM2C melanoma cells.

Metabolic Rescue of Impaired Pathogenicity in MAGL-Disrupted Cancer Cells

MAGL could support the aggressiveness of cancer cells by either reducing the levels of its MAG substrates, elevating the levels of its FFA products, or both. Among MAGs, the principal signaling molecule is the endocannabinoid 2-AG, which activates the CB1 and CB2 receptors (Ahn et al., 2008Mackie and Stella, 2006). The endocannabinoid system has been implicated previously in cancer progression and, depending on the specific study, shown to promote (Sarnataro et al., 2006Zhao et al., 2005) or suppress (Endsley et al., 2007Wang et al., 2008) cancer pathogenesis. Neither a CB1 or CB2 antagonist rescued the migratory defects of shMAGL cancer cells (Figure S5). CB1 and CB2 antagonists also did not affect the levels of MAGs or FFAs in cancer cells (Figure S5).

We then determined whether increased FFA delivery could rectify the tumor growth defect observed for shMAGL cells in vivo. Immune-deficient mice were fed either a normal chow or high-fat diet throughout the duration of a xenograft tumor growth experiment. Notably, the impaired tumor growth rate of shMAGL-C8161 cells was completely rescued in mice fed a high-fat diet. In contrast, shControl-C8161 cells showed equivalent tumor growth rates on a normal versus high-fat diet. The recovery in tumor growth for shMAGL-C8161 cells in the high-fat diet group correlated with significantly increases levels of FFAs in excised tumors (Figure 6D). Collectively, these results indicate that MAGL supports the pathogenic properties of cancer cells by maintaining tonically elevated levels of FFAs.

Figure 6  Recovery of the pathogenic properties of shMAGL cancer cells by treatment with exogenous fatty acids.

MAGL Regulates a Fatty Acid Network Enriched in Pro-Tumorigenic Signals

Studies revealed that neither

  • the MAGL-FFA pathway might serve as a means to regenerate NAD+ (via continual fatty acyl glyceride/FFA recycling) to fuel glycolysis, or
  • increased lipolysis could be to generate FFA substrates for β-oxidation, which may serve as an important energy source for cancer cells (Buzzai et al., 2005), or
  • CPT1 blockade (reduced expression of CPT1 in aggressive cancer cells (data not shown) has been reported previously (Deberardinis et al., 2006))

providing evidence against a role for β-oxidation as a downstream mediator of the pathogenic effects of the MAGL-fatty acid pathway.

Considering that FFAs are fundamental building blocks for the production and remodeling of membrane structures and signaling molecules, perturbations in MAGL might be expected to affect several lipid-dependent biochemical networks important for malignancy. To test this hypothesis, we performed lipidomic analyses of cancer cell models with altered MAGL activity, including comparisons of:

  1. MAGL-OE versus control cancer cells (OVCAR3, MUM2C), and
  2. shMAGL versus shControl cancer cells (SKOV3, C8161).

Complementing these global profiles, we also conducted targeted measurements of specific bioactive lipids (e.g., prostaglandins) that are too low in abundance for detection by standard lipidomic methods. The resulting data sets were then mined to identify a common signature of lipid metabolites regulated by MAGL, which we defined as metabolites that were significantly increased or reduced in MAGL–OE cells and showed the opposite change in shMAGL cells relative to their respective control groups (Figure 7A, B and Table S4).

Figure 7  MAGL regulates a lipid network enriched in pro-tumorigenic signaling molecules.

Most of the lipids in the MAGL-fatty acid network, including several lysophospholipids (LPC, LPA, LPE), ether lipids (MAGE, alkyl LPE), phosphatidic acid (PA), and prostaglandin E2 (PGE2), displayed similar profiles to FFAs, being consistently elevated and reduced in MAGL-OE and shMAGL cells, respectively. Only MAGs were found to show the opposite profile (elevated and reduced in shMAGL and MAGL-OE cells, respectively). Interestingly, virtually this entire lipidomic signature was also observed in aggressive cancer cells when compared to their non-aggressive counterparts (e.g., C8161 versus MUM2C and SKOV3 versus OVCAR3, respectively; Table S4). These findings demonstrate that MAGL regulates a lipid network in aggressive cancer cells that consists of not only FFAs and MAGs, but also a host of secondary lipid metabolites. Increases (rather than decreases) in LPCs and LPEs were observed in JZL184-treated cells (Figure S1 and Table S4). These data indicate that acute and chronic blockade of MAGL generate distinct metabolomic effects in cancer cells, likely reflecting the differential outcomes of short- versus long-term depletion of FFAs.

Within the MAGL-fatty acid network are several pro-tumorigenic lipid messengers, including LPA and PGE2, that have been reported to promote the aggressiveness of cancer cells (Gupta et al., 2007Mills and Moolenaar, 2003). Metabolic labeling studies confirmed that aggressive cancer cells can convert both MAGs and FFAs (Figure S1) to LPA and PGE2 and, for MAGs, this conversion was blocked by JZL184 (Figure S1). Interestingly, treatment with either LPA or PGE2 (100 nM, 4 hr) rescued the impaired migration of shMAGL cancer cells at concentrations that did not affect the migration of shControl cells (Figure 7E).

Heightened lipogenesis is an established early hallmark of dysregulated metabolism and pathogenicity in cancer (Menendez and Lupu, 2007). Cancer lipogenesis appears to be driven principally by FAS, which is elevated in most transformed cells and important for survival and proliferation (De Schrijver et al., 2003;Kuhajda et al., 2000Vazquez-Martin et al., 2008). It is not yet clear how FAS supports cancer growth, but most of the proposed mechanisms invoke pro-tumorigenic functions for the enzyme s fatty acid products and their lipid derivatives (Menendez and Lupu, 2007). This creates a conundrum, since the fatty acid molecules produced by FAS are thought to be rapidly incorporated into neutral- and phospho-lipids, pointing to the need for complementary lipolytic pathways in cancer cells to release stored fatty acids for metabolic and signaling purposes (Prentki and Madiraju, 2008Przybytkowski et al., 2007). Consistent with this hypothesis, we found that acute treatment with the FAS inhibitor C75 (40 μM, 4 h) did not reduce FFA levels in cancer cells (data not shown). Furthermore, aggressive and non-aggressive cancer cells exhibited similar levels of FAS (data not shown), indicating that lipogenesis in the absence of paired lipolysis may be insufficient to confer high levels of malignancy.

Here we show that aggressive cancer cells do indeed acquire the ability to liberate FFAs from neutral lipid stores as a consequence of heightened expression of MAGL. MAGL and its FFA products were found to be elevated in aggressive human cancer cell lines from multiple tissues of origin, as well as in high-grade primary human ovarian tumors. These data suggest that the MAGL-FFA pathway may be a conserved feature of advanced forms of many types of cancer. Further evidence in support of this premise originates from gene expression profiling studies, which have identified increased levels of MAGL in primary human ductal breast tumors compared to less malignant medullary breast tumors (Gjerstorff et al., 2006). The key role that MAGL plays in regulating FFA levels in aggressive cancer cells contrasts with the function of this enzyme in normal tissues, where it mainly controls the levels of MAGs, but not FFAs (Long et al., 2009b). These data thus provide a striking example of the co-opting of an enzyme by cancer cells to serve a distinct metabolic purpose that supports their pathogenic behavior.

Taken together, our results indicate that MAGL serves as key metabolic hub in aggressive cancer cells, where the enzyme regulates a fatty acid network that feeds into a number of pro-tumorigenic signaling pathways.

 

7.7.8 Pirin regulates epithelial to mesenchymal transition and down-regulates EAF/U19 signaling in prostate cancer cells

7.7.8.1  Pirin regulates epithelial to mesenchymal transition independently of Bcl3-Slug signaling

Komai K1Niwa Y1Sasazawa Y1Simizu S2.
FEBS Lett. 2015 Mar 12; 589(6):738-43
http://dx.doi.org:/10.1016/j.febslet.2015.01.040

Highlights

  • Pirin decreases E-cadherin expression and induces EMT.
  • The induction of EMT by Pirin is achieved through a Bcl3 independent pathway.
  • Pirin may be a novel target for cancer therapy.

Epithelial to mesenchymal transition (EMT) is an important mechanism for the initial step of metastasis. Proteomic analysis indicates that Pirin is involved in metastasis. However, there are no reports demonstrating its direct contribution. Here we investigated the involvement of Pirin in EMT. In HeLa cells, Pirin suppressed E-cadherin expression and regulated the expression of other EMT markers. Furthermore, cells expressing Pirin exhibited a spindle-like morphology, which is reminiscent of EMT. A Pirin mutant defective for Bcl3 binding decreased E-cadherin expression similar to wild-type, suggesting that Pirin regulates E-cadherin independently of Bcl3-Slug signaling. These data provide direct evidence that Pirin contributes to cancer metastasis.

Pirin regulates the expression of E-cadherin and EMT markers

In melanoma, Pirin enhances NF-jB activity and increases Slug expression by binding Bcl3 [31], and it may also be involved in adenoid cystic tumor metastasis [23]. Since Slug suppresses E-cadherin transcription and is recognized as a major EMT inducer, we hypothesized that Pirin may regulate EMT through inducing Slug expression. To investigate whether Pirin regulates EMT, we measured E-cadherin expression following Pirin knockdown. As shown in Fig. 1A and B, E-cadherin expression was significantly increased following Pirin knockdown indicating that it may promote EMT. To confirm this, we established Pirin-expressing HeLa cells (Fig. 1C), which inhibited the expression of E-cadherin (Fig. 1D). Additionally, the expression of Occludin, an epithelial marker, was decreased, and several mesenchymal markers, including Fibronectin, N-cadherin, and Vimentin, were increased by Pirin expression (Fig. 1D). These data suggest that Pirin promotes EMT.

Pirin induces EMT-associated cell morphological changes

As mentioned above, cells undergo morphological changes during EMT. Therefore, we next analyzed whether Pirin expression affects cell morphology. Quantitative analysis of morphological changes was based on cell circularity, {4p(area)/(perimeter)2}100, which decreases during EMT-associated morphological changes [34–36]. Indeed, TGF-b or TNF-a exposure induced EMTassociated cell morphological changes in HeLa cells (data not shown). Employing this parameter of circularity, we compared the morphology of our established HeLa/Pirin-GFP cells with control HeLa/GFP cells. Although the control HeLa/GFP cells displayed a cobblestone-like morphology, HeLa/Pirin-GFP cells were elongated in shape (Fig. 2A). Indeed, compared with control cells, the circularity of HeLa/Pirin-GFP cells was significantly decreased (Fig. 2B). To confirm that these observations were dependent on Pirin expression, HeLa/Pirin-GFP cells were treated with an siRNA targeting Pirin. HeLa/Pirin-GFP cells recovered a cobblestone-like morphology (Fig. 2C) and circularity (Fig. 2D) when treated with Pirin siRNA indicating that Pirin expression induces EMT.

Pirin induces cell migration

During EMT cells acquire migratory capabilities. Therefore, we analyzed whether Pirin affects cell migration. HeLa cells were treated with an siRNA targeting Pirin and migration was assessed using a wound healing assay. Although Pirin knockdown had no effect on cell proliferation (data not shown), wound repair was inhibited in Pirin-depleted HeLa cells (Fig. 3A and B) suggesting that Pirin promoted cell migration. Furthermore, camptothecin treatment of HeLa/GFP cells caused decreased cell viability in a dose-dependent manner, whereas HeLa/Pirin-GFP cells were more resistantto drugtreatment (datanot shown).These results suggest that Pirin induces EMT-like phenotypes, such as cell migration and anticancer drug resistance.
Pirin regulates EMT independently of Bcl3-Slug signaling

To investigate whether Pirin controls E-cadherin expression at the transcriptional level, we measured E-cadherin promoter activity with a reporter assay. Indeed, the luciferase reporter analysis indicated that Pirin inhibited E-cadherin promoter activity (Fig. 4A and B). To determine if Bcl3 is involved in Pirin-induced EMT, we tested whether a Pirin mutant defective in Bcl3 binding could inhibit E-cadherin expression. We generated a mutation in the metal-binding cavity of Pirin(E103A) and confirmed that it disrupted Bcl3 binding. In vitro GST pull-down analysis using recombinant Pirin and Bcl3/ARD demonstrated that the Pirin mutant was defective for Bcl3 binding compared to wild-type (Fig. 5A). Interestingly, expression of both wild-type Pirin and the mutant defective in Bcl3 binding reduced E-cadherin gene and protein expression (Fig. 5B and C). Taken together these results indicate that Pirin decreases E-cadherin expression without binding Bcl3, and suggest that Pirin regulates EMT independently of Bcl3-Slug signaling.

Discussion

A characteristic feature of EMT is the disruption of epithelial cell–cell contact, which is achieved by reduced E-cadherin expression. Therefore, revealing the regulatory pathways controlling E-cadherin expression may elucidate the mechanisms of EMT. Several transcription factors regulate E-cadherin transcription. For instance,Snail,Slug,Twist,and Zebact as mastertranscriptional regulators that bind the consensus E-box sequence in the E-cadherin gene promoter and decrease the transcriptional activity [38]. Since Pirin regulates the transcription of Slug [31], we hypothesized that Pirin may also regulate EMT. In this study we demonstrated that Pirin decreases E-cadherin expression, and induces EMT and cancer malignant phenotypes. Since EMT is an initial step of metastasis, Pirin may contribute to cancer progression. We next examined whether the regulation of EMT by Pirin is attributed to Bcl3 binding and the induction of Slug. To this end, we generated a Pirin mutant (E103A) defective for Bcl3 binding (Fig. 5A). Single Fe2+ ion chelating is coordinated by His56, His58, His101, and Glu103 of Pirin, and the N-terminal domain containing these residues is highly conserved between mammals, plants, fungi, and prokaryotic organisms [15,27]. Therefore, it has been predicted that this N-terminal domain containing the metal-binding cavity is important for Pirin function [20,26,31]. Indeed, TPh A inserts into the metal-binding cavity and inhibits binding to Bcl3 suggesting that the interaction occurs with the metal-binding cavity of Pirin [31]. In contrast, Hai Pang suggests that a Pirin–Bcl3– (p50)2 complex forms between acidic regions of the N-terminal Pirin domain at residues 77–82, 97–103 and 124–128 with a basic patch of Bcl3 [27]. In this study, we mutated Glutamic acid 103, a residue common between Hai Pang’s model and Pirin’s metalbinding cavity. Pull-down analysis indicated that an E103A mutant is defectiveinfor Bcl3binding(Fig.5A). Thisis the firstexperimental demonstration showing that Glu103 of Pirin is important Bcl3 binding. However, expression of the E103A mutant suppressed Ecadherin gene expression similarly to wild-type Pirin (Fig. 5B and C). Although the Bcl3–(p50)2 complex participates in oncogene addiction in cervical cells [39,40], expression of Pirin in HeLa cells did not increase Slug expression (data not shown). Therefore, we concludethatPirindecreasesE-cadherinexpressionindependently of Bcl3-Slug signaling. To understand how Pirin suppresses E-cadherin gene expression, we analyzed E-cadherin promoter activity (Fig. 4). Since Pirin decreased the activity of the E-cadherin promoter (995+1), we constructed a series of promoter deletion mutants (795+1, 565+1, 365+1, 175+1) to identify a region important for Pirin-mediated regulation. Expression of Pirin decreased the transcriptional activity of all constructs (Supplementary Fig. S1A), suggesting that Pirin may suppress E-cadherin expression through element(s) in region 175+1. Yan-Nan Liu and colleagues proposed that this region contains four Sp1-binding sites and two E-boxes that regulate E-cadherin expression.

Fig. 1. Pirin regulates E-cadherin gene expression. (A, B) HeLa cells were transfected with siRNA targeting Pirin (siPirin#1 or #2) or control siRNA (siCTRL). Forty-eight hours after transfection, cDNA was used for PCR using primer sets specific against Pirin, E-cadherin and GAPDH (A). Forty-eight hours after transfection, HeLa cells were lysed and the lysates were analyzed by Western blot with the indicated antibodies (B). (C) Lysates from HeLa/Pirin-GFP and HeLa/GFP cells were analyzed by Western blot with the indicated antibodies. (D) cDNA from HeLa/GFP or HeLa/Pirin-GFP cells was used for PCR to determine the effect of Pirin on the expression of EMT marker genes.

Fig. 2. Pirin induces cell morphological changes associated with EMT. (A) Phase contrast and fluorescence microscopic images were taken of HeLa/GFP and HeLa/Pirin-GFP cells. (B) Cell circularity was defined as form factor, {4p(area)/(perimeter)2}100 [%], and calculated using Image J software. A random selection of 100 cells from each condition was measured. (C, D) Phase contrast and fluorescence microscopic images were taken of siRNA-treated HeLa/GFP and HeLa/Pirin-GFP cells. Each cell line was transfected with siPirin#2 or siCTRL. Cells were observed by microscopy 48 h after transfection (C) and circularity was measured (D). Data shown are means ± s.d. ⁄P <0.05, bars 100lm.

Fig. 3. Pirin knockdown suppresses cell migration. (A, B) HeLa cells were transfected with siPirin#2 or siCTRL. An artificial wound was created with a tip 24h after transfection and cells were cultured for an additional 12 h. For quantification, the cells were photographed after 12h of incubation (A) and the area covered by cells was measured using Image J and normalized to control cells (B).

Fig. 4. Pirin regulates E-cadherin promoter activity.(A). HeLacells were transfected with siPirin#2 or siGFP (control) and cultured for 24 h. The E-cadherin promoter construct (995+1) and phRL-TK vectorwere transfected and cellswere cultured for an additional 24 h. Luciferase activities were measured and normalized to Renilla luciferase activity. (B) HeLa cells were transfected with the promoter construct (995+1), phRL-TK vector, and a Pirin expression vector. After 24 h, luciferase activities were measured and normalized to Renilla luciferase activity. Data are the mean ± s.d. ⁄P < 0.05.

Fig. 5. Pirin decreases E-cadherin expression in a Bcl3-independent manner. (A) Purified His6-Pirin and His6-Pirin(E103A) were incubated with Glutathione-Sepharose beads conjugated to GST or GST-Bcl3/ARD. The samples were analyzed by Western blot. (B, C) HeLa cells were transfected with vectors encoding GFP, Pirin-GFP, or Pirin(E103A)GFP. Cells were lysed 48 h after transfection and lysates were analyzed by Western blot (B). RNA collected at 48h was used for RT-PCR with the specified primer sets for each gene (C).

7.7.8.2 1324 PIRIN DOWN-REGULATES THE EAF2/U19 SIGNALING AND RETARDS THE GROWTH INHIBITION INDUCED BY EAF2/U19 IN PROSTATE CANCER CELLS

Zhongjie Qiao, Dan Wang, Zhou Wang
The Journal of Urology Apr 2013; 189(4), Supplement: e541
http://dx.doi.org/10.1016/j.juro.2013.02.2678
EAF2/U19, as the tumor suppressor, has been reported to induce apoptosis of LNCaP cells and suppress AT6.1 xenograft prostate tumor growth in vivo, and its expression level is down-regulated in advanced human prostate cancer. EAF2/U19 is also a putative transcription factor with a transactivation domain and capability of sequence-specific DNA binding. Identification and characterization of the binding partners and regulators of EAF2/U19 is essential to understand its function in regulating apoptosis/survival of prostate cancer cells.

7.7.8.3 Pirin Inhibits Cellular Senescence in Melanocytic Cells

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression

Cellular senescence is a physiological process through which normal somatic cells lose their ability to divide and enter an irreversible state of cell cycle arrest, although they remain viable and metabolically active.1,2The specific molecular circuitry underlying the onset of cellular senescence is dependent on the type of stimulus and on the cellular context. A central role is held by the activation of the tumor suppressor proteins p53 and retinoblastoma susceptibility protein (pRB),3–5 which act by interfering with the transcriptional program of the cell and ultimately arresting cell cycle progression.

In the last decade, senescence has been recognized as a major barrier against the development of tumors in mammals.6–8 One of the most prominent in vivo examples is represented by nevi, in which cells proliferate after oncogene activation and then become senescent. Melanoma is a highly aggressive form of neoplasm often observed to derive from nevi, and the transition implies suppression of the mechanisms that sustain the onset and maintenance of senescence.9 In fact, many of the melanoma-associated tumor suppressor genes identified to date are themselves involved in control of senescence, including BRAF (encoding serine/threonine-protein kinase B-raf), CKD4 (cyclin-dependent kinase 4), and CDKN2A (encoding cyclin-dependent kinase inhibitor 2A isoforms p16INK4a and p19ARF).3,10

Nevi frequently harbor oncogenic mutations of the tyrosine kinase BRAF gene, particularly V600E,11 andBRAFV600E is also found in approximately 70% of cutaneous melanomas.12 Expression of BRAFV600E in human melanocytes leads to oncogene-induced senescence,8 which can be considered as a mechanism that protects from malignant progression. In time, some cells may eventually escape senescence, probably through the acquisition of additional genetic abnormalities, thus favoring transformation to melanoma.13

Pirin (PIR) is a highly conserved nuclear protein belonging to the Cupin superfamily14 whose function is, to date, poorly characterized. It has been described as a putative transcriptional regulator on the basis of its physical association with the nuclear I/CCAAT box transcription factor NFI/CTF115 and with the B-cell lymphoma protein, BCL-3, a regulator of NF-κB/Rel activity. A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG).16 Other reports described quercetinase enzymatic activity,17 and regulation of apoptosis18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.

There is evidence of variations in PIR expression levels in different types of malignancies, but a systematic analysis of PIR expression in human tumors has been lacking. We analyzed PIR expression pattern in a collection of normal and neoplastic human tissues and found that it is expressed in scattered melanocytes, virtually absent in more mature regions of nevi, and present at high levels in a subset of melanomas. Functional studies performed in normal and transformed melanocytic cells revealed that PIR ablation results in cellular senescence, and that PIR levels decrease in response to senescence stimuli. Our results suggest that PIR may be a relevant player in the negative control of cellular senescence in PIR-expressing melanomas.

PIR overexpression in melanoma

Figure 3  PIR overexpression in PIR melanoma cells has no effect on proliferation.
PIR Expression Is Down-Regulated by BRAF Activation and Camptothecin Treatment

BRAF mutations are frequent in nevi, and are directly linked to the induction of oncogene-induced senescence. Variations in PIR expression levels were therefore investigated in an experimental model of senescence induced by oncogenic BRAF. Human diploid fibroblasts (TIG3–hTERT) expressing a conditional form of constitutively activated BRAF fused to the ligand-binding domain of the estrogen receptor (ER) rapidly undergo oncogene-induced senescence on treatment with 4-hydroxytamoxifen (OHT).28,29 PIR protein and mRNA levels were measured in TIG3-BRAF-ER cells at different time points of treatment with 800 nmol/L OHT. PIR expression was significantly repressed both at the mRNA and at the protein level after BRAF activation (Figure 6A), and remained at low levels after 120 hours, suggesting that a significant reduction of PIR expression is associated with the establishment of oncogene-induced senescence in different cell types.

7.7.9 O-GlcNAcylation at promoters, nutrient sensors, and transcriptional regulation

Brian A. Lewis
Biochim et Biophys Acta (BBA) – Gene Regulatory Mechanisms Nov 2013; 1829(11): 1202–1206
http://dx.doi.org/10.1016/j.bbagrm.2013.09.003

Highlights

  • This review article discusses recent advances in the links between O-GlcNAc and transcriptional regulation.
  • Discusses several systems to illustrate O-GlcNAc dynamics: Tet proteins, MLL complexes, circadian clock proteins and RNA pol II.
  • Suggests that promoters are nutrient sensors.

Post-translational modifications play important roles in transcriptional regulation. Among the less understood PTMs is O-GlcNAcylation. Nevertheless, O-GlcNAcylation in the nucleus is found on hundreds of transcription factors and coactivators and is often found in a mutually exclusive ying–yang relationship with phosphorylation. O-GlcNAcylation also links cellular metabolism directly to the proteome, serving as a conduit of metabolic information to the nucleus. This review serves as a brief introduction to O-GlcNAcylation, emphasizing its important thematic roles in transcriptional regulation, and highlights several recent and important additions to the literature that illustrate the connections between O-GlcNAc and transcription.

links between O-GlcNAc and transcriptional regulation.

links between O-GlcNAc and transcriptional regulation.

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr1.sml
links between O-GlcNAc and transcriptional regulation.

systems to illustrate O-GlcNAc dynamics

systems to illustrate O-GlcNAc dynamics

http://ars.els-cdn.com/content/image/1-s2.0-S1874939913001351-gr2.sml
systems to illustrate O-GlcNAc dynamics

7.7.10 O-GlcNAcylation in cellular functions and human diseases

Yang YR1Suh PG2.
Adv Biol Regul. 2014 Jan; 54:68-73
http://dx.doi.org:/10.1016/j.jbior.2013.09.007

O-GlcNAcylation is dynamic and a ubiquitous post-translational modification. O-GlcNAcylated proteins influence fundamental functions of proteins such as protein-protein interactions, altering protein stability, and changing protein activity. Thus, aberrant regulation of O-GlcNAcylation contributes to the etiology of chronic diseases of aging, including cancer, cardiovascular disease, metabolic disorders, and Alzheimer’s disease. Diverse cellular signaling systems are involved in pathogenesis of these diseases. O-GlcNAcylated proteins occur in many different tissues and cellular compartments and affect specific cell signaling. This review focuses on the O-GlcNAcylation in basic cellular functions and human diseases.

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr2.sml
O-GlcNAcylated proteins influence protein phosphorylation and protein-protein interactions

aberrant regulation of O-GlcNAcylation in disease

aberrant regulation of O-GlcNAcylation in disease

http://ars.els-cdn.com/content/image/1-s2.0-S2212492613000717-gr3.sml
aberrant regulation of O-GlcNAcylation in disease

 Comment:

Body of review in energetic metabolic pathways in malignant T cells

Antigen stimulation of T cell receptor (TCR) signaling to nuclear factor (NF)-B is required for T cell proliferation and differentiation of effector cells.
The TCR-to-NF-B pathway is generally viewed as a linear sequence of events in which TCR engagement triggers a cytoplasmic cascade of protein-protein interactions and post-translational modifications, ultimately culminating in the nuclear translocation of NF-B.
Activation of effect or T cells leads to increased glucose uptake, glycolysis, and lipid synthesis to support growth and proliferation.
Activated T cells were identified with CD7, CD5, CD3, CD2, CD4, CD8 and CD45RO. Simultaneously, the expression of CD95 and its ligand causes apoptotic cells death by paracrine or autocrine mechanism, and during inflammation, IL1-β and interferon-1α. The receptor glucose, Glut 1, is expressed at a low level in naive T cells, and rapidly induced by Myc following T cell receptor (TCR) activation. Glut1 trafficking is also highly regulated, with Glut1 protein remaining in intracellular vesicles until T cell activation.

Dr. Aurel,
Targu Jiu

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Graft-versus-Host Disease

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

This piece is a follow up to the article on allogeneic transfusion reactions, which extends into transplantation and transplantation outcomes for hematological diseases, both malignant and nonmalignant. The safety of transfusions in Western countries has improved substantially, and the causes for transfusion mishaps has been reduced to unexpected infectious sources, uncommon immune incompatibilities, and errors in processing the blood products.  The greatest risk is incurred in platelet transfusions because of the short shelf-life of the product, and the time needed for testing prior to release.  This portion of the review is concerned with Graft-versus-Host Disease, which is unique to transfusion and transplanting of blood. In other transplantation, there is graft failure because of host versus graft incompatibility or complications.  The reverse order applies to blood.  In this case, on the contrary, the transfused or grafted donor tissue becomes a pursuer after the recipients hematopoietic cells.

Peter Brian Medawar: Father of Transplantation

Thomas E. Starzl, M.D., PH.D., F.A.C.S.
J Am Coll Surg. 1995 Mar; 180(3): 332–336

Most of the surgical specialities can be tracked to the creative vision of a surgeon. Transplantation is an exception. Here, the father of the field is succinctly defined in the dictionary as: “Peter Brian Medawar: a Brazilian born British Zoologist who at the age of 45 shared a 1960 Nobel Prize for his work on acquired immunologic tolerance”. Medawar was mysteriously overwhelming to many colleagues and observers, even when he was young. He was the son of a Lebanese father and an English mother—tall, athletic, abnormally handsome, hypnotically articulate in public, and politely cordial in his personal relations. In September 1969, at the age of 54, he had the first of a series of strokes. These crippled him physically but not in spirit. Although I saw Medawar often professionally and privately over a 22 year period, before and after he was disabled, this sporadic exposure was not enough to understand him. My sense is that no one did, except perhaps Jean, his wife for nearly 50 years.

Medawar’s dazzling personality before and great courage after his strokes was inspirational, but his fame was based on the unique achievement in 1953 captured by the terse dictionary mention of “acquired immunologic tolerance.” The roots leading to this accomplishment had fed on the blood of war. More than 12 years earlier, the recently wed zoologist Medawar—24 years of age and fresh from graduate studies at Oxford University—was assigned to
the service of the British surgeon, Dr. Thomas Gibson, to determine if skin allografts could be used to treat casualties from the Battle of Britain. First,
in human studies with Gibson, and then with simple and logical rabbit experiments, Medawar showed that rejection of the skin was an immunologic phenomenon. This later was shown  to be analogous to the cell-mediated delayed hypersensitivity that confers immunity to diseases such as tuberculosis. The principal evidence in the early studies was that repetitive grafts from the same donor were rejected more rapidly with each successive attempt —the sensitization and donor specificity confirming an earlier clinical observations by Emil Holman of Stanford in skin-grafted burn victims. Once it was established that rejection was an immune reaction, strategies began to evolve to weaken the recipient immune system. By 1953, total body irradiation and adrenal cortical steroids had been shown to delay skin rejection. However, this immunosuppressive effect was either minor if the animals survived, or lethal to the recipient if the grafts were spared.

Bombshell

In the resulting atmosphere of nihilism about clinical applications, a three and one-half page article by Billingham, Brent, and Medawar in the October 3, 1953 issue of Nature describing acquired tolerance, came as a blinding beacon of hope. The three men had learned that donor splenocytes could be engrafted by their intravenous infusion into immunologically immature mice in utero or perinatally. When these inoculated recipients matured, they could accept skin and other tissues from the donor (but from no other) mouse strain. The immune system of the recipients had been populated by the immunocytes of the donor, meaning that they were now chimeras. The race was on to convert this principle to humans. However, the dark side of their accomplishment soon was revealed by the two younger members of Medawar’s team, Billingham and Brent and by the Dane, Simonsen. The engrafted donor cells could turn the tables and reject the defenseless recipient unless the tissue match was a good one. This was the dreaded graft versus host disease (GVHD) in which transplanted donor cells attacked the recipient skin, gastrointestinal tract, lungs, liver, and the bone marrow itself. Medawar’s dream of 1953 was suddenly a nightmare. Or was it?

On the contrary, the work took a straight line to clinical application, after the demonstration by Prehn and Main that similar tolerance could be induced in adult mice rendered immunologically defenseless by total body irradiation before splenocyte (or later bone marrow) infusion. The recipient conditioning, known as cytoablation, also could be accomplished with myelotoxic drugs. However, as Billingham, Brent, and Medawar had predicted, donor specific tolerance could be induced in humans without GVHD only if there was a good tissue (HLA) match. In 1968, 15 years after the epic Billingham, Brent and Medawar publication, Robert Good and Fritz Bach reported the first two successful human bone marrow transplants. Both recipients of well matched bone marrow from blood relatives are still alive. This was a triumph in which the principal clinicians were internists, as summarized 25 years later in the acceptance speech by the 1990 Nobel Laureate Donnall Thomas.

The growth of bone marrow and whole organ transplantation

The growth of bone marrow and whole organ transplantation

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2681237/bin/nihms-87975-f0001.gif

The growth of bone marrow (right) and whole organ transplantation (left) from the seed planted by Peter Medawar during World War II. GVHD, Graft versus host disease.

Immunological Tolerance: Medawar Nobel Acceptance Lecture

“Immunological tolerance” may be described as a state of indifference or non-reactivity towards a substance that would normally be expected to excite an immunological response. The term first came to be used in the context of tissue transplantation immunity, i.e. of the form of immunity that usually prohibits the grafting of tissues between individuals of different genetic make-up; and it was used to refer only to a non-reactivity caused by exposing animals to antigenic stimuli before they were old enough to undertake an immunological response. For example, if living cells from a mouse of strain CBA are injected into an adult mouse of strain A, the CBA cells will be destroyed by an immunological process, and the A-line mouse that received them will destroy any later graft of the same origin with the speed to be expected of an animal immunologically forearmed. But if the CBA cells are injected into a foetal or newborn A-line mouse, they are accepted; more than that, the A-line mouse, when it grows up, will accept any later graft from a CBA donor as if it were its own. I shall begin by using the term “immunological tolerance” in the rather restricted sense that is illustrated by this experiment, and shall discuss its more general usage later on.

The experiment I have just described can be thought of as an artificial reproduction of an astonishing natural curiosity, the phenomenon of red-cell chimerism in certain dizygotic twins. The blood systems of twin cattle before birth are not sharply distinct from each other, as they are in most other twins; instead, the blood systems make anastomoses with each other, with the effect that the twins can indulge in a prolonged exchange of blood before birth. In 1945, R.D. Owen2 made the remarkable discovery that most twin cattle are born with, and may retain throughout life, a stable mixture – not necessarily a fifty-fifty mixture – of each other’s red cells; it followed, then, that the twin cattle must have exchanged red-cell precursors and not merely red cells in their mutual transfusion before birth. This is the first example of the phenomenon we came to call immunological tolerance; the red cells could not have “adapted” themselves to their strange environment, because they were in fact identified as native or foreign by those very antigenie properties which, had an adaptation occurred, must necessarily have been transformed. A few years later R.E. Billingham and I3, with the help of three members of the scientific staff of the Agricultural Research Council, showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, for skin transplanted from third parties was cast off in the expected fashion.

Some properties of the tolerant state

The main points that emerged from our analysis of the tolerant state were these. In the first place, tolerance must be due to an alteration of the host, not to an antigenic adaptation of the grafted cells, for grafts newly transplanted in adult life have no opportunity to adapt themselves, and the descendants of the cells injected into foetal or newborn animals can be shown by N.A. Mitcbison’s methods to retain their antigenic power10. Once established, the state of tolerance is systemic; if one part of the body will tolerate a foreign graft, so will another; we found no evidence that a tolerated graft builds up a privileged position for itself within its own lymphatic territory. The stimulus that is responsible for instating tolerance is an antigenic stimulus – one which, had it been applied to older animals, would have caused them to become sensitive or immune. A plural stimulus can induce plural tolerance; the donor will usually contain several important antigens that are lacking in the recipient, and long-lasting tolerance must imply tolerance of them all. The state of tolerance is specific in the sense that it will discriminate between one individual and another, for an animal made tolerant of grafts from one individual will not accept grafts from a second individual unrelated to the first; but it will not discriminate between one tissue and another from the same donor.

Tolerance and auto-immunity: 50 years after Burnet.

Martini A1, Burgio GR
Eur J Pediatr. 1999 Oct;158(10):769-75.

Fifty years ago Sir F. Macfarlane Burnet published his first fundamental contribution to the theory of immune tolerance he perfected 10 years later. Since then an impressive amount of new information on the function of the immune system has been gathered. As any original meaningful theory, Burnet’s hypothesis on the development of immune tolerance has undergone extensive modifications to take into account all these new findings. An improved understanding of the mechanisms of tolerance has led to new possibilities for the treatment of auto-immune diseases.

Clonal Selection
http://en.wikipedia.org/wiki/Clonal_selection

Clonal selection theory is a scientific theory in immunology that explains the functions of cells (lymphocytes) of the immune system in response to specific antigens invading the body. The concept was introduced by an Australian doctor Frank Macfarlane Burnet in 1957 in an attempt to explain the formation of a diversity of antibodies during initiation of the immune response. The theory has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens.

The theory states that in a pre-existing group of lymphocytes (specifically B cells), a specific antigen only activates (i.e. selection) its counter-specific cell so that particular cell is induced to multiply (producing its clones) for antibody production. In short the theory is an explanation of the mechanism for the generation of diversity of antibody specificity. The first experimental evidence came in 1958, when Gustav Nossal and Joshua Lederberg showed that one B cell always produces only one antibody. The idea turned out to be the foundation of molecular immunology, especially in adaptive immunity.

The fundamental contribution of Robert A. Good to the discovery of the crucial role of thymus in mammalian immunity

Domenico Ribatti
Immunology. 2006 Nov; 119(3): 291–295.
http://dx.doi.org:/10.1111/j.1365-2567.2006.02484.x

Robert Alan Good was a pioneer in the field of immunodeficiency diseases. He and his colleagues defined the cellular basis and functional consequences of many of the inherited immunodeficiency diseases. His was one of the groups that discovered the pivotal role of the thymus in the immune system development and defined the separate development of the thymus-dependent and bursa-dependent lymphoid cell lineages and their responsibilities in cell-mediated and humoral immunity.  Keywords: bursa of Fabricius, history of medicine, immunology, thymus

Robert Alan Good (May 21, 1922 – June 13, 2003) was an American physician who performed the first successful human bone marrow transplant

Robert A. Good began his intellectual and experimental queries related to the thymus in 1952 at the University of Minnesota, initially with pediatric patients. However, his interest in the plasma cell, antibodies and the immune response began in 1944, while still in Medical School at the University of Minnesota in Minneapolis, with his first publication appearing in 1945.

Idiopathic Acquired Agammaglobulinemia Associated with Thymoma (1953)

  • a markedly deficient ability to produce antibodies and significant deficits of all or most of the cell-mediated immunities
  • in no instance did removal of the thymic tumour restore immunological function or correct the protein deficit

Good syndrome: thymoma with immunodeficiency

  • increased susceptibility to bacterial infections by encapsulated organisms and opportunistic viral and fungal infections
  • immunodeficiencies, leukopenia, lymphopenia and eosinophylopenia
  • severely hypogammaglobulinemic rather than agammaglobulinemic

Good and others found that the patients lacked all of the subsequently described immunoglobulins. These patients were found not to have plasma cells or germinal centers in their hematopoietic and lymphoid tissues. They possessed circulating lymphocytes in normal numbers.

Speculation on the reason for immunological failure following neonatal thymectomy has centered on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunological maturation.

The bursa of Fabricius and the thymus are ‘central lymphoid organs’ in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Studies by Papermaster and co-workers in Good’s laboratory34,35 indicated that bursectomy in the newly hatched chicks did not completely abolish immunological potential in the adult animal but rather produced a striking quantitative reduction insufficient to eliminate the homograft reaction. The failure of thymectomy in newly hatched chicks to alter the immunological potential of the maturing animal probably only reflected the participation of the bursa of Fabricius in the development of full immunological capacity.

Bursectomized and irradiated birds were completely devoid of germinal centers, plasma cells and the capacity to make antibodies yet they had perfectly normal development of thymocytes and lymphocytes elsewhere in the body that mediated cellular immune reactions. On the other hand, thymectomized and irradiated animals were deficient in lymphocytes that mediated cellular immunity as assessed by skin graft rejection, delayed-type hypersensitivity and graft versus host assays, but they still produced germinal centers, plasma cells and circulating immunoglobulins.

 

Graft vs Host Disease

Graft-versus-host disease (GVHD) is a complication that can occur after a stem cell or bone marrow transplant. With GVHD, the newly transplanted donor cells attack the transplant recipient’s body.

Graft-versus-host disease (GVHD) is a common complication following an allogeneic tissue transplant. It is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as “foreign“. The transplanted immune cells then attack the host’s body cells. GVHD can also occur after a blood transfusion if the blood products used have not been irradiated or treated with an approved pathogen reduction system.

http://en.wikipedia.org/wiki/Graft-versus-host_disease

Causes

GVHD may occur after a bone marrow or stem cell transplant in which someone receives bone marrow tissue or cells from a donor. This type of transplant is called allogeneic. The new, transplanted cells regard the recipient’s body as foreign. When this happens, the newly transplanted cells attack the recipient’s body.

GVHD does not occur when someone receives his or her own cells during a transplant. This type of transplant is called autologous.

Before a transplant, tissue and cells from possible donors are checked to see how closely they match the person having the transplant. GVHD is less likely to occur, or symptoms will be milder, when the match is close. The chance of GVHD is:

  • Around 30 – 40% when the donor and recipient are related
  • Around 60 – 80% when the donor and recipient are not related

There are two types of GVHD: acute and chronic. Symptoms in both acute and chronic GVHD range from mild to severe.

  • Acute GVHD usually happens within the first 6 months after a transplant.
  • Chronic GVHD usually starts more than 3 months after a transplant, and can last a lifetime.

Bone marrow transplant

A bone marrow transplant is a procedure to replace damaged or destroyed bone marrow with healthy bone marrow stem cells.  Stem cells are immature cells in the bone marrow that give rise to all of your blood cells.

There are three kinds of bone marrow transplants:

  • Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.
  • Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.
  • Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

  • Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.
  • Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

Histocompatibility antigen:

  • A histocompatibility antigen blood test looks at proteins called human leukocyte antigens (HLAs). These are found on the surface of almost all cells in the human body. HLAs are found in large amounts on the surface of white blood cells. They help the immune system tell the difference between body tissue and substances that are not from your own body.

http://www.nlm.nih.gov/medlineplus/ency/article/001309.htm

Induction of transplantation tolerance in haploidenical transplantation under reduced intensity conditioning: The role of ex-vivo generated donor CD8+ T cells with central memory phenotype

Eran Ophir, Y Eidelstein, E Bachar-Lustig, D Hagin, N Or-Geva, A Lask, , Y Reisner
Best Practice & Research Clinical Haematology 24 (2011) 393–401
http://dx.doi.org:/10.1016/j.beha.2011.05.007

Haploidentical hematopoietic stem cell transplantation (HSCT) offers the advantage of readily available family member donors for nearly all patients. A ‘megadose’ of purified CD34þ hematopoietic stem cells is used to overcome the host’s residual immunity surviving the myeloablative conditioning, while avoiding severe GVHD. However, the number of CD34+ cells that can be harvested is insufficient for overcoming the large numbers of host T cells remaining after reduced intensity conditioning (RIC). Therefore, combining a ‘megadose’ of CD34+ HSCT with other tolerizing cells could potentially support and promote successful engraftment of haploidentical purified stem cell transplantation under a safer RIC. One approach to address this challenge
could be afforded by using Donor CD8 T cells directed against 3rd-party stimulators, bearing an ex-vivo induced central memory phenotype (Tcm). These Tcm cells, depleted of GVH reactivity, were shown to be highly
efficient in overcoming host T cells mediated rejection and in promoting
fully mismatched bone-marrow (BM) engraftment, in HSCT murine models.
This is likely due to the marked lymph node homing of the Tcm, their strong proliferative capacity and prolonged persistence in BM transplant recipients. Thus, combining anti 3rd-party Tcm cell therapy with a ‘megadose’ of purified CD34+ stem cells, could offer a safer RIC protocol for attaining hematopoietic chimerism in patients with hematological diseases and as a platform for organ transplantation or cell therapy in cancer patients.

Induction of tolerance in organ recipients by hematopoietic stem cell transplantation

Eran Ophir, Yair Reisner
International Immunopharmacology 9 (2009) 694–700
http://dx.doi.org:/10.1016/j.intimp.2008.12.009

The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+linhematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism.

Intrinsic unresponsiveness of Mertk/B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Wen-Hai Shao, Y Zhen, FD Finkelman, RA Eisenberg, PL Cohen
Journal of Autoimmunity 39 (2012) 412e419
http://dx.doi.org/10.1016/j.jaut.2012.07.001

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk-/- mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk-/-mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk-/-  mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk-/- mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk-/- mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Kazuki Sakai, Eri Kawata, Eishi Ashihara, Yoko Nakagawa, et al.
Eur. J. Immunol. 2011. 41: 67–75 http://dx.doi.org:/10.1002/eji.200939931

Galectins comprise a family of animal lectins that differ in their affinity for β-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD41 Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca21 influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.

 

GVHD Prevention: An Ounce Is Better Than a Pound

Pavan Reddy, Gerard Socie, Corey Cutler, Daniel Weisdorf
Biol Blood Marrow Transplant 18:S17-S26, 2012  http://dx.doi.org:/10.1016/j.bbmt.2011.10.034

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.

Like all immune responses, certain triggers are critical for induction of acute graft-versus-host disease (aGVHD). These include: (1) Disparities between histocompatibility antigens: antigen disparity can be at the level of major histocompatibility complex (MHC), that is, MHC mismatched or at the level of minor histocompatibility antigens (miHA), that is, MHC matched but miHA mismatched. The severity of aGVHD is directly related to the degree of M HC mismatch. In bone marrow transplants (BMT) that are MHC matched but miHA disparate, donor T cells still recognize MHC-peptide derived from the products of recipient polymorphic genes, the miHAs.

Damage induced by conditioning regimens and underlying diseases: under most circumstances, the initiation of an adaptive immune response is triggered by the innate immune response. The innate immune system is triggered by certain exogenous and endogenous molecules. This is likely the case in the induction of aGVHD. Pattern recognition receptors such as Toll-like receptors (TLR), nucleotide-binding oligomerization domain containing 2 (NOD2) play an essential role in innate immunity and in initiating the cellular signaling pathways that activate cytokine secretion, such as NF-kB. Some of their ligands, such as lipopolysaccharide, CpG, and MDP2, which is recognized by TLR-4, TLR-9, and NOD2, respectively, are released by the preparative regimens and contribute to the induction and enhancement of allo-T cell responses. In this way, the conditioning regimens amplify the secretion of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α,  IL-6, and other interferon family members in a process described as a ‘‘cytokine storm.’’

The triggers that initiate an immune response have to be sensed and presented. APCs might be considered the sensors for aGVHD. The APCs sense the DAMPs, present the MHC disparate or miHA disparate protein, and provide the critical secondary (costimulatory) and tertiary (cytokine) signals for activation of the alloreactive T cells, the mediators of aGVHD. APCs sense allo-disparity through MHC and peptide complexes. Dendritic cells (DCs) are the most potent APCs and the primary sensors of allo-disparity.

APCs provide the critical costimulation signals for turning on the aGVHD process. The interaction between the MHC/allopeptide complex on APCs and the T cell receptor of donor T cells along with the signal via T cell costimulatory molecules and their ligands on APCs is required to achieve T cell activation, proliferation, differentiation, and survival and the in vivo blockade of positive costimulatory molecules (such as CD28, ICOS, CD40, CD30, etc.), or inhibitory signals (such as PD-1 and CTLA-4) mitigate or exacerbate aGVHD, respectively.

Evidence suggests that alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions.

The allo-antigen composition of the host determines which donor T cells subsets differentiate and proliferate. As mentioned previously, in the majority of HLA-matched HCT, aGVHD may be induced by either or both CD41 and CD81 subsets responses to miHAs. The repertoire and immunodominance of the GVHD-associated peptides presented by MHC class I and class II molecules has not been defined. Donor naive CD62L1 T cells are the primary alloreactive T cells that drive the GVHD reaction while the donor effector memory CD62L2 T cells do not. Interestingly, donor regulatory T cells (Tregs) expressing CD62L are also critical to the regulation of GVHD. We now know that it is possible to modulate the alloreactivity of na€ıve T cells by inducing anergy with costimulation blockade, deletion via cytokine modulation, or mixed chimerism. Donor effector memory T cells that are nonalloreactive do not induce GVHD, yet are able to transfer functional memory. In contrast, memory T cells that are alloreactive can cause severe GVHD.

The effector phase that leads to GVHD target organ damage is a complex cascade that involves cytolytic cellular effectors such as CD8 cytotoxic T lymphocytes (CTLs), CD4 T cells, natural killer cells, and inflammatory molecules such as IL-1β, TNF-α, IFN-ϒ, IL-6, and reactive oxygen species. The cellular effectors require cell-cell contact to kill the cells of the target tissues via activation of perforin granzyme, Fas-FasL (CD95-CD95L), or TNFR TRAIL pathways. Other CTLs killing mechanisms such as TWEAK, and LTβ/LIGHT pathways have also been implicated in GVHD. It is important to note that
CTL pathways are essential for GVL effects as well.

All of the above aspects of the biology of aGVHD have been summarized in the mold of a normal immune response. Although this allows for accessing the biology of GVHD, it is important to note that GVHD is a complicated systemic process with as yet still many unknowns and is not a simplified, linear, or cyclical process.

Kinetics of CD4+ and CD8+ T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii

Yasuhiro Shibasakia, H Todaa, Isao kobayashib, T Moritomoa, T Nakanishia
Developmental and Comparative Immunology 34 (2010) 1075–1081
http://dx.doi.org:/10.1016/j.dci.2010.05.009

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8+ T cells in the induction of GVHR, we investigate the kinetics of CD4+ and CD8+ T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8+ T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4+ and CD8+  T cells from sensitized donors greatly

increased in the host trunk kidney, constituting more than 80% of total cells 1–2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4+ T cells was greater and more rapid than that of CD8+ T cells. The number of donor CD4+ and CD8+ T cells was highest in trunk kidney followed by spleen. Increases in donor CD4+ and CD8+ T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8+ T cells. These results suggest that donor-derived CD8+ T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Fludarabine and Exposure-Targeted Busulfan Compares Favorably with Busulfan/Cyclophosphamide-Based Regimens in Pediatric Hematopoietic
Cell Transplantation: Maintaining Efficacy with Less Toxicity

I.H. Bartelink, E.M.L. van Reij, C.E. Gerhardt, E.M. van Maarseveen, et al
Biol Blood Marrow Transplant 20 (2014) 345e353
http://dx.doi.org/10.1016/j.bbmt.2013.11.027

Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu + Bu at a target dose of 80-95 mg-h/L, and between 2005 and 2008, 50 children received Bu targeted to 74-80 mg-h/L þ Cy. In the latter group, Mel was added for patients with myeloid malignancy (n = 12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P,  not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P < .007), veno-occlusive disease (3% versus 28%; P < .003), chronic graft-versus-host disease (9% versus 26%; P < .047), adenovirus infection (3% versus 32%; P < .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P < .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11 days versus 22 days; P < .001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160 mg/m2) with targeted myeloablative Bu (90 mg-h/L) is less toxic than and equally effective
as BuCy (Mel) in patients with similar indications for allo-HCT.

Fibrotic and Sclerotic Manifestations of Chronic Graft-versus-Host Disease

Carrie L. Kitko, Eric S. White, Kristin Baird
Biol Blood Marrow Transplant 18:S46-S52, 2012
http://dx.doi.org:/10.1016/j.bbmt.2011.10.021

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity
and mortality following allogeneic stem cell transplantation (HCT), with approximately 50% to 60% of long-term HCT survivors developing one or more manifestations of the disorder. Although acute GVHD is typically limited to skin, liver, and gastrointestinal involvement, virtually every organ is at risk for the development of cGVHD. Although the pathophysiology of cGVHD remains poorly understood, some of the most severe organ manifestations are linked by end-organ fibrosis. In particular, fibrotic cutaneous and bronchiolar changes, resulting in scleroderma-like changes and bronchiolitis obliterans syndrome (BOS), respectively, are two of the most devastating outcomes for these patients. Both sclerotic GVHD (ScGVHD) and BOS have been reported in 5% to 15% of patients with cGVHD.

Many of the manifestations of cGVHD share clinical characteristics seen in nontransplant conditions, including systemic sclerosis or pulmonary fibrosis. Thus, understanding the pathophysiology underlying these related conditions may help identify potential mechanisms and ultimately new therapeutic options for patients with cGVHD.

Tyrosine kinase inhibitors (TKIs) have been shown to inhibit two different profibrotic pathways (transforming growth factor β [TGF-β] and platelet-derived growth factor [PDGF]) in various mouse models of fibrotic disease and offer a possible novel treatment approach for cGVHD patients suffering from severe sclerosis. Likewise, overexpression of TNF-α has been shown to induce fibrogenesis in experimental hepatocellular disease and has been linked with human scleroderma-associated interstitial pulmonary fibrosis and profibrotic responses in human osteoarthritic hip joint fibroblasts. The use of TNF antagonists has been examined in some clinical situations associated with fibrosis, suggesting they may also be of some benefit to patients with cGVHD; however, this must first be prospectively tested.

Table. Proposed Modifications to NIH BOS Clinical Definition

  • Absence of infection (no change)
  • Another cGVHD manifestation in another organ (no change)
  • FEV1 <75% predicted (no change) or >10% decline from pre-HCT value (modification)
  • Signs of Obstruction
  • FEV1/SVC ratio <0.7 (modification), or
  • RV >120% predicted (no change), or
  • RV/TLC >120% (modification), and
  • HRCT with evidence of air trapping (no change)

SVC indicates slow vital capacity; RV, residual volume; TLC, total lung capacity; HRCT, high-resolution computed tomography

Figure (not shown)
Effect of etanercept on survival in post-HCT patients with subacute lung injury. (A) Overall 5-year survival by pulmonary function testing defect. Patients with an obstructive defect (solid line) had a 5-year survival of 67% compared with 44% in those with a restrictive lung defect (dashed line) (P 5 .19). (B) Overall 5-year survival by response to therapy. Patients who responded to etanercept therapy (solid line) had a 5-year survival of 90% compared with 55% in patients who failed to respond (dashed line) (P 5.07). (Figures reprinted with permission, Biol Blood and Marrow Trans).

Extensive, sclerotic skin changes with superficial or deep subcutaneous or fascial involvement are seen in approximately 4% to 13% of patients with cGVHD and can be a life-threatening manifestation. ScGVHD of the skin includes several cutaneous presentations characterized by inflammation and progressive fibrosis of the dermis and subcutaneous tissues. These changes can resemble morphea, systemic sclerosis, or eosinophilic fasciitis and may or may not occur in the setting of concurrent overlying epidermal GVHD. When severe, ScGVHD can result in contractures, severe wasting, and chest wall restriction.

Development of clinical trials for patients with cGVHD is difficult because of the complexity and heterogeneity of disease, variable approaches to treatment, and the lack of standardized assessments of disease. In particular, the study of ScGVHD lacks universally accepted measures of disease burden and response. Investigators have used several measures to assess ScGVHD involvement including body surface area, magnetic resonance imaging, ultrasound, and range-of-motion measurements. Additionally, investigators have tried to apply the Rodnan score, the standardmeasure for skin involvement in scleroderma. Thus far, none of these measures has proven
to be completely reliable in the setting of ScGVHD, and it is likely that multiple measures will need to be integrated into the assessment of ScGVHD.

Imatinib mesylate (Gleevec in the US; Glivec in Europe, Australia, and Latin America, marketed by Novartis) is a TKI that has biological activity against both PDGF and TGF-β signaling pathways. Both cytokines have been implicated in the pathogenesis of several fibrosing diseases, including hepatic, renal, and lung, as well as in scleroderma, a disease that closely resembles ScGVHD. In addition, stimulatory antibodies specific for the PDGF receptor (PDGFR) were identified in a series of 39 patients with extensive cGVHD with higher levels detected in those patients with skin involvement. Similar stimulatory antibodies targeting PDGFR have been reported in patients with scleroderma, suggesting an important therapeutic target for these fibrosing conditions. Imatinib mesylate has particularly potent activity against PDGF and is FDA approved in the United States for the treatment of several disorders associated with aberrant PDGFR signaling. The side effect profile of the drug is well established in non-HCT patients, which is helpful in the setting of a therapy for allogenic HCT patients, many of whom have multiorgan system symptoms and possible dysfunction and who will require ongoing immunosuppressive therapy.

Through the efforts of the Chronic GVHD Consortium, led by Stephanie Lee at the Fred Hutchinson Cancer Research Center, there is a multicenter, ongoing prospective evaluation of the NIH diagnostic and assessment tools. This effort has already resulted in several publications that have further refined essential criteria for cGVHD evaluation, including organ-specific manifestations such as BOS and ScGVHD. Currently, the Consortium is conducting a multicenter prospective clinical trial of fluticasone propionate, azithromycin, and montelukast for the treatment of BOS (ClinicalTrials.gov NCT01307462); a separate trial of imatinib versus rituximab for treatment of ScGVHD is also enrolling subjects (ClinicalTrials.gov NCT01309997).

Although cGVHD remains a significant problem for many long-term survivors of HCT, critical advances in cGVHD research and treatment can be achieved by cooperative group efforts such as those put forth by the Chronic GVHD Consortium and the Clinical Trials Network.

Hematopoietic stem cell transplantation (HSCT): An approach to autoimmunity

Carmen Alaez, Mariana Loyola, Andrea Murguıa, Hilario Flores, et al.
Autoimmunity Reviews 5 (2006) 167– 179
http://dx.doi.org:/10.1016/j.autrev.2005.06.003

HSCT provides the opportunity to replace a damaged tissue. It is the most important treatment for high risk hematologic malignant and nonmalignant disorders. An important challenge in the identification of matched donors/patients is the HLA diversity. The Mexican Bone Marrow Registry (DONORMO) has nowadays N5000 donors. The prevalent alleles are Amerindian, Mediterranean (Semitic and Spanish genes) and African. In theory, it is possible to find 11% of 6/6 A–B–DR low resolution matches for 70% of patients with Mexican ancestry. We contributed with 39 unrelated, cord blood and autologous HSCT for patients with malignant, genetic and autoimmune disorders. Overall disease survival was 50% (2–7 years) depending on the initial diagnosis, conditioning, disease evolution or other factors. Clinical studies using autologous and unrelated HSC are performed on patients with refractory autoimmune diseases producing mixed results: mainly, T1D, RA, MS, SLE. Improvement has been observed in skin damage and quality of life in SLE and systemic sclerosis. Disease stabilization in 2/3 of MS patients. However, in RA and T1D, initial benefits have been followed by eventual relapse. With growing clinical experience and protocol improvement, treatment-related mortality is decreasing. Proof efficacy will be achieved by comparing HSCT with standard therapy in autoimmunity.

Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor α Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells

Liqing Jin, Erwin M. Lee, Hayley S. Ramshaw, Samantha J. Busfield, et al.
Cell: Stem Cell 5, 31–42, July 2, 2009
http://dx.doi.org:/10.1016/j.stem.2009.04.018

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor α chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing
to bone marrow (BM) and activating innate immunity of nonobese diabetic/ severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival.
Mice with preestablished disease showed reduced AML burden in the BM
and periphery and impaired secondary transplantation upon treatment, establishing that AMLLSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34+ CD38[1] cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Many Days at Home during Neutropenia after Allogeneic Hematopoietic Stem Cell Transplantation Correlates with Low Incidence of Acute Graft-versus-Host Disease

Olle Ringdén, Mats Remberger, Katarina Holmberg, Charlotta Edeskog, et al.
Biol Blood Marrow Transplant 19 (2013) 314e320
http://dx.doi.org/10.1016/j.bbmt.2012.10.011

Patients are isolated in the hospital during the neutropenic phase after allogeneic hematopoietic stem cell transplantation. We challenged this by allowing patients to be treated at home. A nurse from the unit visited and checked the patient. One hundred forty-six patients treated at home were compared with matched hospital control subjects. Oral intake was intensified from September 2006 and improved (P < .002). We compared 4 groups: home care and control subjects before and after September 2006. The cumulative incidence of acute graft-versus-host disease (GVHD) of grades II to IV was 15% in the “old” home care group, which was significantly lower than that of 32% to 44% in the other groups (P <.03). Transplantation-related mortality, chronic GVHD, and relapse were similar in the groups. The “new” home care patients spent fewer days at home (P < .002). In multivariate analysis, GVHD of grades 0 to I was associated with home care (hazard ratio [HR], 2.46; P <.02) and with days spent at home (HR, .92; P < .005) but not with oral nutrition (HR, .98; P = .13). Five year survival was 61% in the home care group as compared with 49% in the control subjects (P < .07). Home care is safe. Home care and many days spent at home were correlated with a low risk of acute GVHD.

Impact on Outcomes of Human Leukocyte Antigen Matching by Allele-Level Typing in Adults with Acute Myeloid Leukemia Undergoing Umbilical Cord Blood Transplantation

Jaime Sanz, Francisco J. Jaramillo, Dolores Planelles, Pau Montesinos, et al.
Biol Blood Marrow Transplant 20 (2014) 106e110
http://dx.doi.org/10.1016/j.bbmt.2013.10.016

This retrospective study analyzed the impact of directional donor-recipient human leukocyte antigen (HLA) disparity using allele-level typing at HLA-A, -B, -C, and -DRB1 in 79 adults with acute myeloid leukemia (AML) who received single-unit umbilical cord blood (UCB) transplant at a single institution. With extended high resolution HLA typing, the donor-recipient compatibility ranged from 2/8 to 8/8. HLA disparity showed no negative impact on nonrelapse mortality (NRM), graft-versus-host (GVH) disease or engraftment. Considering disparities in the GVH direction, the 5-year cumulative incidence of relapse was 44% and 22% for patients receiving an UCB unit matched > 6/8 and < 6/8, respectively (P <.04). In multivariable analysis, a higher HLA disparity in the GVH direction using extended high-resolution typing (Risk ratio [RR] 2.8; 95% confidence interval [CI], 1.5 to 5.1; P ¼.0009) and first complete remission at time of transplantation (RR 2.1; 95% CI, 1.2 to 3.8; P < .01) were the only variables significantly associated with an improved disease-free survival. In conclusion, we found that in adults with AML undergoing single-unit UCBT, an increased number of HLA disparities at allele-level typing improved disease-free survival by decreasing the relapse rate without a negative effect on NRM.

HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection
Cladd E. Stevens, C Carrier, C Carpenter, D Sung, and A Scaradavou

Blood. 2011; 118(14):3969-3978
http://dx.doi.org:/10.1182/blood-2010-11-317271

Donor-recipient human leukocyte antigen mismatch level affects the outcome of unrelated cord blood (CB) transplantation. To identify possible “permissive” mismatches, we examined the relationship between  direction of human leukocyte antigen mismatch (“vector”) and transplantation outcomes in 1202 recipients of single CB units from the New York Blood Center National Cord Blood Program treated in United States Centers from 1993-2006. Altogether, 98 donor/patient pairs had only unidirectional mismatches: 58 in the graft-versus-host (GVH) direction only (GVH-O) and 40 in the host-versus-graft or rejection direction only (R-O). Engraftment was faster in patients with GVH-O mismatches compared with those with 1 bidirectional mismatch (hazard ratio [HR] = 1.6, P < .003). In addition, patients with hematologic malignancies given GVH-O grafts had lower transplantation-related mortality (HR = 0.5, P < .062), overall mortality (HR = 0.5, P < .019), and treatment failure (HR = 0.5, P < .016), resulting in outcomes similar to those of matched CB grafts. In contrast, R-O mismatches had slower engraftment, higher graft failure, and higher relapse rates (HR = 2.4, P < .010). Based on our findings, CB search algorithms should be modified to identify unidirectional mismatches. We recommend that transplant centers give priority to GVH-O-mismatched units over other mismatches and avoid selecting R-O mismatches, if possible.

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation
for acute lymphoblastic leukemia

G Rodríguez-Macías, C Martínez-Laperche, J Gayoso, V Noriega, .., Ismael Buño
Human Pathology (2013) 44, 1696–1699
http://dx.doi.org/10.1016/j.humpath.2013.01.001

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Graft-versus-leukemia in the bone marrow
Blood, 23 JAN 2014; 123(4)
http://imagebank.hematology.org.

63-year-old female with relapsed acute myeloid leukemia (AML) after allogeneic stem cell transplantation reached CR2 after re-induction therapy followed by consolidation with donor lymphocyte infusions: 3 x 107/kg and 3 x 108/kg after 1 and 2.5 months, respectively. No signs of graft-versus-host disease were observed at this time. At 5 months follow-up, her blood count deteriorated: hemoglobin: 6.9 mmol/L, thrombocytes: 58 x 109/L and leukocytes: 1.37 x 109/L. Bone marrow aspirate was not evaluable. Bone marrow trephine biopsy showed relapse AML with hypercellularity in the H&E staining (340 objective lens, panel A) and 20% CD341 blast cells without any signs of maturation (panel B). Also, a high number of CD3 positive T cells (panel C) was noted, intermingling with the CD34 positive blasts, both staining positively with CD43 (panel D). Only supportive care was given. However, normalization of the blood count was observed in the following months and she developed graft-versus-host disease of the lung, which was treated with ciclosporin and prednisone. A bone marrow aspirate performed 3 months after relapse showed a third remission with 0.8% myeloid blasts. In retrospect, one could therefore consider the picture of the bone marrow trephine biopsy at the second relapse as graft-versus-leukemia in the bone marrow.

GVL- panel A

GVL- panel A

GVL - panel B

GVL – panel B

GVL - panel C

GVL – panel C

GVL - panel D

Long-Term Outcomes of Alemtuzumab-Based Reduced-Intensity Conditioned Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndrome and Acute Myelogenous Leukemia Secondary to Myelodysplastic Syndrome

Victoria T. Potter, Pramila Krishnamurthy, Linda D. Barber, ZiYi Lim, et al.
Biol Blood Marrow Transplant 20 (2014) 111e117
http://dx.doi.org/10.1016/j.bbmt.2013.10.021

Allogeneic hematopoietic stem cell transplantation (HSCT) with reduced-intensity conditioning (RIC) offers a potential cure for patients with myelodysplastic syndrome (MDS) who are ineligible for standard-intensity regimens. Previously published data from our institution suggest excellent outcomes at 1 yr using a uniform fludarabine, busulfan, and alemtuzumab-based regimen. Here we report long-term follow-up of 192 patients with MDS and acute myelogenous leukemia (AML) secondary to MDS (MDS-AML) transplanted with this protocol, using sibling (n = 45) or matched unrelated (n = 147) donors. The median age of the cohort was 57 yr (range, 21 to 72 yr), and median follow-up was 4.5 yr (range, 0.1 to 10.6 yr). The 5-yr overall survival (OS), event-free survival, and nonrelapse mortality were 44%, 33%, and 26% respectively. The incidence of de novo chronic graft-versus-host disease (GVHD) was low at 19%, illustrating the efficacy of alemtuzumab for GVHD prophylaxis. Conversely, the 5-yr relapse rate was 51%. For younger patients (age <50 yr), the 5-yr OS and relapse rates were 58% and 39%, respectively. On multivariate analysis, advanced age predicted significantly worse outcomes, with patients age >60 yr having a 5-yr OS of 15% and relapse rate of 66%. Patients receiving preemptive donor lymphocyte infusions had an impressive 5-yr OS of 67%, suggesting that this protocol may lend itself to the incorporation of immunotherapeutic strategies. Overall, these data demonstrate good 5-yr OS for patients with MDS and MDS-AML undergoing alemtuzumab-based RIC-HSCT. The low rate of chronic GVHD is encouraging, and comparative studies with other RIC protocols are warranted.

Natural killer cell activity influences outcome after T cell depleted stem cell transplantation from matched unrelated and haploidentical donors

Peter Lang, Matthias Pfeiffer,  Heiko-Manuel Teltschik, Patrick Schlegel, et al.
Best Practice & Research Clinical Haematology 24 (2011) 403–411
http://dx.doi.org:/10.1016/j.beha.2011.04.009

Lytic activity and recovery of natural killer (NK) cells was monitored in pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n = 18) and mismatched related (haploidentical, n = 29) donors. CD34+ selection with magnetic microbeads resulted in 8 x 103/kg residual T cells. No post-transplant immune suppression was given. NK cells recovered rapidly after transplantation (300 CD56+/mL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/mL at day 90). NK activity was measured as specific lysis of K 562 targets several times (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 2 years, p < 0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 2 years, p < 0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered.

Graft-versus-host disease: Pathogenesis and clinical manifestations of graft-versus-host disease

Sharon R. Hymes, Amin M. Alousi,  and Edward W. Cowen
J Am Acad Dermatol  2012; 66: 515.e1-18.

  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality in patients who undergo allogeneic hematopoietic cell transplantation.
  • Acute graft-versus-host disease manifests as a skin exanthem, liver dysfunction, and gastrointestinal involvement.
  • Chronic graft-versus-host disease of the skin is remarkably variable in its clinical presentation.
  • Chronic graft-versus-host disease is a multisystem disorder that may affect nearly any organ; the most common sites are the skin, oral mucosa, and eyes.

Key points

  • Allogeneic transplantation is in widespread use for hematologic malignancies, but is also increasingly used for marrow failure syndromes, immunodeficiencies, and other life-threatening conditions
  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality after allogeneic hematopoietic cell transplantation
  • Minimizing graft-versus-host disease without losing the graft-versus-tumor effect is an area of active research
  • The skin is the most common organ affected in patients with graft-versus-host disease

Outcomes of Thalassemia Patients Undergoing Hematopoietic Stem Cell Transplantation by Using a Standard Myeloablative versus a Novel Reduced-Toxicity Conditioning Regimen According to a New Risk Stratification

Usanarat Anurathapan, S Pakakasama, P Mekjaruskul, N Sirachainan, et al.
Biol Blood Marrow Transplant 20 (2014) 2056e2075
http://dx.doi.org/10.1016/j.bbmt.2014.07.016

Improving outcomes among class 3 thalassemia patients receiving allogeneic hematopoietic stem cell transplantations (HSCT) remains a challenge. Before HSCT, patients who were > 7 years old and had a liver size > 5 cm constitute what the Center for International Blood and Marrow Transplant Research defined as a very high risk subset of a conventional high-risk class 3 group (here referred to as class 3 HR). We performed HSCT in 98 patients with related and unrelated donor stem cells. Seventy-six of the patients with age < 10 years received the more conventional myeloablative conditioning (MAC) regimen (cyclophos-phamide, busulfan,  + fludarabine); the remaining 22 patients with age > 10 years and hepatomegaly (class 3 HR), and in several instances additional comorbidity problems, underwent HSCT with a novel reduced-toxicity conditioning (RTC) regimen (fludarabine and busulfan). We then compared the outcomes between these 2 groups (MAC versus RTC). Event-free survival (86% versus 90%) and overall survival (95% versus 90%) were not significantly different between the respective groups; however, there was a higher incidence of serious treatment-related complications in the MAC group, and although we experienced 6 graft failures in the MAC group (8%), there were none in the RTC group. Based on these results, we suggest that (1) class 3HRthalassemia patients can safely receive HSCT with our novel RTC regimen and achieve the same excellent outcome as low/standard-risk thalassemia patients who received the standard MAC regimen, and further, (2) that this novel RTC approach should be tested in the low/standard-risk patient population.

Pharmacological Immunosuppression Reduces But Does Not Eliminate the Need for Total-Body Irradiation in Nonmyeloablative Conditioning Regimens for Hematopoietic Cell Transplantation

Marco Mielcarek, Beverly Torok-Storb, Rainer Storb
Biol Blood Marrow Transplant 17: 1255-1260 (2011)
http://dx.doi.org:/10.1016/j.bbmt.2011.01.003

In the dog leukocyte antigen (DLA)-identical hematopoietic cell transplantation (HCT) model, stable marrow engraftment can be achieved with total-body irradiation (TBI) of 200 cGy when used in combination with postgrafting immunosuppression. The TBI dose can be reduced to 100 cGy without compromising engraftment rates if granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are infused with the marrow. T cell-depleting the G-PBMC product abrogates this effect. These results were interpreted to suggest that the additional T cells provided with G-PBMC facilitated engraftment by overcoming host resistance.We therefore hypothesized that the TBI dose may be further reduced to 50 cGy by augmenting immunosupression either by (1) tolerizing or killing recipient T cells, or (2) enhancing the graft-versus-host (GVH) activity of donor T cells. To test the first hypothesis, recipient T cells were activated before HCT by repetitive donor-specific PBMC infusions followed by administration of methotrexate (MTX) (n 5 5), CTLA4-Ig (n = 4), denileukin diftitox (Ontak; n = 4), CTLA4-Ig 1 MTX (n = 8), or 5c8 antibody (anti-CD154) 1 MTX (n = 3). To test the second hypothesis, recipient dendritic cells were expanded in vivo by infusion of Flt3 ligand given either pre-HCT (n = 4) or pre- and post-HCT (n = 5) to augment GVH reactions. Although all dogs showed initial allogeneic engraftment, sustained engraftment was seen in only 6 of 42 dogs (14% of all dogs treated in 9 experimental groups). Hence, unless more innovative pharmacotherapy can be developed that more forcefully shifts the immunologic balance in favor of the donor, noncytotoxic immunosuppressive drug therapy as the sole component of HCT preparative regimens may not suffice to ensure sustained engraftment.

Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control

Usanarat Anurathapan, S Pakakasama, P Rujkijyanont, N Sirachainan, et al.
Biol Blood Marrow Transplant 19 (2013) 1254e1270
http://dx.doi.org/10.1016/j.bbmt.2013.04.023

Patients with class 3 thalassemia with high-risk features for adverse events after high-dose chemotherapy with hematopoietic stem cell transplantation (HSCT) are difficult to treat, tending to either suffer serious toxicity or fail to establish stable graft function. We performed HSCT in 18 such patients age 7 years and hepatomegaly using a novel approach with pretransplant immunosuppression followed by a myeloablative reduced-toxicity conditioning regimen (fludarabine and i.v. busulfan [Flu-IV Bu]) and then HSCT. The median patient age was 14 years (range, 10 to 18 years). Before the Flu-IV Bu þ antithymocyte globulin conditioning regimen, all patients received 1 to 2 cycles of pretransplant immunosuppression with fludarabine and dexamethasone. Thirteen patients received a related donor graft, and 5 received an unrelated donor graft. An initial prompt engraftment of donor cells with full donor chimerism was observed in all 18 patients, but 2 patients developed secondary mixed chimerism that necessitated withdrawal of immunosuppression to achieve full donor chimerism. Two patients (11%) had acute grade III-IV graft-versus-host disease, and 5 patients had limited chronic graft-versus-host disease. The only treatment-related mortality was from infection, and with a median follow-up of 42 months (range, 4 to 75), the 5-year overall survival and thalassemia-free survival were 89%. We conclude that this novel sequential immunoablative pretransplant-ation conditioning program is safe and effective for patients with high-risk class 3 thalassemia exhibiting additional comorbidities.

Profiling antibodies to class II HLA in transplant patient sera

Curtis McMurtrey, D Lowe, R Buchli, S Daga, D Royer, A Humphrey, et al.
Human Immunology 75 (2014) 261–270
http://dx.doi.org/10.1016/j.humimm.2013.11.015

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3 lg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to patient antibody concentra-tions did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Reduced-intensity conditioning and HLA-matched hemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicenter study

Tayfun Güngör, P Teira, M Slatter, G Stussi, P Stepensky, D Moshous, et al.
Lancet 2014; 383: 436–48
http://dx.doi.org/10.1016/S0140-6736(13)62069-3

Background In chronic granulomatous disease allogeneic hemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.       Methods This prospective study was done at 16 centers in ten countries worldwide. Patients aged 0–40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centers. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m² [infants <9 kg 1∙2 mg/kg]; one dose per day on days –8 to –3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days –4 to –1; or thymoglobulin 2·5 mg/kg, one dose per day on days –5 to –3]; or low-dose alemtuzumab [<1 mg/kg on days –8 to –6]), and low-dose (50–72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45–65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days –5 to –3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centers (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. Findings 56 patients (median age 12∙7 years; IQR 6·8–17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14–39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16–22) for neutrophils and 21 days (IQR 16–25) for platelets. At median follow-up of 21 months (IQR 13–35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86∙46–99∙09) and of EFS was 91% (79∙78–96∙17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III–IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. Interpretation This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.

Refinement of the Definition of Permissible HLA-DPB1 Mismatches with Predicted Indirectly ReCognizable HLA-DPB1 Epitopes

Kirsten A. Thus, MTA Ruizendaal, TA de Hoop, Eric Borst, et al.
Biol Blood Marrow Transplant 20 (2014) 1705e1710
http://dx.doi.org/10.1016/j.bbmt.2014.06.026

Hematopoietic stem cell transplantation with HLA-DPB1emismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DPe derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLADPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P <.05; and HR, 4.07; 95% CI, .97 to 17.19; P < .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P < .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Treosulfan-Thiotepa-FludarabineeBased Conditioning Regimen for
Allogeneic Transplantation in Patients with Thalassemia Major: A
Single-Center Experience from North India

Dharma Choudhary, SK Sharma, N Gupta,…, Satyendra Katewa
Biol Blood Marrow Transplant 19 (2013) 492e503
http://dx.doi.org/10.1016/j.bbmt.2012.11.007

Hematopoietic stem cell transplantation (HSCT) is the definite treatment
for patients with thalassemia major. A busulfan (Bu) and cyclophosphamide
(Cy)ebased regimen has been the standard myeloablative chemotherapy,
but it is associated with higher treatment-related toxicity, particularly in
patients classified as high risk by the Pesaro criteria. Treosulfan-based
conditioning regimens have been found to be equally effective and less
toxic. Consequently, we analyzed the safety and efficacy of treosulfan/
thiotepa/fludarabine (treo/thio/flu)-based conditioning regimens for
allogeneic HSCT in patients with thalassemia major between February
2010 and September 2012. We compared those results retrospectively
with results in patients who underwent previous HSCT with a Bu/Cy/
antithymocyte globulin (ATG)ebased conditioning regimen. A treo/thio/
flu-based conditioning regimen was used in 28 consecutive patients with
thalassemia major. The median patient age was 9.7 years (range, 2-18
years), and the mean CD34+ stem cell dose was 6.18 x 106/kg. Neutrophil
and platelet engraftment occurred at a median of 15 days (range, 12-23
days) and 21 days (range, 14-34 days), respectively. Three patients
developed veno-occlusive disease, 4 patients developed acute graft
versus-host disease (GVHD), and 2 patients had chronic GVHD. Treatment-
related mortality (TRM) was 21.4%. Two patients experienced secondary
graft rejection. We compared these results with results in patients who
underwent previous HSCT using a Bu/Cy/ATG-based conditioning regimen.
Twelve patients were treated with this protocol, at a median age of 7.2
years (range, 2-11 years). One patient had moderate veno-occlusive disease,
2 patients developed acute GVHD, 2 patients had chronic GVHD, and 2
patients experienced graft rejection. There was no TRM in this group. We
found no significant differences between the 2 groups (treo/thio/flu vs Bu/
Cy/ATG) in terms of the incidence of acute GVHD, chronic GVHD, TRM,
and graft failure, although a trend toward higher TRM was seen with the
treo/thio/flu regimen.

Graft-versus-Host Disease
James L.M. Ferrara, John E. Levine, Pavan Reddy, and Ernst Holler
Lancet. 2009 May 2; 373(9674): 1550–1561
http:dx.doi.org:/10.1016/S0140-6736(09)60237-3

The number of allogeneic hematopoietic cell transplantations (HCT)
continues to increase with more than 25,000 allogeneic transplantations
performed annually. The graft-versus leukemia/ tumor (GVL) effect during
allogeneic HCT effectively eradicates many hematological malignancies.
The development of novel strategies that use donor leukocyte infusions,
non-myeloablative conditioning and umbilical cord blood (UCB)
transplantation have helped expand the indications for allogeneic HCT
over the last several years, especially among older patients. Improvements
in infectious prophylaxis, immunosuppressive medications, supportive care
and DNA-based tissue typing have also contributed to improved outcomes
after allogeneic HCT. Yet the major complication of allogeneic HCT, graft-
versus-host disease (GVHD), remains lethal and limits the use of this
important therapy. Given current trends, the number of transplants from
unrelated donors is expected to double within the next five years,
significantly increasing the population of patients with GVHD. In this
seminar we review advances made in identifying the genetic risk
factors and pathophysiology of this major HCT complication, as well
as its prevention, diagnosis and treatment.

Non-HLA Genetics—Despite HLA identity between a patient and donor,
approximately 40% of patients receiving HLA-identical grafts develop
acute GVHD due to genetic differences that lie outside the HLA loci,
or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY
and HA-3, are expressed on all tissues and are targets for both GVHD
and GVL. Other minor HAs, such as HA-1 and HA-2, are expressed most
abundantly on hematopoietic cells (including leukemic cells) and may
therefore induce a greater GVL effect with less GVHD. Polymorphisms
in both donors and recipients for cytokines that are involved in the
classical `cytokine storm’ of GVHD have been implicated as risk factors
for GVHD. Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10),
Interferon-γ (IFNγ) variants have correlated with GVHD in some, but
not all, studies. Genetic polymorphisms of proteins involved in innate
immunity, such as nucleotide oligomerization domain 2 and Keratin 18
receptors, have also been associated with GVHD.

Future strategies to identify the best possible transplant donor will
probably incorporate both HLA and non-HLA genetic factors. Skin is most
commonly affected and is usually the first organ involved, often coinciding
with engraftment of donor cells. The characteristic maculopapular rash is
pruritic and can spread throughout the body, sparing the scalp. In severe
cases the skin may blister and ulcerate. Apoptosis at the base of epidermal
rete pegs is a characteristic pathologic finding. Other features include
dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent
to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic
infiltration in the dermis.

Gastrointestinal tract involvement of acute GVHD usually presents as
diarrhea but may also include vomiting, anorexia, and/or abdominal pain
when severe. The diarrhea of GVHD is secretory and often voluminous
(greater than two liters per day). Bleeding, which carries a poor prognosis,
occurs as a result of mucosal ulceration but patchy involvement of the
mucosa often leads to a normal appearance on endoscopy.

The incidence of the severity of acute GVHD is determined by the extent
of involvement of  three principal target organs. The overall grades are
classified as I (mild), II (moderate), III (severe) and IV (very severe). Severe
GVHD carries a poor prognosis, with 25% long term survival for grade III and
5% for grade IV. The incidence of acute GVHD is directly related to the
degree of mismatch between HLA proteins and ranges from 35-45% in
recipients of full matched sibling donor grafts to 60-80% in recipients of
one-antigen HLA mismatched unrelated donor grafts. The same degree
of mismatch causes less GVHD using UCB grafts and incidence of acute
GVHD is lower following the transplant of partially matched UCB units
and ranges from 35-65%.

Two important principles are important to consider regarding the
pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated
but normal inflammatory mechanisms mediated by donor lymphocytes infused
into the recipient where they function appropriately, given the foreign
environment they encounter. Second, the recipient tissues that stimulate
donor lymphocytes have usually been damaged by underlying disease,
prior infections, and the transplant conditioning regimen. As
a result, these tissues produce molecules (sometimes referred to as
“danger” signals) that promote the activation and proliferation of donor
immune cells.  Based largely on experimental models, the development
of acute GVHD can be conceptualized in three sequential steps or phases:
(1) activation of the APCs; (2) donor T cell activation, proliferation,
differentiation and migration; and (3) target tissue destruction.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein
expressed on a broad spectrum of leukocytes including lymphocytes,
monocytes, and dendritic cells. Its use in GVHD prophylaxis in a
Phase II trial decreased the incidence of acute and chronic GVHD
following reduced intensity transplant.98 In two prospective studies,
patients who received alemtuzumab rather than methotrexate showed
significantly lower rates of acute and chronic GVHD, but experienced
more infectious complications and higher rates of relapse, so that there
was no overall survival benefit. Alemtuzumab may also contribute to
graft failure when used with minimal intensity conditioning regimens.

An alternative strategy to TCD attempted to induce anergy in donor
T cells by ex vivo antibody blockade of co-stimulatory pathways prior
to transplantation. A small study using this approach in haploidentical
HCT recipients was quite encouraging, but has not yet been replicated.
Thus the focus of most prevention strategies remains  pharmacological
manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis
has also been extensively tested. The best studied drugs are anti-
thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations.
These sera, which have high titers of polyclonal antibodies, are made
by immunizing animals (horses or rabbits) to thymocytes or lymphocytes,
respectively. A complicating factor in determining the role of these
polyclonal sera in transplantation is the observation that even different
brands of the same class of sera exert different biologic effects. However,
the side effects of ATG/ALG infusions are common across different
preparations and include fever, chills, headache, thrombocytopenia
(from cross-reactivity to platelets), and, infrequently, anaphylaxis. In
retrospective studies, rabbit ATG reduced the incidence of GVHD in
related donor HSCT recipients without appearing to improve survival.
In recipients of unrelated donor HSCT, addition of ALG to standard
GVHD prophylaxis effectively prevented severe GVHD, but did not
result in improved survival because of increased infections. In a long
term follow-up study, however, pretransplant ATG provided significant
protection against extensive chronic GVHD and chronic lung dysfunction.

As allogeneic transplantation becomes an increasingly attractive therapeutic
option, the need for novel approaches to GVHD has accelerated. The
number of patients receiving transplants from unrelated donors is
expected to double in the next five years, significantly increasing
the population of patients with GVHD. The advent of RIC regimens
has reduced transplant-related mortality and lengthened the period
during which acute GVHD may develop (many new cases present up
to day 200) and the need for close monitoring of patients in this period
has increased. Patients have often returned to the care of their primary
hematologists by this time, increasing the need for these physicians to
collaborate with transplant specialists in the management of GVHD and
its complications.

Identification of biomarkers for GVHD with diagnostic (and possibly
prognostic) significance may eventually make the treatment of GVHD
preemptive rather than prophylactic. The use of cellular component therapy,
such as regulatory T cells that have been expanded ex vivo. will also
enter clinical trials in the near future, but the extensive infrastructure
required for such cellular approaches will likely limit their use initially.

Immunomodulatory Effects of Palifermin (Recombinant Human
Keratinocyte Growth Factor) in 
an SLE-Like Model of Chronic
Graft-Versus-Host Disease

C. A. Ellison, Y. V. Lissitsyn, I. Gheorghiu & J. G. Gartner
Scandinavian Journal of Immunology 2011; 75, 69–76
http://dx.doi.org:/10.1111/j.1365-3083.2011.02628.x

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation
and survival. Recombinant human KGF, also known as palifermin, protects
epithelial cells from injury induced by chemicals, irradiation and acute murine
graft versus-host disease (GVHD). Findings from our studies and others
have shown that palifermin also has immunomodulatory properties. In a
model of acute GVHD, we showed that it shifts the immune response
from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine
profile. Using the DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-hybrid model of chronic,
systemic lupus erythematosus-like GVHD, we showed that palifermin
treatment is associated with higher levels of Th2 cytokines, the production
of anti-nuclear antibodies, cryoglobulinemia and the development of more
severe pathological changes in the kidney. The aim of our current study
was to gain a better understanding of the immunobiology of KGF by
further characterizing the palifermin-mediated effects in this model of
chronic GVHD. Because the pathological changes we observed resemble
those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we
had originally hypothesized that palifermin might augment TSLP levels.
Surprisingly, we did not observe an increase in thymic

TSLP mRNA expression in palifermin-treated recipients. We did, however,
observe some differences in the percentages of CD4+CD25+Foxp3+
regulatory T cells in the spleen at some time points in palifermin-treated
recipients. Most importantly, we found that TGFβ levels were higher in
palifermin-treated recipients early in the GVH reaction, raising the
possibility that KGF might indirectly induce the development of fibrosis
and glomerulonephritis through a pathway involving TGFβ.

Keratinocyte growth factor (KGF) is an epithelial cell growth factor that is
produced by both mesenchymal cells and intraepithelial cdT cells. It is
also known as fibroblast growth factor 7. Its receptor, (KGFR⁄FGF7R), an
alternatively spliced form of FGFR2 ⁄ bek, is found on epithelial cells in
the intestine, mammary glands, ovaries and urinary tract, and on
hepatocytes, keratinocytes and alveolar type II cells. Previously, it
was shown that recombinant human KGF, also known as palifermin,
can protect the lung, bladder or intestine from chemical- or irradiation-
induced injury. This has been attributed to the ability of KGF to reduce
oxidative damage and enhance DNA repair.

Our own studies have provided a better understanding of the immuno-
biological properties of KGF in pathologically distinct models of systemic
disease driven by intense immunological and inflammatory responses.
The acute GVHD that develops in the C57BL ⁄ 6 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model is characterized by the activation of alloreactive donor T cells,
the production of Th1 cytokines and tissue injury in the skin, gastrointestinal
tract, liver, thymus and lung, where epithelia are present. Injury to the
intestinal mucosa permits the translocation of endotoxin into the system,
which, if untreated, leads to the development of endotoxemic shock. We
showed that palifermin treatment protects recipients from epithelial
cell injury, endotoxemia and morbidity in GVH mice. Palifermin also
shifts the immune response away from one that is predominated by Th1
cytokines towards a profile of mixed Th1 and Th2 cytokines, with a
preponderance of Th2 cytokines. The DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model of chronic GVHD is characterized by pathological changes
resembling those seen in systemic lupus erythematosus (SLE). Using this
model, we showed that palifermin treatment augments the production of Th2
cytokines such as IL-4, IL-5 and IL-13 and obviates IFN-c production. Both
untreated and palifermin-treated recipients developed pathological changes
in the kidney, but these changes were more severe in palifermin-treated
recipients. Some of the changes that developed in the palifermin-treated
recipients resemble those seen in thymic stromal lymphopoietin (TSLP)
transgenic mice. These similarities include the presence of ANA in the
sera, the development of cryoglobulinemia and the development of
glomerulonephritis featuring the deposition of immune complexes
consisting of IgG, IgA, IgM and C3 in the mesangium and the glomerular
capillaries. This led us to hypothesize that treating the recipient mice with
palifermin might induce TSLP expression in this model.

In this study, we were interested in determining whether palifermin
treatment was indeed associated with increased TSLP expression.
We were also interested in knowing whether palifermin treatment
changes the percentage of CD4+CD25+FoxP3+ cells in the spleen,
because palifermin treatment has been associated with increased
percentages of CD4+CD25+FoxP3+ cells in other studies including
our own. Lastly, we wished to study the effect of palifermin treatment
on TGFb levels, because this cytokine is known to play a pivotal role
in the development of glomerulonephritis.

We studied the histopathological changes to confirm that the pathological
changes seen in the kidney in this study were the same as those reported
by us previously.We examined kidney sections from both untreated and
palifermin-treated recipients. In these experiments, we were able to
reproduce findings from an earlier study that showed that palifermin-
treated recipients mice in this model of chronic GVHD develop a severe,
extracapillary proliferative glomerular nephritis characterized by epithelial
crescents and hyaline thrombi. These changes were associated with higher
levels of protein in the urine and the development of ascites, presumably
related to the development of nephrotic syndrome, as a consequence
of glomerular injury.

Pathological changes in the kidney

Pathological changes in the kidney. (A) shows a section from a BDF1-hybrid control
mouse that did not receive a graft. (B) shows increased epithelial cellularity within a
glomerulus from an untreated recipient with chronic graft-versus-host disease, on
day 50. No crescents were observed in sections from this group of recipients.
(C and D) show examples of pathological changes observed in kidneys from
palifermin-treated recipients on day 50. Arrows indicate examples of crescentic
glomerulonephritis and the development of protein casts within tubular lumena.
(E and F) show examples of the hyaline thrombi (arrows) seen in the glomeruli
in kidney sections from palifermin-treated recipients on day 50. All sections
were stained with haematoxylin and eosin except for that shown in (F), which
was stained with Masson Trichrome. The concentration of protein measured in
the urine is shown in the lower left corner of each photomicrograph. Original
magnification: ·200 (B–E) and ·400 (A and F).

TGFβ is a highly pleiotropic cytokine with three isoforms, TGFβ1, TGFβ2 and
TGFβ3 . Nearly, all cells have receptors for at least one of these isoforms,
but cells of the immune system primarily express TGFβ1. This cytokine
was implicated in the development of experimental glomerulonephritis in
experiments in which rats were treated with antiserum directed against
TGFβ1. The ability of palifermin to induce TGFβ release and reverse
limited airflow was demonstrated in a mouse model of emphysema. The
authors further showed that palifermin induced the release of TGFβ1
from primary cultures of mouse alveolar type 2 cells. Our results show
that palifermin treatment is associated with a rise in splenic TGFβ levels
during the first month of the GVH reaction. It is possible that by inducing
TGFβ production shortly after transplantation, palifermin treatment is able
to promote the development of the severe, crescentic glomerulonephritis
that we observed at later time points. As such, our findings raise the
possibility that endogenous KGF might play a role in the development
of glomerulonephritis and ⁄ or other autoimmune phenomena associated
with chronic GVHD and ⁄ or SLE.

T cells, murine chronic graft-versus-host disease and autoimmunity

Robert A. Eisenberg, Charles S. Via
Journal of Autoimmunity 39 (2012) 240e247
http://dx.doi.org:/10.1016/j.jaut.2012.05.017

The chronic graft-versus-host disease (cGVHD) in mice is characterized by
the production of autoantibodies and immunopathology characteristic of
systemic lupus erythematosus (lupus). The basic pathogenesis involves
the cognate recognition of foreign MHC class II of host B cells by alloreactive
CD4 T cells from the donor. CD4 T cells of the host are also necessary for
the full maturation of host B cells before the transfer of donor T cells.
CD8 T cells play critical roles as well. Donor CD8 T cells that are highly
cytotoxic can ablate or prevent the lupus syndrome, in part by killing
recipient B cells. Host CD8 T cells can reciprocally downregulate donor
CD8 T cells, and thus prevent them from suppressing the autoimmune
process. Thus, when the donor inoculum contains both CD4 T cells and
CD8 T cells, the resultant syndrome depends on the balance of activities
of these various cell populations. For example, in one cGVHD model
(DBA/2 (C57BL/6xDBA/2)F1, the disease is more severe in females, as
it is in several of the spontaneous mouse models of lupus, as well as in
human disease. The mechanism of this female skewing of disease
appears to depend on the relative inability of CD8 cells of the female host
to downregulate the donor CD4 T cells that drive the autoantibody response.
In general, then, the abnormal CD4 T cell help and the modulating roles
of CD8 T cells seen in cGVHD parallel the participation of T cells in
genetic lupus in mice and human lupus, although these spontaneous
syndromes are presumably not driven by overt alloreactivity.

Systemic lupus erythematosus (SLE) is characterized by a spectrum of auto-
antibodies that targets multiple normal cellular components, particularly
nucleic acids or proteins that are physiologically bound to nucleic acids.
Although SLE is highly diverse in its manifestations, a common theme
is the loss of B cell tolerance to these cellular autoantigens. More than
for any other human condition, several spontaneously arising mouse
models for SLE have been described, beginning with the New Zealand
strains in 1959. These models are largely genetic. In some cases, an
individual gene such as fas or Yaa plays a major role in driving the loss
of tolerance. However, in general the genetic contribution is complex and
involves multiple loci, which are not yet fully defined.

Despite extensive investigations, the failures in immunoregulation that
underlie the genetic SLE models remain poorly understood. It is not known
for sure which B cell tolerance checkpoints are breached in a given model,
and why. The autoantibody response to DNA, Sm, and other autoantigens
resembles the normal response to exogenous antigens: it involves clonal
expansion, somatic mutation, and a pattern of isotype use characteristic of
a T-cell dependent immunization. Thus the cellular dynamics of the response
may be basically normal. Yet the B-cell repertoire is abnormally autoreactive.

In this review we wish to focus more on the role of the T cell in SLE. As
stated above, the loss of B cell tolerance in SLE does appear in general
to require the participation of T cells. Multiple T cells abnormalities have
been described in human and in murine SLE, although in most cases it is
not clear if these are primary or secondary manifestations. Nevertheless,
it is striking how difficult it has been to demonstrate definitively the specificity
of the T cells that provide help for autoantibody production.

The key cellular mechanism in the cGVHD that results in the loss of B cell
tolerance and the production of the autoantibodies typical of SLE is the
cognate interaction of CD4 T cells with an MHC class II determinant on
the B cell surface. A variety of protocols have achieved this interaction.
In general, either the donor/recipient strains are paired in such away
that they only differ at the MHC class II loci, or the CD4 cells are isolated
free of CD8 cells that would recognize MHC class I. If the allorecognition
involves both CD4 T cell interaction with MHC II and CD8 interaction with
MHC I, an acute GVHD occurs, which is immunosuppressive, rather than
immunostimulatory. The DBA/2 (C57BL/6 DBA/2)F1 (B6D2F1) and the
BALB/c (BALB/c A/J)F1 models are exceptions to this rule. The former
has been investigated extensively for a deficiency in CD8 cytotoxic
lymphocytes.

The MHC class II recognition may be at either the I-A or the I-E locus.
However, the autoantibody specificities seen and the degree of immuno-
pathology differ depending on the locus targeted. In one set of experiments,
F1 mice were bred between either B6 or coisogenic bm12 mice and
B10.A(2R) or B10.A(4R) MHC recombinant congenics. The MHC class II
of B6 is I-Ab, while that of bm12 is I-Abm12. These two alleles differ by
only three amino acids, which is sufficient for a full strength MLR (mixed
lymphocyte reaction) between the two strains. Otherwise B6 and bm12
are identical. B10.A(2R) and B10.A(4R) differ only by the expression of
I-E in the former strain, but not in the latter strain. Thus, donor/recipient
combinations could be employed that provided for allogeneic differs only
at I-A, only at I-E, or at both loci.

Results from Busser et al. delineate requirements for this MHC class II
recognition. Utilizing several transgenic mouse strains that express a
more or less constricted CD4 autoreactive repertoire, they showed that
a diverse repertoire was essential to the production of SLE autoantibodies
by MHC II recognition. On the other hand, the non-specific, early polyclonal
B cell activation phase of cGVHD occurred even with a limited CD4 repertoire.

Figure not shown. Chronic GVHD in bm12 C57BL/6 mice. The MHC of the
bm12 donor differs from the MHC of the C57BL/6 recipient just in three
amino acids in the I-A class II molecule. Thus donor CD4 T cells recognize
MHC IIþ B cells as foreign. Donor CD8 T cells see only self MHC I. All T
cells do not express MHC II. Polyclonal activation and specific lupus
autoantibody responses ensue..

Lupus can result from unchecked CD4 T cell cognate help to a polyclonal
population of B cells. CD8 T cells can downregulate this CD4 driven B-cell
hyperactivity through CD8 CTL effectors and can maintain remission,
possibly through memory CD8 T cells. Whether CD8 CTL actually prevent
lupus in normals and fail in lupus prone individuals is not known; however,
data from the P F1 model suggest that therapeutic induction of CD8 CTL
and possibly long term memory cells may be beneficial in preventing or
limiting disease expression. The potential major role played by either
IFNa and IL-21 in both lupus expression and CD8 CTL function remains
to be further defined, but already these cytokines are being targeted in
human or murine lupus.

It is not surprising that the T cells have been shown to have diverse roles in
the autoimmune cGVHD in mice. Donor CD4 T cells drive the host B cell
activation, while host CD4 T cells are required to mature these B cells prior
to their encounter with donor T cells. Donor CD4 T cells also help activate
donor CD8 T cells, which in turn can downregulate or even ablate the
autoimmune response. Donor CD4 T cells license host DC cells, which in
turn can interact with donor CD8 T cells. Host CD8 T cells can suppress
the activity of donor CD8 T cells, and thereby favor the development of
the lupus syndrome. Although the precise mechanisms of T cell participation
in spontaneous lupus are still being defined, it seems reasonable to probe
these syndromes in humans and in mice for T cell mechanism that have
been shown to participate in cGVHD, CD4-B cell interactions almost
certainly are central to the pathogenesis of spontaneous lupus, and they
have been a target of investigation for several decades. If we understood
the peptide specificity of the alloreactive CD4 T cells that drive the formation
of the characteristic lupus autoantibodies, we would have a much clearer
idea where to look for such epitopes in spontaneous disease. Much less
is known about the other T cell activities defined in cGVHD, particularly
those that involve CD8 T cells. This area should invite further detailed
investigation. For example, the striking role of CD8 T cells in the stronger
female disease in the DBA BDF1 model clearly demands that similar
mechanisms be sought for in spontaneous disease.

Understanding Chronic GVHD from Different Angles

Bruce Blazar, Eric S. White, Daniel Couriel
Biol Blood Marrow Transplant 18:S184-S188, 2012
http://dx.doi.org:/10.1016/j.bbmt.2011.10.025

Whereas acute graft-versus-host disease (aGVHD) rates have decreased
with more intensive GVHD preventive agents and use of single and double
umbilical cord blood units as a source of donor cells in adult recipients,
significant chronic GVHD (cGVHD) rates unexpectedly have remained high.
Moreover, granulocyte colony stimulating factor mobilized peripheral blood
stem cell grafts have been associated with an increased overall risk of
cGVHD. As such, cGVHD has emerged as a primary cause of morbidity
and mortality following allogeneic hematopoietic stem cell transplantation.
Progress in developing cGVHD interventional strategies has been hampered
by variable onset and clinical and pathological manifestations of cGVHD, now
better defined by the National Institutes of Health (NIH) consensus conference,
and a dearth of preclinical models that closely mimic the conditions in which
cGVHD is generated and manifested. Although the exact causes of cGVHD
remain unknown, higher antibody levels have been associated with auto-
immunity and implicated in cGVHD. Newly diagnosed patients with
extensive cGVHD had elevated soluble B cell activating factor levels and
anti-double-strand DNA antibodies were found, which was associated with
higher circulating levels of pregerminal center (GC) B cells and post-GC
plasmablasts. B cells from cGVHD patients were hyperresponsive to Toll-like
receptor-9 signaling and have up-regulated CD86 levels.

By using a Cy and low doses of donor T cells, aGVHD was avoided and
cGVHD with BO favored. Histologic changes were similar to the findings in
human cGVHD with peribronchiolar and perivascular cuffing and infiltration
of the airway epithelium. The liver had inflammation and lymphocytic
infiltration, along with collagen deposition. The parotid and submandibular
salivary glands displayed lymphocytic infiltrates in both the bone marrow
and cGVHD groups, likely because of transplantation conditioning.

Treatment of steroid refractory cGVHD patients with rituximab, a B cell–
depleting anti-CD20 monoclonal antibody, has shown a beneficial role in
resolution of the autoimmune disorders such as systemic lupus erythmatosus
and rheumatoid arthritis, andcGVHD, with overall response rates of 29%
to 36% for oral, hepatic, gastrointestinal, and lung cGVHD, and 60% for
cutaneous cGVHD in aggregate data from multiple trials. Thus, we recently
undertook studies to identify the presence of CD41 T helper cells and B2201
B cells in the airways of mice that had BO, tissue-specific antibodies from sera,
and alloantibody deposition in the lung and liver of cGVHD recipients. cGVHD
development was associated with IgG2c deposition in the lung and liver,
abrogated if the donor bone marrow was deficient in mature B cells or
incapable of producing antihost reactive IgG. Robust GC formation was
seen in mice with cGVHD. Alleviation of symptoms in mice that received
B cell–deficient bone marrow confirms the requirement of B cells for lung
dysfunction and inflammation and fibrosis in the lung and liver.

Given a role for IgG antibodies, allo- or auto-Ab binding to the cGVHD organs
could enable tissue destruction or the pathology could be defined by the
specific function of these secreted antibodies. Pathogenic antibody production
therefore is likely to be an important inducer of cGVHD, and targeting this
specific function of the B cells is an attractive strategy for cGVHD. Because
GC B cells display lower susceptibility to rituximab-mediated clearance, probably
because they reside in a nonoptimal environment for antibody-based depletion,
our observation that GC B cells are critical to the development of cGVHD
suggests that agents that are more effective at disrupting the GC might be
more clinically useful. Treatment with LTbR-Ig, a fusion protein that blocks
interactions between LTbR and its ligands, had a direct effect on the
symptoms of cGVHD, at least in part by blocking GC formation and suggest
that LTbR-Ig could be a potential clinical interventional strategy for prevention
and therapy of cGVHD.

Fibrosis is the end result of a number of inflammatory and other injurious events,
resulting in replacement of normal tissue with a dense extracellular matrix (ECM)
scar composed primarily of collagens. While some degree of tissue fibrosis is
considered protective (e.g. in the setting of cutaneous wound healing),
exaggerated or unrelenting ECM deposition with replacement of the normal
tissue architecture is considered pathologic. Fibroproliferative disorders as
a class involving multiple organs (e.g. cGVHD following hematopoietic stem
cell transplant [affecting up to 30% of recipients surviving more than 100 days,
scleroderma [estimated to affect 70,000 in the US], idiopathic pulmonary fibrosis
[estimated to affect 200,000 in the US], hepatic cirrhosis [estimated to affect
up to 400,000 in the US], and renal fibrosis due to diabetic nephropathy and
other causes [estimated to affect over 400,000 in the US]) are a major cause
of morbidity and mortality. Combined, these disorders alone are conservatively
estimated to affect approximately 1 in 300 persons in the United States. When
coupled with a host of other disorders in which tissue fibrosis contributes to
morbidity (e.g. fibroproliferative acute respiratory distress syndrome,
hypersensitivity pneumonitis, solid organ transplant rejection), that estimate
is likely to be much greater.

Wound healing occurs by a highly orchestrated, complex process that has
been well defined. In general, wound repair occurs in 4 stages which overlap
considerably: clotting/coagulation, inflammation, fibroproliferation, and tissue
remodeling. The initial injury leads to a local disruption of epithelial and
endothelial barriers resulting in the elaboration of inflammatory mediators and
extravasation of cells and plasma proteins that serve to achieve hemostasis
and provide a provisional fibrin-rich matrix for the influx of inflammatory and
other reparative cells. Simultaneously, platelet degranulation provides a local
“boost” of vasodilators, growth factors, and ECM proteins that aid in the wound
healing response. Inflammatory cell influx occurs next, with polymorphonuclear
leukocytes (PMNs) arriving first. Following PMN degranulation, mononuclear
cells (macrophages and lymphocytes) arrive next and, along with PMN derived
products, sterilize and remove foreign materials from the wound. This process
also results in the elaboration of cytokines and chemokines designed to
augment the inflammatory response, to promote angiogenesis (allowing for
enhanced nutrient and oxygen delivery to the wound bed), and to recruit
fibroblasts to the wound bed. Fibroblast recruitment and transdifferentiation to
myofibroblasts (or recruitment of already-differentiated myofibroblasts or
fibroblast precursors; this point is still controversial) marks the fibroproliferative
stage, with the result being the elaboration of ECM proteins (collagens,
fibronectins) to repair the tissue defect.

Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host
disease after related-donor reduced-intensity conditioning allogeneic
hemopoietic 
stem-cell transplantation: a phase 1/2 trial

Sung Won Choi, T Braun, L Chang, JLM Ferrara, A Pawarode, et al.
Lancet Oncol 2014; 15: 87–95
http://dx.doi.org/10.1016/S1470-2045(13)70512-6

Background Acute graft-versus-host disease (GVHD) remains a barrier to more
widespread application of allogeneic hemopoietic stem-cell transplantation.
Vorinostat is an inhibitor of histone deacetylases and was shown to attenuate
GVHD in preclinical models. We aimed to study the safety and activity of
vorinostat, in combination with standard immunoprophylaxis, for prevention of
GVHD in patients undergoing related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation. Methods Between March 31, 2009,
and Feb 8, 2013, we did a prospective, single-arm, phase 1/2 study at two
centers in the USA. We recruited adults (aged ≥18 years) with high-risk
hematological malignant diseases who were candidates for reduced-intensity
conditioning hemopoietic stem-cell transplantation and had an available 8/8
or 7/8 HLA matched related donor. All patients received a conditioning regimen
of fl udarabine (40 mg/m² daily for 4 days) and busulfan (3·2 mg/kg daily for
2 days) and GVHD immunoprophylaxis of mycophenolate mofetil (1 g three
times a day, days 0–28) and tacrolimus (0·03 mg/kg a day, titrated to a goal
level of 8–12 ng/mL, starting day –3 until day 180). Vorinostat (either 100 mg
or 200 mg, twice a day) was initiated 10 days before haemopoietic stem-cell
transplantation until day 100. The primary endpoint was the cumulative
incidence of grade 2–4 acute GVHD by day 100. This trial is registered with
ClinicalTrials.gov, number NCT00810602.
Findings 50 patients were assessable for both toxic effects and response;
eight additional patients were included in the analysis of toxic effects. All
patients engrafted neutrophils and platelets at expected times after
hemopoietic stem-cell transplantation. The cumulative incidence of grade
2–4 acute GVHD by day 100 was 22% (95% CI 13–36). The most common
non-hematological adverse events included electrolyte disturbances (n=15),
hyperglycemia (11), infections (six), mucositis (four), and increased activity
of liver enzymes (three). Non-symptomatic thrombocytopenia after
engraftment was the most common hematological grade 3–4 adverse
event (nine) but was transient and all cases resolved swiftly.
Interpretation Administration of vorinostat in combination with standard
GVHD prophylaxis after related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation is safe and is associated with a
lower than expected incidence of severe acute GVHD. Future studies
are needed to assess the effect of vorinostat for prevention of GVHD in
broader settings of hemopoietic stem-cell transplantation.

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Intracoronary Transplantation of Progenitor Cells after Acute MI

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

Transcoronary Transplantation of Progenitor Cells after Myocardial Infarction

Birgit Assmus, M.D., Jörg Honold, M.D., Volker Schächinger, M.D., Martina B. Britten, M.D., Ulrich Fischer-Rasokat, M.D., et al.
From the Division of Cardiology and Mo­lecular Cardiology, Department of Medi­cine III (B.A., J.H., V.S., M.B.B., U.F.-R., R.L., C.T., K.P., S.D., A.M.Z.), Division of He­matology, Department of Medicine II (H.M.), and the Department of Diagnos­tic and Interventional Radiology (N.D.A.), Johann Wolfgang Goethe University; and the Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service, Baden–Württem-berg–Hessen (T.T.) — both in Frankfurt, Germany.

N Engl J Med 2006;355:1222-32.

Background

Pilot studies suggest that intracoronary transplantation of progenitor cells derived from bone marrow (BMC) or circulating blood (CPC) may improve left ventricular function after acute myocardial infarction. The effects of cell transplantation in patients with healed myocardial infarction are unknown.

METHODS

After an initial pilot trial involving 17 patients, we randomly assigned, in a controlled crossover study, 75 patients with stable ischemic heart disease who had had a myo­cardial infarction at least 3 months previously to receive either no cell infusion (23 patients) or infusion of CPC (24 patients) or BMC (28 patients) into the patent coro­nary artery supplying the most dyskinetic left ventricular area. The patients in the control group were

  • subsequently randomly assigned to receive CPC or BMC, and
  • the patients who initially received BMC or CPC crossed over to receive CPC or BMC, respectively, at 3 months’ follow-up.

RESULTS

The absolute change in left ventricular ejection fraction was significantly greater among patients receiving BMC (+2.9 percentage points) than among those receiving CPC (−0.4 percentage point, P = 0.003) or no infusion (−1.2 percentage points, P<0.001). The increase in global cardiac function was related to significantly

  • en­hanced regional contractility in the area targeted by intracoronary infusion of BMC.

The crossover phase of the study revealed that intracoronary infusion of BMC was associated with a significant increase in global and regional left ventricular func­tion, regardless of whether patients crossed over from control to BMC or from CPC to BMC.

CONCLUSIONS

Intracoronary infusion of progenitor cells is safe and feasible in patients with healed myocardial infarction. Transplantation of BMC is associated with moderate but significant improvement in the left ventricular ejection fraction after 3 months. (ClinicalTrials.gov number, NCT00289822.)

Introduction

HRONIC HEART FAILURE IS COMMON, and its prevalence continues to increase.1 Ischemic heart disease is the principal cause of heart failure.2 Although myocardial salvage due to early reperfusion therapy has significantly re­duced early mortality rates,3

  • postinfarction heart failure resulting from ventricular remodeling re­mains a problem.4

One possible approach to re­versing postinfarction heart failure is

  • enhance­ment of the regeneration of cardiac myocytes as well as
  • stimulation of neovascularization within the infarcted area.

Initial clinical pilot studies have suggested that

  • intracoronary infusion of pro­genitor cells is feasible and may
  • beneficially af­fect postinfarction remodeling processes in pa­tients with acute myocardial infarction.5-9

However, it is currently unknown whether such a treatment strategy may also be associated with

  • improvements in cardiac function in patients with persistent left ventricular dysfunction due to healed myocardial infarction with established scar formation.

Therefore, in the prospective TOPCARE-CHD (Transplantation of Progenitor Cells and Recovery of LV [Left Ventricular] Function in Patients with Chronic Ischemic Heart Disease) trial, we inves­tigated

  • whether intracoronary infusion of pro­genitor cells into the infarct-related artery at least 3 months after myocardial infarction improves global and regional left ventricular function.

Patient Outcome Criteria

The primary end point of the study was the absolute change in global left ventricular ejection fraction (LVEF) as measured by quantitative left ventricular angiography 3 months after cell infu­sion. Secondary end points included quantitative variables relating to the regional left ventricular function of the target area, as well as left ven­tricular volumes derived from serial left ventric­ular angiograms. In addition, functional status was assessed by NYHA classification. Finally, event-free survival was defined as freedom from death, myocardial infarction, stroke, or rehospi­talization for worsening heart failure. Causes of rehospitalization during follow-up were verified by review of the discharge letters or charts of hospital stays.

DETECTION OF VIABLE MYOCARDIUM

All patients underwent low-dose dobutamine stress echocardiography, combined thallium sin­gle-photon-emission computed tomography and [18F]fluorodeoxyglucose positron-emission tomog­raphy, or both, as previously described.6 It was pos­sible to analyze regional left ventricular viability in 80 patients (87%).

RESULTS

BASELINE CHARACTERISTICS OF THE PATIENTS

A total of 92 patients were enrolled in the study. Of these, 35 patients received BMC as their ini­tial treatment (in phases 1 and 2 of the trial), 34 patients received CPC (in phases 1 and 2), and 23 patients received no intracoronary cell infu­sion (in phase 2, as the control group). Table 1 illustrates that the three groups of patients were well matched.

EFFECTS OF PROGENITOR-CELL INFUSION

Quantitative Characteristics of Left Ventricular Function

Patients with an adverse clinical event (six), sub­total stenosis of the target vessel at follow-up (three), an intraventricular thrombus precluding performance of left ventricular angiography (one), or atrial flutter or fibrillation at follow-up (one) were excluded from the exploratory analysis. In addition, of the 81 eligible patients, left ventricu­lar angiograms could not be quantitatively ana­lyzed in 4 because of inadequate contrast opaci-fication, in 1 because of ventricular extrasystoles, and in 4 because of the patients’ refusal to un­dergo invasive follow-up. Thus, a total of 72 of 81 serial paired left ventricular angiograms were available for quantitative analysis (28 in the BMC group, 26 in the CPC group, and 18 in the control group).

Table 2 summarizes the angiographic charac­teristics of the 75 patients included in the ran­domized phase of the study. At baseline, the three groups did not differ with respect to global LVEF, the extent or magnitude of regional left ventricu­lar dysfunction, left ventricular volumes, or stroke volumes.

The absolute change in global LVEF from base­line to 3 months did significantly differ among the three groups of patients. Patients receiving BMC had a significantly larger change in LVEF than patients receiving CPC (P = 0.003) and those in the control group (P<0.001). Similar results were ob­tained when patients from the first two phases of the study (the pilot phase and the randomized phase) were pooled. The results did not differ when patients without evidence of viable myo­cardium before inclusion were analyzed sepa­rately. The change in LVEF was −0.3±3.4 percent­age points in the control group (9 patients), +0.4±3.0 percentage points in the CPC group (18 patients), and +3.7±4.0 percentage points in the BMC group (18 patients) (P = 0.02 for the com­parison with the control group and P = 0.02 for the comparison with the CPC group).

In the subgroup of 35 patients who underwent serial assessment of left ventricular function by MRI, MRI-derived global LVEF increased signifi­cantly, by 4.8±6.0% (P = 0.03) among those receiv­ing BMC (11 patients) and by 2.8±5.2% (P = 0.02) among those receiving CPC (20 patients), where­as no change was observed in 4 control patients (P = 0.14). Thus, MRI-derived assessment of left ventricular function further corroborated the re­sults obtained from the total patient population.

Analysis of regional left ventricular function revealed that BMC treatment significantly in­creased contractility in the center of the left ven­tricular target area (Table 2). Likewise, MRI-derived regional analysis of left ventricular function re­vealed that the number of hypocontractile seg­ments was significantly reduced, from 10.1±3.6 to 8.7±3.6 segments (P = 0.02), and the number of normocontractile segments significantly in­creased, from 3.8±4.5 to 5.4±4.6 segments (P = 0.01), in the BMC group, whereas no significant changes were observed in the CPC group. MRI-derived infarct size, as measured by late enhance­ment volume normalized to left ventricular mass, remained constant both in the CPC group (25± 18% at baseline and 23±14% at 3 months,13 patients) and in the BMC group (20±10% at both time points, 9 patients). Thus, taken together, the data suggest that intracoronary infusion of BMC is associated with significant improvements in global and regional left ventricular contractile function among patients with persistent left ven­tricular dysfunction due to prior myocardial in­farction.

To identify independent predictors of improved global LVEF, a stepwise multivariate regression analysis was performed; it included classic deter­minants of LVEF as well as various baseline characteristics of the three groups (Table 3). The multivariate analysis identified the type of pro­genitor cell infused and the baseline stroke vol­ume as the only statistically significant indepen­dent predictors of LVEF recovery.

Functional Status

The functional status of the patients, as assessed by NYHA classification, improved significantly in the BMC group (from 2.23±0.6 to 1.97±0.7, P = 0.005). It did not improve significantly either in the CPC group (class, 2.16±0.8 at baseline and 1.93±0.8 at 3 months; P = 0.13) or in the control group (class, 1.91±0.7 and 2.09±0.9, respectively; P = 0.27).

RANDOMIZED CROSSOVER PHASE

Of the 24 patients who initially were randomly assigned to CPC infusion, 21 received BMC at the time of their first follow-up examination. Likewise, of the 28 patients who initially were randomly assigned to BMC infusion,

  • 24 received CPC after 3 months.

Of the 23 patients of the control group, 10 patients received CPC and 11 received BMC at their reexamination at 3 months (Fig. 1). As illustrated in Figure 2, regardless of whether patients received BMC as initial treatment, as crossover treatment after CPC infusion, or as crossover treatment after no cell infusion,

  • glob­al LVEF increased significantly after infusion of BMC. In contrast,
  • CPC treatment did not significantly alter LVEF when given either before or after BMC.

Thus, the intrapatient comparison of the dif­ferent treatment strategies not only documents the superiority of intracoronary infusion of BMC over the infusion of CPC for improving global left ventricular function, but also corroborates our findings in the analysis of data according to initial treatment assignment. The

  • preserved im­provement in cardiac function observed among patients who initially received BMC treatment and
  • then crossed over to CPC treatment demon­strates that the initially achieved differences in cardiac function persisted for at least 6 months after intracoronary infusion of BMC.
 Table 1. Baseline Characteristics of the Patients.* (not copied)  

Table 2. Quantitative Variables Pertaining to Left Ventricular Function, as Assessed by Left Ventricular Angiography.*

copy protected

Figure 2. Absolute Change in Quantitative Global Left Ventricular Ejection Fraction (LVEF) during the Crossover Phase of the Trial.

Data at 3 and 6 months are shown for all patients crossing over from BMC to CPC infusion (18 patients), from CPC to BMC infusion
(18 patients), and from no cell infusion to either CPC infusion (10 patients) or BMC infusion (11 patients). I bars represent standard
errors.

Table 3. Stepwise Linear Regression Analysis for Predictors of Improvement in Global Left Ventricular Ejection Fraction.*

Variable Nonstandardized Coefficient B

95% CI for B

P Value

Treatment group

1.49

0.53 to 2.46

0.003
Baseline stroke volume

−0.13

−0.22 to –0.05

0.002
No. of cardiovascular risk factors 0.76
Time since most recent MI 0.48
Concomitant PCI 0.60
Age 0.82
Baseline ejection fraction 0.72
Baseline end-diastolic volume 0.88

* Values are shown only for significant differences. MI denotes myocardial infarc­tion, and PCI percutaneous coronary intervention. For the overall model, the ad­justed R2 was 0.29; P<0.001 by analysis of variance.

 

DISCUSSION

Intrapatient comparison in the crossover phase of the trial rules out the possibility that differences in the patient populations studied may have affected outcomes. However, the mechanisms involved in mediating improved contractile function after intracoronary progenitor-cell infusion are not well understood.

Experimentally, although there is no definitive proof that cardiac myocytes may be regenerated, BMC were shown to contribute to functional re­covery of left ventricular contraction when in­jected into freshly infarcted hearts,13-15 whereas CPC profoundly stimulated ischemia-induced neovascularization.16,17 Both cell types were shown to prevent cardiomyocyte apoptosis and reduce the development of myocardial fibrosis and there­by improve cardiac function after acute myocar­dial infarction.18,19 Indeed, in our TOPCARE-AMI (Transplantation of Progenitor Cells and Regen­eration Enhancement in Acute Myocardial Infarc­tion) studies,6,7,9 intracoronary infusion of CPC was associated with functional improvements similar to those found with the use of BMC im­mediately after myocardial infarction. In the cur­rent study, however, which involved patients who had had a myocardial infarction at least 3 months before therapy,

  • transcoronary adminis­tration of CPC was significantly inferior to administration of BMC in altering global left ven­tricular function.

CPC obtained from patients with chronic ischemic heart disease show pro­found functional impairments,20,21 which might limit their recruitment, after intracoronary infu­sion, into chronically reperfused scar tissue many months or years after myocardial infarction. Thus, additional studies in which larger numbers of functionally enhanced CPC are used will be re­quired to increase the response to intracoronary infusion of CPC.

The magnitude of the improvement after in-tracoronary infusion of BMC, with absolute increases in global LVEF of approximately 2.9 percentage points according to left ventricular angiography and 4.8 percentage points accord­ing to MRI, was modest. However, it should be noted that the improvement in LVEF occurred in the setting of full conventional pharmacologic treatment: more than 90% of the patients were receiving beta-blocker and angiotensin-convert-ing–enzyme inhibitor treatment. Moreover, results from trials of contemporary reperfusion for the treatment of acute myocardial infarction, which is regarded as the most effective treatment strat­egy for improving left ventricular contractile per­formance after ischemic injury, have reported in­creases in global LVEF of 2.8% (in the CADILLAC [Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications] trial) and 4.1% (in the ADMIRAL [Abciximab before Direct Angioplasty and Stenting in Myocardial Infarction Regarding Acute and Long-Term Fol­low-up] trial).22,23

The number of patients, as well as the dura­tion of follow-up, is not sufficient to address the question of whether the moderate improvement in LVEF associated with one-time intracoronary BMC infusion is associated with reduced mortal­ity and morbidity among patients with heart fail­ure secondary to previous myocardial infarction. We conclude that intracoronary infusion of BMC is associated with persistent improvements in regional and global left ventricular function and improved functional status among patients who have had a myocardial infarction at least 3 months previously. Given the reasonable short-term safety profile of this therapeutic ap­proach, studies on a larger scale are warranted to examine its potential effects on morbidity and mortality among patients with postinfarction heart failure.

REFERENCES (1-8/23)

  1. 2001 Heart and stroke statistical up­date. Dallas: American Heart Association, 2000.
  2. Braunwald E. Cardiovascular medicine at the turn of the millennium: triumphs, concerns, and opportunities. N Engl J Med 1997;337:1360-9.
  3. Lange RA, Hillis LD. Reperfusion ther­apy in acute myocardial infarction. N Engl J Med 2002;346:954-5.
  4. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation 2000;101:2981-8.
  5. Strauer BE, Brehm M, Zeus T, et al. Re­pair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circula­tion 2002;106:1913-8.
  6. Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myo­cardial Infarction (TOPCARE-AMI). Circu­lation 2002;106:3009-17.
  7. Britten MB, Abolmaali ND, Assmus B, et al. Infarct remodeling after intra-coronary progenitor cell treatment in pa­tients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003;108: 2212-8.
  8. Wollert KC, Meyer GP, Lotz J, et al. In-tracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet 2004;364:141-8.

 

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Neoangiogenic Effect of Grafting an Acellular 3-Dimensional Collagen Scaffold Onto Myocardium

Author: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

This is Part 3 of a series of contributions on cardiac regeneration after myocardial infarct with stem cells.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)
Larry H. Bernstein, MD, FCAP, and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-27/larryhbern/Progenitor-Cell-Transplant-for-MI,-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold  Induces Neo-angiogenesis (Part 3)
Larry H. Bernstein, MD, FCAP, and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

This series of articles discusses the difficulties we have encountered in adopting stem cell research to clinical therapeutics in regeneration of cardiac tissue damaged post myocardial infarct.  Enormous problems have been encountered in the selection of progenitor cells, the growth into compatible and functional myocardial tissue, and the survival of the myocardium.  Part I went into some detail on a method of obtaining suitable cells, growing them in sheets, and transferring the sheets to the surface for regeneration and repair, which is now going into clinical trials.  Part I will be confined to the importance of source of progenitor cells, whether adult stem cells or umbilical cord blood.

These are issues that need to be considered

  • Adult stem cells
  • Umbilical cord tissue sourced cells
  • Sheets of stem cells
  • Available arterial supply at the margins
  • Infarct diameter
  • Depth of ischemic necrosis
  • Distribution of stroke pressure
  • Stroke volume
  • Mean Arterial Pressure (MAP)
  • Location of infarct
  • Ratio of myocytes to fibrocytes
  • Coexisting heart disease and, or
  • Comorbidities predisposing to cardiovascular disease, hypertension
  • Inflammatory reaction against the graft

Despite successes in pre-clinical animal models with stem cells, a problem arises with respect to the biology of the transplanted progenitor cells.  In Part II, we discovered that neo-angiogenesis occurs without evidence of myocyte generation.  That is the topic we discuss here.

Grafting an Acellular 3-Dimensional Collagen Scaffold Onto a Non-transmural Infarcted Myocardium Induces Neo-angiogenesis and Reduces Cardiac Remodeling

MA Gaballa,a JNE Sunkomat,a H Thai,a,b E Morkin,a G Ewy,a and S Goldman,a,b
From the aSection of Cardiology, University of Arizona Sarver Heart Center, Tucson, Arizona and bSouthern Arizona Veterans Administration Health Care System, Tucson, Arizona.
J Heart Lung Transplant 2006; 25: 946–54.

Background: This study was designed to determine whether tissue engineering could be used to reduce ventricular remodeling in a rat model of non-transmural, non–ST-elevation myocardial infarction.

Methods: We grafted an acellular 3-dimensional (3D) collagen type 1 scaffold (solid porous foam) onto infarcted myocardium in rats. Three weeks after grafting, the scaffold was integrated into the myocardium and retarded cardiac remodeling by reducing left ventricular (LV) dilation. The LV inner and outer diameters, measured at the equator at zero LV pressure, decreased (p < 0.05) from 11,040 ± 212 to 9,144 ± 135 pm, and 13,469 ± 187 to 11,673 ± 104 pm (N = 12), after scaffold transplantation onto infarcted myocardium. The scaffold also shifted the LV pressure–volume curve to the left toward control and induced neo-angiogenesis (700 ± 25 vs 75 ± 11 neo-vessels/cm2, N = 5, p < 0.05). These vessels (75 ± 11%) ranged in diameter from 25 to 100 pm and connected to the native coronary vasculature. Systemic treatment with granulocyte-colony stimulating factor (G-CSF), 50 pg/kg/day for 5 days immediately after myocardial injury, increased (p < 0.05) neo-vascular density from 700 ± 25 to 978 ± 57 neo-vessels/cm2.

Conclusions: A 3D collagen type 1 scaffold grafted onto an injured myocardium induced neo-vessel formation and reduced LV remodeling. Treatment with G-CSF further increased the number of vessels in the myocardium, possibly due to mobilization of bone marrow cells.

Introduction

Despite advances in the treatment of heart failure after myocardial infarction, the incidence and prevalence of this disease is increasing steadily. This is due in part to recent advances in treatment of the acute ischemic event; however, even when patients survive a large myocardial infarction, they are left with damaged ventricles, often leading to heart failure without another ischemic event. Cardiomyocytes may not possess sufficient regenerative capacity after birth because loss of these cells in the acute setting results in a fibrous scar and associated regional contractile dysfunction. Transplantation of exogenous cardiac or stem/progenitor cells has been proposed as treatment for heart failure.1,2 Despite the apparent success of stem-cell therapy, there are conflicting reports about the fate of these cells and their effects on cardiac function.3–6 Previous studies in tissue engineering show that grafting an alginate or collagen scaffold seeded with either fetal cardiomyocytes or fibroblasts on injured myocardium induces neovascularization, but the morphology of these new vessels is abnormal.7–9 Our hypothesis is that grafting of a biodegradable 3D collagen type 1 scaffold onto infarcted rat myocardium would provide temporary mechanical support for the ventricular wall, induce neovascularization, and reduce cardiac remodeling.

We used the cryoinjury approach to create a model of a non–ST-elevation myocardial infarction (NON-STEMI). Our model of a relatively small non-transmural injury is similar to what is seen clinically in patients with acute ischemic injury. We speculate that the scaffold provides an initial mechanical support to retard myocardial dila¬tion after acute MI and a later collateralization between native viable blood vessels in the injured myocardium and the newly formed vessels within the scaffold. Because mobilization of progenitor/stem cells using granulocyte-colony stimulating factor (G-CSF) has been reported to increase vascularization and improve car diac function after MI,10 G-CSF treatment is performed at the time of grafting to further enhance neo-vascular-ization in our model. This study was designed as a “proof of concept” with the ultimate clinical goal to surgically graft a matrix onto the heart in patients with acute MI. This could be done using a minimally invasive approach, such as video-assisted thoracic surgery (VATS) with or without robotics.

METHODS

Six-month-old adult male Fisher 344 rats (inbred strain) were used in this study.6 Fifty infarcted and 10 non-injured rats were used. Six groups of rats were studied:
(1) sham (n = 5);
(2) cryoinjury (n = 14);
(3) sham scaffold (n = 5);
(4) infarction scaffold (n = 12);
(5) infarction G-CSF (n = 12); and
(6) infarction scaffold G-CSF (n = 12).

Scaffolds were grafted immediately after cryoinjury. Six weeks after grafting –

  • hemodynamics,
  • LV pressure–volume,
  • vascular density,
  • immunohistochemistry and
  • LV remodeling measurements were performed.

The experimental protocol was carried out as described in what follows.

Experimental Myocardial Infarction Model

Myocardial infarction was produced as previously de-scribed.11 In brief, after anesthesia with ketamine, xylazine, acepromazine and atropine, a left lateral thoracotomy was performed through the third and fourth intercostal space. A 4-mm stainless-steel probe was submersed in liquid nitrogen and placed on the LV free-wall for 10 seconds. The heart was assessed visibly for viability, and the injury procedure was repeated once at the same site. Before closing the chest, the collagen scaffold was engrafted onto the injured myocardium as described in what follows. The lungs were inflated, the chest was closed, and the animal was allowed to recover. In the sham rats the chest was opened but the cold probe was not placed on the myocardium.

Grafting of 3D Collagen Type 1 Scaffold

Immediately after infarction, while the chest was still open, the scaffold was sutured to the injured myocardium at four different points along the outer boundary of the scaffold. The scaffold, a circular collagen type 1 foam disk 5 mm in diameter and 0.5 mm in thickness (Suwelack Co., Billerback, Germany), was prepared from porcine skin collagen. The scaffold is highly flexible with a porosity of 70% and an average pore diameter of 30 to 60 m as determined by scanning electron microscopy. Di-isocyanate was used to elimi-nate toxic residue after cross-linking, which is common with the use of glutaraldhyde polymers. Before engrafting, the scaffold was washed with distilled water, allowed to dry overnight, treated with rat serum, and again allowed to dry overnight.

Cytokine Treatment

Immediately after scaffold grafting, rats in Groups 5 and 6 received subcutaneous injection of G-CSF (50 g/kg/ day; Amgen Biologicals) for 5 days. Control rats and Groups 2 and 4 were treated with saline. Rats were studied 6 weeks after grafting.

All of the following measurements were performed at 6 weeks after grafting and in all groups, unless otherwise specified.

Hemodynamics

Six weeks after grafting, rats were anesthetized with inactin (100 mg/kg intraperitoneal injection) and placed on a specially equipped operating table with a heating pad to maintain constant body temperature. After endotracheal intubation and placement on a rodent ventilator (Harvard Instruments), a 2F solid-state micromanometer-tipped catheter with two pressure sensors (Millar) was inserted via the right femoral artery, with one sensor located in the left ventricle and another in the ascending aorta. The pressure sensor was equilibrated in 37°C saline before obtaining baseline pressure measurements. After a period of stabilization, LV and aortic pressures and heart rate were recorded and digitized at a rate of 1,000 Hz using a PC equipped with an analog-to-digital converter and customized soft-ware. From these data, LV dP/dt was calculated.

Left Ventricular Pressure–Volume Relationships

Six weeks after grafting and after completing the hemodynamic measurements, LV pressure–volume relations were measured in four randomly selected rats from each group as outlined in our previous publications.12 In brief, a catheter, consisting of PE-90 tubing with telescoped PE-10 tubing inside and a water-filled bal-loon attached, was inserted in the left ventricle via the left atrial appendage. Previous testing of the balloon showed essentially infinite compliance up to 0.68 ml of volume; thus, all LV infusions were kept at 0.68 ml. One end of the double-lumen LV catheter was con-nected to a volume infusion pump (Harvard Apparatus) while the other end was connected to a pressure transducer zeroed at the level of the heart. A drainage tube was also placed in the LV cavity, and the right ventricle was partially incised to prevent loading on the LV. After 2 minutes of perfusion with phosphate-buffered saline (PBS), the LV was filled (0.6 ml/min) and unfilled while pressure was recorded onto a physiologic recorder (Gould). Volume infused is a function of filling rate. The ventricle was infused to 60 mm Hg for all experiments and recordings were done in triplicate.

Communication Between the Newly Formed Vessels Within the Scaffold and Native Vessels in Surviving Myocardium

To determine whether the newly formed vessels within the scaffold were connected to the native circulation in the surviving myocardium, isolated hearts were perfused with Evans blue at the aortic root. In brief, the hearts were perfused at 100 mm Hg with PBS just to clear the blood from the coronary circulation. The hearts were then perfused at 100 mm Hg with 4 mg/ml Evans blue in PBS for 30 seconds. Standard 35-mm photographs were taken within the first 1 to 1.5 minutes after starting Evans blue perfusion. The hearts were than washed with PBS and used for morphologic and histologic analysis as described in what follows.

Morphology, Histology and Immunohistochemistry

LV remodeling (morphology).
After completing all the aforementioned measurements, the hearts were per-fused-fixed with glutaraldhyde at 100 mm Hg via the coronary circulation at zero LV pressure. In the in-farcted group, the lesion area, which is slightly larger than the steel-probe cross-sectional area, was visually measured using standard techniques developed in our laboratory to measure infarct size.13,14 However, in the infarction scaffold groups, it was difficult to measure the lesion area 6 weeks after grafting because the scaffolds were absorbed by the heart tissue and it was difficult to distinguish between the scaffold tissue and the scarred area. Each heart was cut in the short axis to five segments from the apex to the base. The inner and outer diameters were measured in the segment located at the short-axis equator using a computer attached to a digital camera.14
Vascular density within the scaffold.
The perfused-fixed hearts were dehydrated and embedded in paraffin. Five-micron-thick transverse sections, which included the scaffold, were processed for hematoxylin–eosin staining. Selected sections were stained using Factor VIII–like antigen (von Willebrand factor) to identify the endothelial cells. Sections were re-hydrated and antigen retrieval was accomplished by incubation twice in 10 mmol/liter citric acid (pH 6.0) at 95°C for 5 minutes. Endogenous peroxidase activities were removed by incubation for 10 minutes in a PBS solution containing 0.6% H2O2. Slides were incubated with primary antibody and biotinylated rabbit anti-rat IgG (Dako) as secondary antibody. After rinsing with PBS, 0.05% diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide were applied for 5 minutes and washed with water. Muscle sections were examined for positive (brown color) staining. Vascular density was measured by light microscopy at x40 magnification. The number of cross-section vessels per field was counted. Average measurements from six different fields were recorded for each value. Knowing the area of the optical field, data were reported as number of vessels/mm2.
Vascular smooth muscle cells.
Vascular smooth muscle cells were detected by immunohistochemical (IHC) analysis in selected myocardial sections, using antibody directed against -smooth-muscle actin (M0951, Dako).15 Tissue fixation and antibody incubation and detection were performed as described earlier for the vascular density measurements.

Cardiac myocytes.

To determine whether the cells that migrated into the scaffold exhibit a cardiomyocyte-like phenotype, myocardial sections including the scaffold were incubated with mouse monoclonal IgGs primary antibody against either sarcomeric myosin heavy chain, MF20 (1:100 dilution, hybridoma supernatant; Hybrid-oma Bank, University of Iowa) or cardiac troponin T-C (Santa Cruz Biotechnology). Immunostaining on deparaffinized sections was performed using peroxidase standard protocols (as described earlier).

Statistical Analysis

Data are presented as mean +/- SD.  p  < 0.05 indicates statistical significance. For Groups 1, 2, 3 and 4, a 2-way analysis of variance (ANOVA; injury and scaffold-graft¬ing as the 2 factors) was performed, followed by multiple comparisons using Student–Newman–Keuls test. A second 2-way ANOVA (scaffold grafting and G-CSF treatment as the 2 factors), followed by multiple comparisons using Student–Newman–Keuls test, was performed on Groups 2, 4, 5 and 6.

RESULTS

A total of 50 infarcted and 10 non-infarcted rats were used in this study. Immediately after injury, the 3D collagen type 1 scaffold was grafted onto the infarcted myocardium. All major findings of this study were obtained at 6 weeks after scaffold grafting. In a small sub-set of animals, the scaffold was examined at 3 weeks after grafting and was found to be integrated (i.e., attached to the underlying myocardium), not only at the four suture points along the perimeter of the scaffold, but also in the middle section of the scaffold (n =2). Six weeks after grafting, when all subsequent measurements were obtained, the scaffold was mostly absorbed by the underlying myocardial tissue and the distinction between the scaffold tissue and the underlying scar became difficult to identify (n = 36).

Hemodynamics and LV Remodeling After Scaffold Grafting

Non-transmural injury resulted in LV dilation. Six weeks after infarction the LV lumen and the outer diameters, measured at the equator at zero LV pressure, were increased (p < 0.05) from 8,726 ± 189 to 11,041 ± 212 um, and 12,006 ± 99 to 13,469 ± 189 um, respectively (N = 12). Six weeks after scaffold transplantation onto infarcted myocardium, reduced myocardial dilation was detected. The LV inner lumen diameter (Di) and the outer diameter (Do) measured at the short-axis equator at zero LV pressure were decreased from 11,041 ± 212 to 9,144 ± 135 um (N = 12, p < 0.05) and from 13,469 ± 187 to 11,673 ± 104 um (N = 12, p < 0.05), respectively. The scaffold also improved cardiac remodeling by shifting of the LV pressure–volume curve to the left toward the sham (control) curve (Figure 1). In this study, the extent of damage by cryoinjury was small, with no changes in hemodynamic parameters in the injured rats with or without the scaffold at 6 weeks after grafting. Specifically, there were no changes  (nopt shown)

  • in LV end-diastolic pressure,
  • mean arterial pressure or
  • LV dP/dt (Table 1).

Figure 1. Pressure–volume curves for the four groups

Figure 1. Pressure–volume curves for the four groups. Solid line: treatment group (infarcted rats with collagen scaffold); dashed line: untreated sham and sham treated with collagen scaffold; dotted line: untreated infarcted groups (cryoinjury). The curve for sham with collagen scaffold is superimposed upon the untreated sham curve. Note that, 6 weeks after transplantation, the P-V curve is shifted to the left. N = 4 for each group. *p < 0.05.

Induction of Large Vessel Formation 6 Weeks After 3D Collagen Type 1 Scaffold Transplantation

Six weeks after grafting the collagen scaffold onto the infarcted myocardium, large vessels within the graft were observed (Figure 2A). These vessels differed from the typical angiogenesis achieved during wound heal¬ing, which is characterized by thin-walled, leaky vessels (Figure 2B). Vascular density was measured by counting the number of Factor VIII positively stained cells (Figure 3). This microscopic evaluation was carried out by a histologist without knowledge of the intervention. The scaffold induced neo-angiogenesis (700 ± 25 vs 75 ± 11 neo-vessels/cm2, N = 5). These vessels (75 ± 11%) ranged in diameter from 25 to 100 um. Note the presence of mural cells within the vessel wall, which were positive for a-smooth-muscle actin (Figure 4). Finally, the scaffold transplantation onto infarcted hearts decreased (p < 0.05) the scar area (12 ± 3% vs 21 ± 8%, N = 8) compared with infarction alone. However, it was difficult to distinguish the scar tissue from the scaffold at 6 weeks after grafting.

Figure 2. Engrafted scaffold showing vessels

Figure 2. (A) High magnification (original magnification 40) of the H&E stain of the engrafted scaffold showing large vessel (arrows). These vessels are thick-walled and have multiple cell layers. (B) Same magnification as (A) of the H&E staining of infarcted myocardium without the scaffold. Note that the ischemia-induced vascularization is characterized by thin-walled vessels.

Figure 3.  Neovascularization in scaffold

Figure 3. (A) A typical Factor VII staining for endothelial cells in cryoinjured heart with scaffold shows neo-vascularization in the scaffold (top) at 6 weeks after grating onto native myocardium (bottom). (B) A different field from the same section.

Figure 4. Smooth muscle actin staining

Figure 4. Vascular a-smooth-muscle actin staining. (A) Native (non-injured) myocardium (control). (B) Scaffold. Brown staining within the neo-vessels indicates the presence of mural cells 6 weeks after grafting.

Effects of Cytokine Treatment on Vessel Formation 6 Weeks After Scaffold Engraftment

Comparing infarcted + scaffold to infarcted + scaffold + G-CSF rats, systemic treatment with G-CSF 50 ug/kg/ day for 5 days started immediately after cryoinjury increased (p < 0.05) neo-vascular density within the scaffold from 700 ± 25 to 978 ± 57 neo-vessels/mm2 (Figure 5). No effects were observed for systemic treatment with G-CSF in LV remodeling or pressure– volume (P-V) curves when infarcted + scaffold + G-CSF were compared with untreated infarcted + scaffold rats.

Figure 5. Effects of scaffold grafting with and without G-CSF

Figure 5. Effects of scaffold grafting, with and without G-CSF administration, on vascular density. Vascular density increased by 8-fold with the scaffold alone and by 40% with scaffold and G-CSF treatment. *p < 0.05 infarcted scaffold compared with infarcted alone. **p < 0.05 infarcted scaffold G-CSF compared with infarcted scaffold. N  = 5 for each group.

Communication Between Newly Formed Vessels Within the Scaffold and Native Coronary Circulation

In both infarcted + scaffold and cryoinjured + scaffold + G-CSF groups, 6 weeks after grafting, isolated hearts, perfused with Evans blue at the aortic root, showed that the newly formed vessels within the scaffold were connected to the native vessels in the surviving myo-cardium, as indicated by the presence of blue dye within the scaffold (Figure 6B). To confirm if the newly formed vessels within the scaffold are connected to the native coronary circulation, hearts perfused with Evans blue were sectioned (5 um), hematoxylin–eosin (H&E)-stained, and examined under a fluorescence micros-copy. Evans blue showed red under fluorescence (white arrows, Figure 7).

Figure 6. coronary artery prfusion of isolated hearts

Figure 6. Coronary artery perfusion of isolated hearts with Evans blue. (A) Infarcted heart without scaffold (control). (B) Infarcted heart with scaffold. Note the neovasculature within the scaffold that perfuses blue, indicating a connection to the coronary arteries.

not shown

Figure 7. Micrographs showing Evans blue within myocardial vessels (white arrows, red color). H&E myocardial sections examined under fluorescence microscopy (original magnification 40). (A) Non-infarcted myocardium. (B) 3D scaffold.

Induction of Myofibril-like Tissue Within the Scaffold 6 Weeks After Scaffold Engraftment

Six weeks after collagen scaffold grafting onto infarcted myocardium and after treatment with G-CSF for 5 days, there was some evidence of a limited number of myofibril-like cells identified within the scaffold (Figure 8A). These myofibril-like cells were positive for the sarcomeric myosin heavy chain antibody (Figure 8A), MF20 (Hybridoma Bank, University of Iowa) and car-diac troponin T-C (sc-8121, Santa Cruz Biotechnology; Figure 8B). In that section, where these myofibril-like cells are found, there was < 0.01% per field.

Figure 8. cardiac myofibril bundle in scaffold

Figure 8. (A) Detection of cardiac myofibril bundle within the scaffold (left) by MF20 (A) and cardiac troponin T (B) immunohistochemical staining (brown, arrows) in the infarcted scaffold groups. Native myocardium is shown on the right. (C) Control staining for sham rats (uninjured myocardium).

DISCUSSION

In the present study we have shown that, in the presence of a non-transmural MI:

(1) grafting of a 3D extracellular matrix scaffold onto injured myocardium results in neo-vascularization and reduces cardiac re-modeling;

(2) mobilization of bone marrow cells using cytokine treatment further increases this neo-vascularization; and

(3) the resulting vasculature consists of large vessels, which are connected to the native coronary circulation in the surviving myocardium.

The further increase in neo-vascularization by G-CSF treatment suggests that bone marrow cells may contribute to this process. This report shows that, as a “proof of principle,” it is possible to graft a biodegradable scaffold matrix onto an injured heart to promote neo-vascularization and to possibly provide a stable platform in which circulating and/or resident progenitor cells can flourish.
We used an infarcted non-transmural MI model to examine neovascularization at the early stages of an ischemic injury that occurs without severe hemodynamic insult. The clinical correlate of our model is the non–ST-elevation MI (NON-STEMI). Interestingly, similar to our experimental model, in this clinical infarction model

  • there is LV remodeling with chamber dilation without changes in hemodynamics.

The finding that the collagen scaffold prevents this remodeling suggests that this type of approach may have a role in the treatment of early stages of ischemic injury.
Our extracellular matrix scaffold

  • induced large vessels containing vascular smooth muscle cells as evidenced by α-smooth-muscle actin (α-SMA)-positive staining.

These data differ from a previous report, which showed that grafting a scaffold based on a 3D human fibroblast patch on infarcted myocardium induced thin-walled vessels.8 The difference between these two approaches may be due in part to the scaffold itself. The scaffold we used is highly flexible with a moderate pore size (30 to 60 m) and high porosity (70%), thus allowing for cell attachment, migration, delivery of nutrients and waste removal. It also has the advantage that cells and/or growth factors can be delivered in a controlled setting before grafting. More importantly, cyclic stretch applied to the scaffold in vivo, during the cardiac cycle, may help explain the induction of large vessels within the scaffold.
In our NON-STEMI model, adverse remodeling occurs without major hemodynamic insult. The grafted scaffold

  • prevents LV dilation and thinning of the infarcted myocardium.

Preservation of LV geometry may be the main mechanism of the improved P-V relationship after grafting. The scaffold may act as a temporary mechanical support for the injured ventricular wall. Theoretically, the scaffold could also act as a homing site for the injury-mobilized cells that may reduce cardiac remodeling by induction of neovascularization. This is consistent with a previous report in which neovascularization has been suggested to improve cardiac remodeling and function.16
Several different types of myocyte preparations have been directly injected into the myocardium, such as

  • smooth muscle cells,
  • skeletal muscle cells and
  • satellite skeletal muscle cells,

all of which have been shown to enhance cardiac function.

  1. autologous transplantation of skeletal muscle has been shown to reverse LV remodeling.17–19
    1. this approach has been complicated by the induction of arrhythmias that may be due to the lack of electromechanical coupling between the injected skeletal muscle cells and the native myocardium.20
    2. repopulate the infarcted myocardium by direct injection with the patient’s own bone marrow progenitor cells.2,21–23

While these reports have even led to preliminary clinical trials,23 the fate of exogenously delivered cells directly into the myocardium is still unclear.5,6 It is beyond the scope of this report to reconcile this debate. Recently, intramyocardial transplantation of a pouch containing a mixture of collagen type 1 gel and embryonic stem cells was reported to restore infarcted myocardium.24 The approach outlined here for the heart is analogous to the reports of bone marrow cells contributing to the endothelialization of vascular autografts.25
In the present study, we grafted a 3D collagen type 1 scaffold onto the myocardium immediately after cryoinjury. This model was purposely chosen over infarction by coronary artery ligation because cryoinjury with our technique creates a well-defined, non-transmural, reproducible, similarly sized scar every time, as opposed to the coronary ligation model, in which the infarct size is transmural and variable depending upon how proximal the ligature is on the coronary artery. Cryoinjury also results in a non-transmural necrosis, potentially

  • allowing the still-viable native circulation in the surviving myocardium underneath the scar to connect to the newly formed vessels within the scaffold.

The scaffold was applied soon after infarction because we believe that injury is a strong stimulus for recruiting cells into the scaffold in vivo. After acute MI, mRNA expressions of cytokines, such as vascular endothelial growth factor (VEGF), flk-1 and flt-1, are elevated initially throughout the entire heart.27 Our finding of increased density of neo-vessels in the infarcted myocardium with the scaffold is consistent with data showing that the number of circulating endothelial progenitor cells increases after myocardial injury.28
The fact that we could increase the level of neovascularization with G-CSF suggests that mobilization of bone marrow cells and possible migration to the injured myocardium may be responsible for the increase in neovascularization. Although this relationship has not been directly tested in the current study, it is consistent with previous studies demonstrating that exogenous administration of cytokines such as VEGF, stromal cell–derived factor (SDF-1) and fibroblast growth factor (FGF-1)

  1. increase the number of circulating endothelial progenitor cells,
  2. their recruitment to sites of active inflammation, and
  3. induction of angiogenesis.29,30

Taken together, this study has shown that a 3D collagen type 1 scaffold immediately grafted onto an acutely injured myocardium

  • integrates with the tissue;
  • allows for cell population,
  • growth and differentiation;
  • induces large-vessel formation within the graft; and
  • retards LV remodeling.

The further increase in neovascularization after cytokine treatment with G-CSF suggests that mobilized bone marrow cells contribute to this process. Several pre-clinical and clinical trials reported beneficial effects of cell-based therapy after MI. However, due to lack of standardization (i.e., different cell type, cell number, route of administration, etc.) in both clinical and pre-clinical studies, the efficacy of these trials is still unclear. We have shown that grafting of a biodegradable scaffold may be an effective approach for cardiac re-vascularization. Our scaffold could provide a supporting structure with the appropriate milieu for new blood vessel growth.

REFERENCES

1. Sunkomat JN, Gaballa MA. Stem cell therapy in ischemic heart disease. Cardiovasc Drug Rev 2003;21:327–42.

2. Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–5.

3. Murry CE, Soonpaa MH, Reinecke H, et al. Haematopoi-etic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 2004;428:664–8.

4. Balsam LB, Wagers AJ, Christensen JL, et al. Haematopoi-etic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428:668–73.

5. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J Mol Cell Cardiol 2001;33:907–21.

6. Kajstura J, Rota M, Whang B, et al. Bone marrow cells differentiate in cardiac cell lineages after infarction inde-pendently of cell fusion. Circ Res 2005;96:127–37.

7. Leo J, Aboulafia-Etzion S, Dar A, et al. Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 2000;102(suppl III):III-56–61.

8. Kellar RS, Landeen LK, Shepherd BR, Naughtom GK, Ratcliffe A, Willams SK. Scaffold-based 3-D human fibro¬blast culture provides a structural matrix that support angiogenesis in infarcted heart tissue. Circulation 2001; 104:2063–8.

9. Nugent HM, Edelman ER. Tissue engineering therapy for cardiovascular disease. Circ Res 2003;92:1068–78.

10. Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci USA 2001;98:10344–9.

11. Li RK, Jia ZQ, Weisel RD, et al. Cardiomyocyte transplan¬tation improves heart function. Ann Thorac Surg 1996; 62:654–61.

12. Raya TE, Gaballa M, Anderson P, Goldman S. Left ventric¬ular function and remodeling after myocardial infarction in aging rats. Am J Physiol 1997;273:H2652–8.

13. Gaballa MA, Raya TE, Goldman S. Large artery remodeling after myocardial infarction. Am J Physiol 1995;268: H2092–3.

 

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Source of Stem Cells to Ameliorate Damaged Myocardium (Part 2)

Author and Curator: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

This is Part 2 of a 3 part series of perspectives on stem cell applications to regenerating damaged myocardium.

Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-27/larryhbern/Progenitor-Cell-Transplant-for-MI,-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold  Induces Neo-angiogenesis (Part 3)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

This series of articles discusses the difficulties we have encountered in adopting stem cell research to clinical therapeutics in regeneration of cardiac tissue damaged post myocardial infarct.  Enormous problems have been encountered in the selection of progenitor cells, the growth into compatible and functional myocardial tissue, and the survival of the myocardium.  Part I went into some detail on a method of obtaining suitable cells, growing them in sheets, and transferring the sheets to the surface for regeneration and repair, which is now going into clinical trials.  Part I will be confined to the importance of source of progenitor cells, whether adult stem cells or umbilical cord blood.

Do Adult Stem Cells Ameliorate the Damaged Myocardium? Human Cord Blood as a Potential Source of Stem Cells

Elise M.K. Furfaro and Mohamed A. Gaballa
Dept Internal Med, Sarver Heart Center, University of Arizona, College of Medicine, Tucson, AZ
Current Vascular Pharmacology, 2007, 5, 27-44  © 2007 Bentham Science Publishers Ltd.

Abstract: The heart does not mend itself after infarction. Cell-based strategies have promising therapeutic potential. Recent clinical and pre-clinical studies demonstrate varying degrees of improvement in cardiac function using different adult stem cell types such as bone marrow (BM)-derived progenitor cells and skeletal myoblasts. However, the efficacy of cell therapy after myocardial infarction (MI) is inconclusive and the cellular source with the highest potential for regeneration is unclear. Clinically, BM and skeletal muscle are the most commonly used sources of autologous stem cells. One major pitfall of using autologous stem cells is that the number of functional cells is generally depleted in the elderly and chronically ill. Therefore, there is an urgent need for a new source of adult stem cells. Human umbilical cord blood (CB) is a candidate and appears to have several key advantages. CB is a viable and practical source of progenitor cells. The cells are naïve and what’s more, CB contains a higher number of immature stem/progenitor cells than BM.

We review recent clinical experience with adult stem cells and explore the potential of CB as a source of cells for cardiac repair following MI. We conclude that there is a conspicuous absence of clinical studies utilizing CB-derived cells and there is a pressing need for large randomized double-blinded clinical trials to assess the overall efficacy of cell-based therapy.

Keywords: Umbilical cord blood, adult stem cell, myocardial infarction, congestive heart failure, human bone marrow, skeletal muscle, angiogenesis

 INTRODUCTION

There is an urgent need for new and effective therapy for congestive heart failure (CHF). Heart cells may have a limited capacity to regenerate after myocardial infarction (MI), therefore the use of stem cells for cardiac repair is a logical option. In the past three years, clinical and pre-clinical stud-ies examined the potential of a variety of adult stem cells from different sources as therapy for cardiac disease [1-40]. Adult stem cells are typically chosen in clinical studies be-cause their use avoids the ethical problems associated with embryonic cells. Furthermore, adult stem cells were reported to be pluripotent, capable of differentiating to different cell types [41-45]. Bone marrow-derived hematopoietic stem cells, for example, appear to differentiate into brain cells, skeletal muscle cells, liver cells and cardiomyocytes [42-45]. However, the conclusions of the studies have been recently challenged [10-21, 45].

Regardless of the source, stem cells are difficult to iden-tify because they are hard to distinguish from other cells. No techniques are available to reliably identify stem cells other than surface markers. However, cell surface markers are fickle in that none of them appear to be unique to stem cells. For example, stem and progenitor cells of a varying degree of maturity all express the CD34+ surface marker.. Stem cells are typically recovered by isolation of mononuclear cells (MNCs) and subsequent enrichment for a subset of cells that express certain surface markers such as CD34+ or CD133+, etc. These precursors are commonly sorted using the fluores-cence activated sorting system [1-45].

Direct intramyocardial injection of stem cells into the myocardium is the common route of delivery during surgical intervention. This technique of local delivery of stem/ pro-genitor cells to the myocardium has been shown to be feasi-ble and safe in patients with heart disease [1-4, 10-12, 13, 20, 22, 28]. Other than open-heart surgery, the intra-coronary route appears to be the preferred approach in clinical studies because the stem cells are delivered directly to the affected area without traumatizing the myocardium or submitting the body to the systemic side effects of stem cell mobilization [5-9, 14-19, 21]. A complementary approach to increase the efficiency of progenitor cell transplantation is to enhance cell recruitment and retention in the infarcted heart. For example, stromal cell-derived factor (SDF-1α) has recently been shown to play a critical role in stem cell recruitment to the heart after MI [46].

Although there are other sources of adult stem cells such as adipose tissue [47, 48] and cardiac tissue [49, 50], this review briefly discusses clinical trials using BM stem cells and skeletal muscle myoblasts and pre-clinical studies that used cord blood (CB) cells for heart repair carried out during the past three years. This time period was chosen due to the plethora of excellent published reviews that serve as a foun-dation for this work [51-54]. In addition, the reader may re-fer to several recently published reviews [55-63]. Current clinical experience purports the safety and feasibility of BM stem cells and skeletal muscle myoblasts as autologous cell-therapy for cardiac disease [1-20, 22-30]. However, these cell sources have limitations. For example, recovering sufficient numbers of functional BM progenitor cells is a problem in the elderly and ill [64]. Cardiovascular diseases such as diabetes are associated with BM cell dysfunction [64]. Cardiac calcifications were reported in patients following BM stem cell transplantation [64]. Bone marrow-derived mesenchymal cells (MSCs) have been suggested to play a role in myocardial scarring [64]. Skeletal myoblasts have been associated with arrhythmias and have failed to establish gap junctions with native myocardial cells [64]. Furthermore, the efficacy of these cells in repairing damaged myocardium in clinical settings is still not clear partially due to the lack of protocol standardization as well as the use of adjunct treatment. Different diseases, cell types, cell numbers, routes of cell delivery, end point measurements, and the small number of patients included in these studies make it difficult to draw conclusions about the efficacy of stem cell therapy. Larger clinical trials are now underway to assess the risks and benefits of cell-transplantation using stem cells from BM and skeletal muscle [65].

Another emerging source of stem cells is human umbilical CB. CB has the advantage of being readily available. Numerous CB banks already exist and their number is on the rise [64, 66]. CB is obtained by a non-invasive procedure, and contains a larger portion of immature and non-committed cells than BM. Stem cells derived from CB are expandable ex vivo, appear to be more resistant to apoptosis and the risk of transmission of infection is low [64, 67]. In addition, transplantation of CB cells is associated with a lower incidence and risk of graft-versus-host disease [68, 69]. Similar to previous studies that reported beneficial effects of stem cells isolated from BM and skeletal muscle, CB stem cells also show promise for cardiac repair [1, 3-9, 10¬12, 14, 15, 17-23, 25, 27-29]. Over four thousand CB transplants worldwide have been performed for the treatment of other diseases such as leukemia and immune deficiencies [70]. In contrast, to date, no clinical trials using CB-derived stem cells for transplant after MI have been reported.

The following is an update on recent clinical trials that used BM and skeletal muscle stem cells and preclinical studies that used CB cells to repair the injured myocardium. The emphasis is to evaluate CB as a potential and practical source of stem cells for heart repair after MI.

SKELETAL STEM CELLS

Being the first cell type used clinically, it seems logical to start by discussing the use of skeletal myoblasts, or skeletal muscle satellite cells, as cell therapy after MI. The advantages of these cells are that they are readily available from muscle biopsies, they are contractile cells, and they can be expanded ex vivo before delivery into the myocardium. Moreover, they appear to have an increased resistance to ischemia [55, 71]. Cell transplantation was usually performed concomitant to revascularization or in patients with previous revascularization [1, 2, 4-6]. Most of the studies used direct injection as the delivery route [1-4]. The number of patients in each study ranged from five to 30 and patients were followed up from 68 days to four years. Except for one study, transplantation of satellite cells was shown to improve left ventricular ejection fraction (LVEF) in all recent clinical studies [1, 3-6].
Several of these studies showed improvement in New York Heart Association (NYHA) class. Interestingly, Pagani et al. showed enhanced angiogenesis after cell transplantation, but they did not measure cardiac function or ventricular remodeling. Unfortunately, it appears that the incidence of arrhythmia and ventricular tachycardia, necessitating the implementation of prophylactic amiodarone or implanted cardioverter defibrillator as an adjunct treatment, is commonplace among these trials [2-6]. Further undermining the clinical use of skeletal myoblasts is the reported lack of cardiomyogenesis and electrical coupling with native cardiac cells that would be necessary to maintain a healthy and functioning heart [55, 72]. Detailed descriptions of these most recent clinical studies using skeletal muscle satellite cells are included in Table 1 (not shown).

[It is not surprising to this reader that the inadequacy of skeletal muscle donor cells is found to be inadequate for maintaining normal cardiac contractility.  Even though contraction of skeletal muscle, smooth muscle, and heart muscle share a basic motif involving CaMKII, the generation of a calcium spark triggering contraction involves a specific relationship between CaMKIIδ and the RyR2 receptor.   CaMKIIδ is specific to the cardiomyocyte.  The other consideration is that the heart is a syncytium, and it has a relationship to neurohumoral control, distinctly different than that in skeletal muscle  This is perhaps the most telling observation in the observed lack of cardiomyogenesis and electrical coupling with native cardiac cells that would be necessary to maintain a healthy and functioning heart [55, 72]].

BM CLINICAL TRIALS

To date, only small-scale clinical trials, including five to 69 patients, have been performed using bone marrow-derived stem cells (BM-SCs) for transplantation. Three different types of BM-SCs are typically used in recent clinical trials, namely un-fractionated MNCs, CD34+ cells and MSCs. These cells were proposed to treat acute or old MI as well as heart failure [7-21]. Intracoronary injection is the delivery route of choice for these cells [7-9, 14-21]. Revascularization with percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) is commonly used concomitant to cell treatment [13, 15, 16, 18-21]. Several recent trials purported improvement in cardiac function and/or ventricular remodeling three to 12 months after cell treatment [7-11, 15, 17, 18-21]. Some of these studies reported additional enhancement in clinical parameters such as

  • end diastolic (EDV),
  • end systolic volume (ESV)
  • and/or myocardial perfusion [7-9, 10, 17-20].

A small number of studies reported no benefits from BM transplantation [12-14, 16]. In one study, bone marrow transplantation was complicated by coronary artery re-occlusion [21]. The primary endpoint of most of these trials was to assess the safety and feasibility of BM-SC transplantation as a treatment for ischemic heart disease, however these studies are underpowered. In addition, the efficacy of bone marrow cell therapy is difficult to ascertain from clinical studies, at least in part, due to common utilization of adjunct therapy such as revascularization. More detailed descriptions of bone marrow clinical studies are found in Tables 2-5 (not shown).

MOBILIZATION OF BM-DERIVED CELLS

Since transplantation of autologous BM-SCs leads to improvement in cardiac function, mobilization of BM-SCs using cytokines to increase the number of circulating cells was utilized in succeeding studies. Granulocyte colony stimulating factor (G-CSF) is the most common cytokine used to mobilize BM-SCs in clinical studies [22-31]. The feasibility and safety of G-CSF has been reported by several investigators. The number of patients in the G-CSF studies ranged from five to 114 and they were followed for up to 52 weeks. Clinical studies in the last three years have shown that cardiac function improved in about half of the trials using G-CSF to mobilize BM-SCs [22, 23, 27-29]. The remaining half of G-CSF studies reported no effects on cardiac function [24-26, 30, 31]. In one study, an unexpected reduction in LVEF was reported [31]. Adverse effects of G-CSF treatment were reported in almost all the recent clinical studies [22, 24-27, 29, 31]. Detailed descriptions of G-CSF stud¬ies are shown in Tables 6-7.

HUMAN UMBILICAL CORD BLOOD: NO LONGER A WASTE PRODUCT

Amidst the flurry of clinical studies utilizing BM and skeletal muscle SCs, it is a wonder why no trials are reported using CB cell transplantation in humans. However, several pre-clinical studies using various animal models demonstrated the potential use of CB stem cells for cardiac repair after MI [32-40]. Conserved commonalities of cardiac function improvement exist in these studies despite dissimilarity of protocols used [32-40]. The following is a description of the pre-clinical studies which used different subsets of CB-derived stem cells to treat MI. In this review, the pre-clinical studies are categorized according to the type of stem cell administered.

We first start with studies that used CB-derived MNCs. Ma et al. reported that intravenous injection of six million CB-MNCs into non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice 24 h post-MI resulted in an increase in capillary density and decrease in both infarct size and collagen deposition three weeks after treatment [38]. No myogenesis was observed. Human DNA was identified in 10 out of the 19 mice that underwent induction of MI. Direct myocardial injection of one-sixth of the amount of cells used in the above study in rats also reduced infarct size and increased both ventricular wall thickness and LVdP/dt and ejection fraction up to six months after treatment [34].

Similar to CB-MNCs, transplantation of two hundred thousand CD34+ cells, a subset of MNCs, within 20 min after MI

  • increased vascular density,
  • reduced LV dilation, and
  • improved cardiac function four weeks after treatment [35].

However, only about one percent of the injected cells were incorporated into the vessels of the rat myocardium, which suggests that angiogenic factors released by these cells may contribute to the observed angiogenesis [35]. A subset of CD34+, CD34+ KDR+ cell fraction, was proposed to be the subset of cells responsible for angiogenesis induction and improvement in cardiac function after treatment with either MNCs or CD34+ cells [32]. Two hundred thousand of either CD34+ or MNCs, or two thousand of either CD34+ KDR+ or CD34+ KDR- cells were injected in a NOD/SCID mouse model of MI. Compared to transplantation of MNCs or PBS, CD34+ cells

  • increased LVdP/dt,
  • decreased LV end diastolic pressure and
  • infarct size up to five months after MI.

Treatment with two thousand CD34+KDR+ cells, which is two log less than the number of CD34+ cells, resulted in more
angiogenesis compared to either MNC or CD34+ [32].

An immature subset of CB-MNCs, CD133+ cells, were also reported to improve cardiac function after transplantation into MI mice [37]. One to two million CD133+ cells were intravenously injected into athymic nude rats seven days after MI. Four weeks after transplantation,

  • reduction in both scar thinning and
  • LV systolic dilation, and
  • increase in LV fractional shortening were observed.

In contrast to other studies, vessel density did not differ between the cell-treated and control rats [37]. Similarly, transplantation of a subset of these immature CD133+ cells, CD34+ CD133+ cells, into a mouse model of hindlimb ischemia resulted in angiogenesis induction [40]. Transplantation of one hundred thousand CD34+ CD133+ cells into ischemic limbs of immunosup-pressed mice increased both vessel and muscle fiber densities fourteen days after injection. In contrast, administration of CD34+ cells resulted in increased vessel density only. Neither of these findings was observed after administration of CD34- cells [40].

An alternative subset of progenitor cells, called endothelial progenitor cells (EPCs) from either CB or adult peripheral blood (PB), was also shown to induce angiogenesis in ischemic hindlimb [39]. EPCs were derived from MNC CD34+ cells and identified in culture as attaching cells that exhibit spindle-shape. These cells

  1. incorporated acetylated-low density lipoprotein,
  2. released nitric oxide, and
  3. expressed KDR, VE-cadherin, CD31, and vW factor and CD45-.

Not only were the CB-derived EPCs more abundant (10 fold increase) than those derived from PB, they also further in-creased capillary density when injected into ischemic tissue [39].

Finally, another CB-derived cell subset, denoted as human unrestricted somatic stem cells (USSCs), was shown to engraft in the infarcted heart and improve cardiac perfusion [36]. USSCs were defined as negative for the following surface markers:

  • CD14, CD31, CD33, CD34, CD45, CD56, CD133 and human leukocyte antigen class II and
  • positive for CD13, CD29, CD44, and CD49e.

In a porcine model of MI, one hundred million USSCs were directly injected into the infarcted heart four weeks after MI.

  1. Regional perfusion,
  2. LVEF,
  3. scar thickness, and
  4. wall motion increased four weeks after transplantation [36].

In addition to cell transplantation alone, the combination of gene and cell therapy was shown to be a potential treatment for MI [33]. For example,

CD34+ cells transduced with the adeno associated viral vector that encoded either human angiopieotin-1 or vascular endothelial growth factor (VEGF) were intramyocardially injected in a mouse model of MI. Improved cardiac function and increased capillary density were observed with CD34+ cells alone.

However, exaggerated improvements were obtained with the combined therapy of CD34+ cells transfected with Angiopieotin-1 and or VEGF. Compared with CD34+ treatment alone,

  • the combined therapy further increased capillary density and decreased infarct size [33].

Taken together, based on the pre-clinical studies, a common feature of transplantation of human CB-derived cells is

  • induction of angiogenesis and cardiac function improvement in animal models of ischemia.

Myogenesis does not seem to be a mechanism of the beneficial effects of CB transplantation.

Compared with adult stem cells, CB cell treatment has limitations. The practical and crucial difference between stem cells obtained from adult human donors and from CB is quantitatively, not qualitatively based. It is uncommon that more than several million stem cells can be isolated from CB. That amount may be too small for transplantation to an adult. Children appear to be ideal recipients when utilizing this source of stem cells since they are smaller patients and require fewer cells per kilogram of body weight [71]. However, ex vivo expansion of these cells may overcome this limitation [73, 74]. There is another concern that the use of CB for transplantation presents a higher risk of transmitting opportunistic infections [75]. The human herpes viruses are common pathogens found in transplant recipients. Currently, it is routine to test for the presence of anti-cytomegalovirus immunoglobin M. However, screening prospective CB donors for these pathogens reduces the risk of transmission of infection [75].

(Tables from published document are to be viewed in that document.)

CONCLUSIONS

Although early clinical studies suggest that bone marrow and skeletal myoblast transplantation into the infarcted heart improves cardiac perfusion and function, there is an urgent need for large randomized double-blinded clinical trials that assess the overall efficacy of cell-based therapy. In addition, little is known about the mechanisms by which stem cells render their positive effects. Cardiac regeneration by bone marrow cells is an obvious mechanism. However, a small number of experimental studies have purported the occurrence of myocardial regeneration by bone marrow cells. Furthermore, substantial evidence demonstrates that cell types other than cardiomyocytes improve cardiac function, suggesting that the beneficial effects of cell therapy may be independent of cardiac regeneration [76-89]. Enhanced vascularization, on the other hand, is a common finding after bone marrow cell transplantation. Cell engrafment to the vascular wall as well as angiogenic factors released by transplanted cells may be responsible for the enhanced vascularization. Obviously, there remain a considerable number of unanswered questions that must be addressed in basic science laboratories before stem cell therapy becomes standard practice. For example, what are the mechanisms of improvement in cardiac function? Which cell type is best-suited for transplantation? What is the optimal cell concentration that should be used for transplant and what is the most effective route of delivery?

The target patient population which would draw clinical benefit from cell-based therapy must also be defined and the optimal time of injection after the onset of infarction has to be determined. Currently, it is difficult to assess the efficacy of stem cell treatment of MI. This is in part due to lack of standardization among clinical as well as pre-clinical studies. Therefore, in order to accomplish these objectives, there is great need for communication among the various research groups concerned with stem cells and clinical studies.

Here we add yet another source of stem cells, namely the umbilical CB. This source of stem cells had many advantages mentioned in the preceding sections. In addition, pre-clinical studies indicate the efficacy of CB cells in myocardial repair. However, the fate and benefits of these cells need to be tested in clinical settings.

REFERENCES

[1]  Herreros J, Prosper F, Perez A, Gavira JJ, Garcia-Velloso MJ, Barba J, et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003; 24: 2012-20.

[2]  Pagani FD, DerSimonian H, Zawadzka A, Wetzel K, Edge AS, Jacoby DB, et al. Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol 2003; 41: 879-88.

[3]  Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onder-water EE, Lee CH, et al. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol 2003; 42: 2063-9.

[4]  Dib N, Michler RE, Pagani FD, Wright S, Kereiakes DJ, Lengerich R, et al. Safety and feasibility of autologous myoblast transplantation in patients with ischemic cardiomyopathy: four-year follow-up. Circulation 2005; 112: 1748-55.

[5]  Siminiak T, Kalawski R, Fiszer D, Jerzykowska O, Rzezniczak J, Rozwadowska N, et al. Autologous skeletal myoblast transplantation for the treatment of postinfarction myocardial injury: phase I clinical study with 12 months of follow-up. Am Heart J 2004; 148: 531-7.

[6]  Siminiak T, Fiszer D, Jerzykowska O, Grygielska B, Rozwadowska N, Kalmucki P, et al. Percutaneous trans-coronary-venous trans-plantation of autologous skeletal myoblasts in the treatment of post-infarction myocardial contractility impairment: the POZNAN trial. Eur Heart J 2005; 26: 1188-95.

[7]  Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002; 106: 3009-17.

[8]  Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004; 44: 1690-9.

[9]  Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003; 108: 2212-8.

[10]  Perin EC, Dohmann HFR, Borojevic R, Silva SA, Sousa ALS, Mesquita CT, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003; 107: 2294–302.

Human umbilical cord blood stem cells, myocardial infarction (and stroke)

Nathan Copeland, David Harris and Mohamed A Gaballa
Nathan Copeland, Research Associate and Medical Student, University of Arizona Medical School, Tucson, Arizona; David Harris, Professor of Microbiology and Immunology, University of Arizona, Tucson, Arizona; Mohamed A Gaballa, Director, Center for Cardiovascular Research, Sun Health Research Institute, Sun City, Arizona; Section Chief of Basic Science, Cardiology Section, Banner GoodSam Medical Center, Phoenix, Arizona
Clinical Medicine 2009, Vol 9, No 4: 342–5

ABSTRACT – Myocardial infarction (MI) and stroke are the first and third leading causes of death in the USA accounting for more than 1 in 3 deaths per annum. Despite interventional and pharmaceutical advances, the number of people diagnosed with heart disease is on the rise. Therefore, new clinical strategies are needed. Cell-based therapy holds great promise for treatment of these diseases and is currently under extensive preclinical as well as clinical trials. The source and types of stem cells for these clinical applications are questions of great interest. Human umbilical cord blood (hUCB) appears to be a logical candidate as a source of cells. hUCB is readily available, and presents little ethical challenges. Stem cells derived from hUCB are multipotent and immunologically naive. Here is a critical literature review of the beneficial effects of hUCB cell therapy in preclinical trials.
KEY WORDS: animal models, cerebral infarction, myocardial infarction, stem cells, umbilical cord blood

Introduction

The study of stem cell therapies to address some of the most daunting medical challenges, including heart disease and stroke, has advanced steadily over the last three years. The majority of preclinical studies of stem cells as a potential therapy for either myocardial or cerebral ischaemia were positive on average. Small clinical trials, however, show either no or modest improvement in cardiac function after myocardial infarction (MI). Currently, there are two major types of autologous cells that are clinically used for MI and stroke. The first is skeletal myoblasts, harvested from skeletal muscle. These cells can be expanded in culture. Positive outcomes were recently reported in a phase 1 clinical trial using catheter-based injection of myoblasts to the endocardium (CAUSMIC, American Heart Association (AHA) Scientific Sessions 2007). The second is bone marrow cells (BMCs). Intracoronary injection of BMCs improve global left ventricular function (IC-BMC, AHA Scientific Sessions 2007). However, direct injection of BMC administration into scarred myocardium does not alter cardiac contractility of the injured area (IC/IM-BMC, AHA Scientific Sessions 2007). The effects of stem cell therapy can only be addressed using clinical trials that:

•             are randomised, blinded, placebo controlled and adequately sized

•             use standardisation of autologous stem cell processing protocols

•             use robust endpoints of efficacy and safety

•             ensure that follow-up is complete and of adequate duration.

It is becoming clear that realisation of the full potential of the therapeutic benefit of stem cells will require understanding the biology of these undifferentiated cells. A successful therapy will require a source with plentiful supply of multipotent stem cells with minimal or no immune rejection. Several sources of stem cells were explored such as

  • adipose tissue,1–3
  • cardiac tissue,4
  • skeletal muscle biopsies,5,6 and
  • hUCB.

Whether these subpopulations of cells are best suited to treat a disease is still unanswered.

Currently, the only confirmed source for totipotential cells is embryonic. However, there are ethical and scientific obstacles to unbridled use of such cells. For clinical application, autologous adult stem cells are the obvious choice. To date, only adult stem cells derived from a patient’s own bone marrow are being used in clinical trials.

Autologous BMC therapy is not without problems. The majority of instances of MI and cerebral ischaemia (CI) occur in the elderly. Since the quantity and function of BMCs decrease with age, an allogeneic younger donor may be used to source BMCs. This may hinder the efficiency of such a treatment and suffer rejection, therefore another source of stem cells is needed.

Cryopreserved stem cells derived from human leukocyte antigen (HLA)-matched and unmatched unrelated donor hUCB were realised as a sufficient source of transplantable hematopoietic stem cells with high donor-derived engraftment and low risk of refractory acute graft-versus-host disease. However, the use of hUCB cells as treatment for either MI or CI has only been recently investigated in preclinical models.

There are several outstanding review articles on stem cells derived from cord blood in MI7–11 and stroke.12–17 This article adds depth to the debate by providing an updated review as well as presenting an integrated overview of studies involving MI and CI cell-based therapy. In the preparation of this review, every effort was made to include all relevant publications since 2005. Due to space limitations, the number of articles cited has been limited.

Cardiovascular disease

Since 2005, several studies have explored the use of various sub-populations of hUCB stem cells for regenerative therapy. Five types of UCB-derived stem cells were investigated: umbilical cord derived stem (UCDS), unrestricted somatic stem cells (USSC), mononuclear progenitor cells (MNCs), CD133+ and CD34+ subpopulations. The experimental parameters of the studies varied. The majority of studies, however, were performed using the rat animal model and utilising the left antero-lateral descending (LAD) coronary artery ligation model of MI with intramyocardial injection of the stem cells. The laboratory used a similar model to determine the efficacy of stem cell derived from hUCB to improve cardiac function after ischemia and reperfusion. The data indicated that intracoronary administration of mononuclear or CD34+ cells derived from hUCB improved cardiac function after MI by inducing neovascularisation and retarding left ventricular (LV) remodelling.37

The majority of reported studies using hUCB cells showed improvement in the outcomes.18–25 Cardiac functional improvements were almost universally reported as evaluated by:

  • increased ejection fraction;
  • improved wall motion;
  • lowered LV end-diastolic pressure; and
  • increased cardiac contraction as determined by the maximum slope of LV pressure.18–21,23–25

There were conflicting reports on the effects of stem cells on LV fractional shorting. One study reported improved shortening while another reported that BM but not UCB cells produced improved shortening.22,23 Improvements in

  • myocardial perfusion, evaluated by increased capillary density, were repeatedly demonstrated as were
  • reductions in infarct size and the number of apoptotic cells.18–25

Retardation or reduction in LV remodelling were also reported.18,21,22 Although the vast majority of studies showed positive outcomes, HLA matching and further study are still needed before UCB stem cell therapies can become safe and effective treatments in humans. A prime example of the need for further elucidation of these emerging therapies can be illustrated by the findings in a study by Moelker.26 This study used intracoronary administration of unrestricted somatic stem cells (USSCs) in a balloon left circumflex artery (LCX) occlusion ischaemia-reperfusion porcine model of MI. They found that treatment did not improve outcome and actually increased infarct size. Their histological analysis revealed that the injected cells worsened the infarct by obstructing vessels downstream.

Furthermore, the mechanisms of the observed benefits of UCB stem cell therapy in MI are under investigation:

  • improved myocardial perfusion,
  • attenuation of cardiac remodelling,
  • reduction of inflammatory responses by
    • limiting expression of TNF-a, MCP-1, MIP and INF–y, and cardiac regeneration.18–5

Tissue regeneration may be mediated by incorporation of delivered cells in the target tissue.18–21,23 An in vitro study confirmed that mononuclear cells were migrated toward homogenised infarcted myocardium and that the greatest migration occurred at two and 24 hours post-MI.20 Paracrine effect, ie the delivered cells release factors that promote neovascularisation, was also reported. Indeed, the study laboratory has shown that hUCB cells release angiogenic factors in vitro under hypoxic conditions. The data are consistent with a previous report that showed

  • increased expression of VEGF 164 and 188 accompanied by
  • angiogenesis and improved remodelling after administration of hUCB mononuclear cells into the myocardium.21

Identifying subpopulations of progenitor cells with the highest potential for tissue repair is another unanswered ques¬tion prior to widespread application of this therapy in clinical settings. Previous studies showed that UCB-derived endothe¬lial progenitor cells (EPC) to be a promising subset of stem cells for treatment of MI; however their number may be insufficient to treat adult patients. This problem can be addressed by expanding these cells in culture prior to transplant. Techniques are being developed to culture clinically significant quantities (60 population doublings) of EPCs from UBC CD.25 Transplantation of these expanded cells improved ejection fraction (EF) and vascular density in vivo, demonstrating that such a culture method may be a viable option to produce EPCs for future use in humans. Another study evaluated the use of gene therapies in conjunction with UCB stem cell therapy.24 CD34+ cells were transfected with AAV-Ang1 and/or AAV-VEGF 165. The gene-modified stem cells resulted in greater increases in capillary density and cardiac performance along with larger reduction in infarct size compared to CD34+ cell therapy alone.

References

1 Valina C, Pinkernell K, Song YH et al. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. Eur Heart J 2007;28:2667–77.

2 Zhang DZ, Gai LY, Liu HW et al. Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction. Chin Med J (Engl) 2007;120:300–7.

4 Hoogduijn MJ, Crop MJ, Peeters AM et al. Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities. Stem Cells Dev 2007;16:597–604.

5 Payne TR, Oshima H, Okada M et al. A relationship between vascular endothelial growth factor, angiogenesis, and cardiac repair after muscle stem cell transplantation into ischemic hearts. J Am Coll Cardiol 2007;50:1677–84.

6 Herreros J, Prósper F, Perez A et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003;24:2012–20.

7 Goldberg JL, Laughlin MJ, Pompili VJ. Umbilical cord blood stem cells: implications for cardiovascular regenerative medicine. J Mol Cell Cardiol 2007;42:912–20.

8  Wu KH, Yang SG, Zhou B et al. Human umbilical cord derived stem cells for the injured heart. Med Hypotheses 2007;68:94–7.

9 Zhang L, Yang R, Han ZC. Transplantation of umbilical cord blood-derived endothelial progenitor cells: a promising method of therapeutic revascularisation. Eur J Haematol 2006;76:1–8.

18 Wu KH, Zhou B, Yu CT et al. Therapeutic potential of human umbil¬ical cord derived stem cells in a rat myocardial infarction model. Ann Thorac Surg 2007;83:1491–8.

19 Kim BO, Tian H, Prasongsukarn K et al. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation 2005;112:I96–104.

20 Henning RJ, Burgos JD, Ondrovic L et al. Human umbilical cord blood progenitor cells are attracted to infarcted myocardium and sig-nificantly reduce myocardial infarction size. Cell Transplant 2006;15:647–58.

21 Hu CH, Wu GF, Wang XQ et al. Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction. Chin Med J (Engl)  2006;119:1499–506.

22 Ma N, Ladilov Y, Moebius JM et al. Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells. Cardiovasc Res 2006;71:158–69.

23 Leor J, Guetta E, Feinberg MS et al. Human umbilical cord blood-derived CD133+ cells enhance function and repair of the infarcted myocardium. Stem Cells 2006;24:772–80.

24 Chen HK, Hung HF, Shyu KG et al. Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac perfor-mance in mouse after acute myocardial infarction. Eur J Clin Invest 2005;35:677–86.

 

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Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1

Author and Curator: Larry H. Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
This article is Part I of a review of three perspectives on stem cell transplantation onto a substantial size of infarcted myocardium to generate cardiogenesis in tissue that is composed of both repair fibroblasts and cardiomyocytes, after essentially nontransmural myocardial infarct.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/28/progenitor-cell-transplant-for-mi-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold Induces Neo-angiogenesis
 (Part 3)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

The same approach is considered for stroke in one of these studies.  These are issues that need to be considered
  1. Adult stem cells
  2. Umbilical cord tissue sourced cells
  3. Sheets of stem cells
  4. Available arterial supply at the margins
  5. Infarct diameter
  6. Depth of ischemic necrosis
  7. Distribution of stroke pressure
  8. Stroke volume
  9. Mean Arterial Pressure (MAP)
  10. Location of infarct
  11. Ratio of myocytes to fibrocytes
  12. Coexisting heart disease and, or
  13. Comorbidities predisposing to cardiovascular disease, hypertension
  14. Inflammatory reaction against the graft

Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function

L Zakharova1, D Mastroeni1, N Mutlu1, M Molina1, S Goldman2,3, E Diethrich4, and MA Gaballa1*
1Center for Cardiovascular Research, Banner Sun Health Research Institute, Sun City, AZ; 2Cardiology Section, Southern Arizona VA Health Care System, and 3Department of Internal Medicine, The University of Arizona, Tucson, AZ; and 4Arizona Heart Institute, Phoenix, AZ
Cardiovascular Research (2010) 87, 40–49   http://dx.doi.org/10.1093/cvr/cvq027

Abstract

Aims

Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet.

Methods and results

Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 ± 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI.

Conclusion

The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival.These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascular-ization but also inducing a significant level of cardiomyogenesis
Keywords  Myocardial infarction • Cardiac progenitor cells • Cardiospheres • Cardiac regeneration • Contractility

Introduction

Despite advances in cardiac treatment after myocardial infarction (MI), congestive heart failure remains the number one killer world-wide. MI results in an irreversible loss of functional cardiomyocytes followed by scar tissue formation. To date, heart transplant remains the gold standard for treatment of end-stage heart failure, a procedure which will always be limited by the availability of a donor heart. Hence, developing a new form of therapy is vital.
A number of adult non-cardiac progenitor cells have been tested for myocardial regeneration, including skeletal myoblasts,1 bone-marrow2, and endothelial progenitor cells.3,4 In addition, several cardiac resident stem cell populations have been characterized based on the expression of stem cell marker proteins.5–8 Among these, the c-Kit+ population has been reported to promote myocardial repair.5,9 Recently, an ex vivo method to expand cardiac-derived progenitor cells from human myocardial biopsies and murine hearts was developed.10 Using this approach, undifferentiated cells (or cardiospheres) grow as self-adherent clusters from postnatal atrium or ventricular biopsy specimens.11
To date, the most common technique for cell delivery is direct injection into the infarcted myocardium.12 This approach is inefficient because more than 90% of the delivered cells die by apoptosis and only a small number of the survived cells differentiated into cardiomyocytes.13 An alternative approach to cell delivery is a biodegradable scaffold-based engineered tissue.14,15 This approach has the clear advantage in creating tissue patches of different shapes and sizes and in creating a beating heart by decellularization technology.16 Advances are being made to overcome the issue of small patch thickness and to minimize possible toxicity of the degraded substances from the scaffold.15 Recently, scaffold-free cell sheets were created from fibroblasts, mesenchymal cells, or neonatal myocytes.17,18 Transplantation of these sheets resulted in a limited improvement in cardiac function due to induced neovascularization and angiogenesis through secretion of angiogenic factors.17–19 However, few of those progenitor cells have differentiated into cardiomyocytes.17 The need to improve cardiac contractile function suggests focusing on cells with higher potential to differentiate to cardiomyocytes with an improved delivery method.
In the present study, we report a cell-based therapeutic strategy that surpasses limitation inherent in previously used methodologies. We have created a scaffold-free sheet composed of cardiac progenitor cells (cardiospheres) incorporated into a layer of cardiac stromal cells. The progenitor cells survived when transplanted as a cell sheet onto the infarcted area, improved cardiac contractile functions, and supported recovery of damaged myocardium by promoting not only vascularization but also a significant level of cardiomyogenesis. We also showed that cells from a sheet can be recruited to the site of injury driven, at least partially, by the stromal-derived factor (SDF-1) gradient.

Methods

Detailed methods are provided in the Supplementary Methods

Animals

Three-month-old Sprague Dawley male rats were used. Rats were randomly placed into four groups:
(1) sham-operated rats, n = 12;
(2) MI, n = 12;
(3) MI treated with rat sheet, n = 10; and
(4) MI treated with human sheet, n = 10.

Myocardial infarction

MI was created by the ligation of the left coronary artery.20 Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. The extent of infarct was verified by measuring the area at risk: heart was perfused with PBS containing 4 mg/mL Evans Blue as previously described by our laboratory.20 The area at risk was estimated by recording the size of the under-perfused (pale-colored) area of myocardium (see Supplementary material online, Figure S1). Only animals with an area at risk >30% were used in the present study. Post-mortem infarct size was measured using triphenyl tetrazolium chloride staining as previously described by our laboratory.20

Isolation of cardiosphere-forming cells

Cardiospheres were generated as described10 from atrial tissues obtained from:
(1) human atrial resection samples obtained from patients (aged from 53 to 73 years old) undergoing cardiac bypass surgery at Arizonam Heart Hospital (Phoenix, AZ) in compliance with Institutional Review Board protocol (n = 10),
(2) 3-month-old SD rats (n = 10). Briefly, tissues were cut into 1–2 mm3 pieces and tissue fragments were cultured ‘as explants’ in a complete explants medium for 4 weeks (Supplementary Methods).
Cell sheet preparation, labelling, handling, and transplantation
Cardiosphere-forming cells (CFCs) combined with cardiac stromal cells were seeded on double-coated plates (poly-L-lysine and collagen type IV from human placenta) in cardiosphere growing medium (Supplementary Methods). The sheets created from the same cell donors were divided into two groups,
one for transplantation and the other for characterization by immunostaining and RT–PCR (Supplementary Methods).
Prior to transplantation, rat cell sheets were labelled with 2 mM 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, DiI, for tracking transplanted cells in rat host myocardium (Molecular Probes, Eugene, OR). Sheets created using human cells were transplanted unlabelled. Sheets were gently peeled off the collagen-coated plate and folded twice to form four layers. The entire sheet with 200 ml of media was
  • gently aspirated into the pipette tip,
  • transferred to the supporting polycarbonate filter (Costar) and
  • spread off by adding media drops on the sheet (Figure 2A).
Polycarbonate filter was used as a flexible mechanical support for cell sheet to facilitate handling during the transplantation. Immediately after LAD occlusion, the cell sheet was transplanted onto the infarcted area, allowed to adhere to the ventricle for 5–7 min, and the filter was removed before closing the chest (Figure 2A).

Cardiac function

Three weeks after MI, closed-chest in vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar Instruments, Houston, TX) (Supplementary Methods).

Cell sheet survival, engraftment, and cell migration

Rat host myocardium and cell sheet composition after transplantation were characterized by immunostaining (Supplementary Methods). Rat-originated cells were traced by DiI, while human-originated cells were identified by immunostaining with anti-human nuclei or human lamin antibodies.
  1. To assess sheet-originated cardiomyocytes within the host myocardium, the number of cells positive for both human nuclei and myosin heavy chain (MHC) (human sheet); or both DiI and MHC (rat sheet) were counted.
  2. To assess sheet-originated capillaries within the rat host myocardium, the number of cells positive for both human nuclei and von Willebrand factor (vWf) (human sheet); or both DiI and vWf (rat sheet) were counted. Cells were counted in five microscopic fields within cell sheet and area of infarct (n = 5). The number of cells expressing specific markers was normalized to the total number of cells determined by 40,6-diamidino-2-phenylindole staining of the nuclei DNA.
  3. To assess the survival of transplanted cells, sections were stained with Ki-67 antibody followed by fluorescent detection and caspase 3 primary antibodies followed by DAB detection (Supplementary Methods).
  4. To evaluate human sheet engraftment, sections were stained with human lamin antibody followed by fluorescent detection (Supplementary Methods).
  5. Rat host inflammatory response to the transplanted human cell sheet 21 days after transplantation was evaluated by counting tissue mononuclear phagocytes and neutrophils (Supplementary Methods).

Imaging

Images were captured using Olympus IX70 confocal microscope (Olympus Corp, Tokyo, Japan) equipped with argon and krypton lasers or Olympus IX-51 epifluorescence microscope using excitation/emission maximum filters: 490/520 nm, 570 /595 nm, and 355 /465 nm. Images were processed using DP2-BSW software (Olympus Corp).

Statistics

All data are represented as mean ± SE Significance (P < 0.05) was deter-mined using ANOVA (StatView).

Results

Generation of cardiospheres

Cardiospheres were generated from atrial tissue explants. After 7–14 days in culture, a layer of stromal cells arose from the attached explants (Supplementary material online, Figure S2a). CFCs, small phase-bright single cells, emerged from explants and bedded down on the stromal cell layer (Supplementary material online, Figure S2b).
  • After 4 weeks, single CFCs, as well as cardiospheres (spherical colonies generated from CFCs) were observed (Supplementary material online, Figure S2c).
Cellular characteristics of cardiospheres in vitro
Immunocytochemical analysis of dissociated cardiospheres revealed that
  • 30% of cells were c-Kitþ indicating that the CFCs maintain multi-potency. About
  • 22 and 28% of cells expressed a, b-MHC and cardiac troponin I, respectively.
These cells represent an immature cardiomyocyte population because they were smaller (10–15 pm in length vs. 60–80 pm for mature cardiomyocytes) and no organized structure of MHC was detected. Furthermore
  • 17% of the cells expressed a-smooth muscle actin (SMA) and
  • 6% were positive for vimentin,
    • both are mesenchymal cell markers (Supplementary material online, Figure S3a and b).
  • Less then 5% of cells were positive for endothelial cell marker; vWf.
Cell characteristics of human cardiospheres are similar to those from rat tissues (Supplementary material online, Figure S3c).
Cardiospheres were further characterized based on the expression of c-Kit antigen. RT–PCR analysis was performed on both c-Kitþ and c-Kit2 subsets isolated from re-suspended cardiospheres. KDR, kinase domain protein receptor, was recently identified as a marker for cardiovascular lineage progenitors in differentiating embryonic stem cells.21 Here, we found that
  • the c-Kitþ cells were also Nkx2.5 and GATA4-positive, but were low or negative for KDR (Supplementary material online, Figure S3d). In contrast,
  • c-Kit2 cells strongly expressed KDR and GATA4, but were negative for Nkx2.5.
  • Both c-Kitþ and c-Kit2 subsets did not express Isl1, a marker for multipotent secondary heart field progenitors.22
Characteristics of cell sheet prior to transplantation
The cell sheet is a layer of cardiac stromal cells in which the cardiospheres were incorporated at a frequency of 21 ± 0.5 spheres per 100,000 viable cells (Figure 1A). The average diameter of cardiospheres within a sheet was 0.13 ± 0.02 mm and their average area was 0.2 ± 0.06 mm2 (Figure 1A). After sheets were peeled off the plate, it exhibited a heterogeneous thickness ranging from 0.05– 0.1 mm (n 1/4 10), H&E staining (Figure1B) and Masson’s Trichrome staining (Figure 1C) of the sheet sections revealed tissue-like organized structures composed of muscle tissue intertwined with streaks of collagen with no necrotic core. Based on the immunostaining results, sheet compiled of several cell types including
  • SMAþ cardiac stromal cells (50%),
  • MHCþ cardiomyocytes (20%), and
  • vWfþ endothelial cells (10%) (Figure 1D and E).
  • 15% of the sheet-forming cells were c-Kitþ suggesting the cells multipotency (Figure 1E).
  • Cells within the sheet expressed gap-junction protein C43, an indicator of electromechanical coupling between cells (Figure 1D).
  • 40% of cells were positive for the proliferation marker Ki-67 suggesting an active cell cycle state (Figure 1D, middle panel).
Human sheet expressed genes
  1. known to be upregulated in undifferentiated cardiovascular progenitors such as c-Kit and KDR;
  2. cardiac transcription factors Nkx2.5 and GATA4; genes related to adhesion, cell homing, and
  3. migration such as ICAM (intercellular adhesion molecule), CXCR4 (receptor for SDF-1), and
  4. matrix metalloprotease 2 (MMP2).
No expression of Isl1 was detected in human sheet (Figure 1F).
sheet transplant on MI_Image_2
Figure 1 Cell sheet characteristics. (A) Fully formed cell sheet. Arrow indicates integrated cardiosphere. (B) H&E staining; pink colour (arrowhead) indicates cytosol and blue (arrows) indicates nuclear stain. Note that there is no necrotic core within the cell sheet. (C) Masson’s Trichrome staining of sheet section. Arrowhead indicates collagen deposition within the sheet. (D and E) Sheet sections were labelled with antibodies against following markers: (D) vWf (green), Ki-67 (green), C43 (green); (E) c-Kit (green), MHC (red), SMA (red) as indicated on top of each panel. Nuclei were labelled with blue fluorescence of 40,6-diamidino-2-phenylindole (DAPI). (F) Gene expression analysis of the cell sheet. Scale bars, 200 pm (A) or 50 pm (B–E).

Cell sheet survival and proliferation

Two approaches were used to track transplanted cells in the host myocardium.
  • rat cell sheets were labelled with red fluorescent dye, DiI, prior to the transplantation.
  • the sheet created from human cells (human sheet) were identified in rat host myocardium by immunostaining with human nuclei antibodies.
DiI-labelling together with trichrome staining showed engraftment of the cardiosphere-derived cell sheet to the infarcted myocardium (Figure 2B–D). In vivo sheets grew into a stratum with heterogeneous thickness ranging from 0.1–0.5 mm over native tissue. The percentage of Ki-67þ cells within the sheet was 37.5 ± 6.5 (Figure 2F) whereas host tissue was mostly negative (except for the vasculature).
To assess the viability of transplanted cells, the heart sections were stained with the apoptosis marker, caspase 3. A low level of caspase 3 was detected within the sheet, suggesting that the majority of transplanted cells survived after transplantation (Figure 2G).
sheet transplant on MI_Image_3
Figure 2 Transplantation and growth of cell sheet after transplantation.
(A) Sheet transplantation onto infarcted heart. Detached cell sheet on six-well plate (left); cell sheet folded on filter (middle); and transplanted onto left ventricle (right). Scale bar 2 mm. DiI-labelled cell sheets grafted above MI area at day 3
(B) and day 21
(C) after transplantation.
(D) LV section of untreated MI rat at day 21 showing no significant red fluorescence background.
Bottom row (B–D) demonstrates the enlargement of box-selected area of corresponding top panels.
(E) Similar sections stained with Masson’s Trichrome. Section of rat (F) or human (G) sheet treated rat at day 21 after MI.
(F) Section was stained with antibody against Ki-67 (green). Cell sheet was pre-labelled with DiI (red). Nuclei stained with blue fluorescence of DAPI.
(G) Section was double stained with human nuclei (blue) and caspase 3 (brown, arrows) antibodies and counterstained with eosin.
Asterisks (**) indicate cell sheet area. Scale bars 200 mm (B–D, top row), 100 mm (B–D, bottom row, and E) or 50 mm (F, G).
Identification of inflammatory response
Twenty-one days after transplantation of human cell sheet, inflammatory response of rat host was examined. Transplantation of human sheet on infarcted rats reduced the number of mononuclear phagocytes (ED1-like positive cells) compared with untreated MI control (Supplementary material online, Figure S4a–e and l). In addition, the number of neutrophils was similar in both control untreated MI and sheet-treated sections (Supplementary material online, Figure S4f–k and m). These data suggest that at 21 days post transplantation, human cell sheet was not associated with significant infiltration of host immune cells.

Cell sheet engraftment and migration

Development of new vasculature was determined in cardiac tissue sections by co-localization of DiI labelling and vWf staining (Figure 3C). Three weeks after transplantation, the capillary density of ischaemic myocardium in the sheet-treated group significantly increased compared with MI animals (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05, Figure 3A and B). The capillaries originated from the sheet ranged in diameter from 10 to 40 jim (n 1/4 30). A gradient in capillary density was observed with higher density in the sheet area which was decreased towards underlying infarcted myocardium. Mature blood vessels were identified within the sheet area and in the underlying myocardium in close proximity to the sheet evident by vWf and SMA double staining (Figure 3D).
sheet transplant on MI_Image_4
Figure 3 Neovascularization of infarcted wall. (A) Frozen tissue sections stained with vWf antibody (green). LV section of control (sham), infarcted (MI), and MI treated with cell sheet (sheet) rats. Scale bar, 100 jim. (B) Capillary density decreased in the MI compared with sham (*P < 0.05) and improved after cell sheet treatment (#P < 0.05). (C) Neovascularization within cell sheet area was recognized by co-localization of DiI- (red) and vWf (green) staining. Scale bar 100 jim. (D) Mature blood vessels (arrows) were identified by co-localization of SMA (red) and vWf (green) staining. Scale bar 50 jim.
Furthermore, 3 weeks after transplantation, a large number of labelled human nuclei positive or DiI-labelled cells were detected deep within the infarcted area indicating cell migration from the epicardial surface to the infarct (Figure 4A, B, and D). Minor or no migration was detected when the cell sheet was transplanted onto non-infarcted myocardium, sham control (Figure 4C). To evaluate engraftment of sheet-originated cells, sections were labelled with anti-human nuclear lamin antibody. Quantification of engraftment was performed using two approaches: fluorescence intensity and cell counting. Fluorescence intensity of the signal was analysed and compared for different areas of myocardium (Figure 4E–J). Since the transplanted sheets are created by human cells and are stained with human nuclear lamin-labelled with green fluorescence, the signal intensity of the sheet is set to 100% (100% of cells are lamin-positive). Myocardial area with no or limited number of labelled cells had the lowest level of fluorescence signal (13%, or 3.2 ± 1.4% of total number of cells), while
  1. the area where the cell migrated from the sheet to the infarcted myocardium had higher signal intensity (47%, or 11.9 ± 1.7% of total number of cells), indicating a higher number of sheet-originated cells are engrafted in the infarcted area.) (Figure 4K and L).
  2. Migrated cells were positive for KDR (Supplementary material online, Figure S5).
sheet transplant on MI_Image_5
Figure 4 Engraftment quantification of cells migrated from the sheet into the infarcted area of MI. Animals were treated with rat (A) or human (B–F) sheets. Cardiomyocytes were labelled with MHC antibody (A, green or B, red). Rat sheet-originated cells were identified with DiI-labelling, red (A). Arrows indicate the track of migrating cells. Human sheet-originated cells were identified by immunostaining with human nuclei antibody followed by secondary antibodies conjugated with either Alexa 488 (B, E and F, green) or AP (C, D, blue). No migration was detected when the cell sheet was transplanted onto non-infarcted myocardium (C). Heart sections were counterstained with eosin, pink (C–D). Higher magnification of area selected in the box is presented (D, right). Immunofluorescence of sheet (green) grafted to the myocardium surface (E) or cells migrated to the infarction area (F). Fluorescence profiles acrossthe cell sheet itself(G, box 1), area underlying cell sheet (I, box 2) and infarction areawith migrated cells (F, box 3). Mean fluorescence intensityofthe grafted human (K) cells was determined by outlining the region of interest (ROI) and subtracting the background fluorescence for the same region. Fluorescence intensity was normalized to the area of ROI (ii 1/4 6). (L) Percent engraftment was defined as number of lamin-positive cells divided by total number of cells per ROI. ‘M’, myocardium,’S’ sheet, ‘I’ infarction. Scale bars 100 mm (A–C, D, left, E and F), or 50 mm (D, right).
To elucidate a possible mechanism of cell migration, sections were stained to detect SDF1 and its unique receptor CXCR4. The migration patterns of cells from the sheet coincided with SDF-1 expression. Within 3 days after MI, SDF-1 was expressed in the injured myocardium (Figure 5A). At 3 weeks after MI and sheet transplantation, SDF-1 was co-localized with the migrated labelled cells (Figure 5B). PCR analysis revealed CXCR4 expression in cell sheet before transplantation (Figure 1F). However, after transplantation only a fraction of migrated cells expressed CXCR4 (Figure 5C).
sheet transplant on MI_Image_6
Figure 5 Migration of sheet-originated cells into the infarcted area. Confocal images of MI animals treated with sheets from rats (A and B) or human (C). SDF1 (green) was detected at border zone of the infarct at day 3 (A) and day 21 (B). Rat sheet-originated cells were identified with DiI-labelling (red). Note co-localization of DiI-positive sheet-originated cells with SDF1 at 21 days after MI (B). Human cells were identified by immunostaining with human nuclei antibody, red, (C). Note human cells that migrated to the area of infarct express CXCR4 (green) (C). Scale bar, 200 mm (A, B) or 50 mm (C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

3.7 Cardiac regeneration

The differentiation of migrating cells into cardiomyocytes was evident by the co-localization of MHC staining with either human nuclei (Figure 6A) or DiI (Figure 6B and C). In contrast to the immature cardiomyocyte-like cells within the pre-transplanted cell sheet, the migrated and newly differentiated cells within the myocardium were about 30–50 mm in size and co-expressed C43 (see Supplementary material online, Figure S6). Cardiomyogenesis within the infarcted myocardium was observed in the sheets created from either rat or human cells.
sheet transplant on MI_Image_6
Figure 6 Cardiac regeneration. Sections of MI animals treated with human (A) or rat (B, C) sheets. Human sheet was identified by immunostaining with human nuclei antibody (green). Section was double-stained with MHC (red) antibody. Newly formed cardiomyocytes was identified by co-localization of human nuclei and MHC (yellow, arrow). (B) Rat sheet-originated cells were identified by DiI labelling (red). Section was double-stained with MHC (green) antibody. Newly formed cardiomyocytes were detected by co-localization of DiI with MHC (yellow, arrows). (C) Higher magnification of area selected in the boxes (B). Scale bars 200 mm (B), or 20 mm (A, C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

Cell sheet improved cardiac contractile function and retarded LV remodelling after MI

Closed-chest in vivo cardiac function was derived from left ventricle (LV) pressure–volume loops (PV loops), which were measured using a solid-state Millar conductance catheter system. MI resulted in a characteristic decline in LV systolic parameters and an increase in diastolic parameters (Table 1). Cell sheet treatment improved both systolic and diastolic parameters (Table 1). Specifically, load-dependent parameters of systolic function: ejection fraction (EF), dP/dTmax, and cardiac index (CI) were decreased in MI rats and increased towards sham control with the cell sheet treatment (Table 1). Diastolic function parameters, dP/dTmin, relaxation constant (Tau), EDV, and EDP were increased in the MI rats and returned towards sham control parameters after sheet treatment (Table 1). However, load-independent systolic function, Emax, was decreased after MI. Treatment with human sheet improved Emax, while treatment with rat sheet had no effect (Table 1). Treatment with either rat or human sheets retarded LV remodelling; as such that it increased the ratio of anteriolateral wall thickness/LV inner diameter (t/Di) and wall thickness/LV outer diameter (t/Do) (see Supplementary material online, Table S3). However, human sheets appear to further improve LV remodelling compared with rat sheets as indicated by increased ratio of wall thickness to ventricular diameter and decreased both EDV and EDP (Table 1 and see Supplementary material online, Table S3).
Table 1 Hemodynamic parameters
Table 1. hemodynamic parameters

Discussion

The majority of the cardiac progenitor cells delivered using our scaffold-free cell sheet survived after transplantation onto the infarcted heart. A significant percentage of transplanted cells migrated from the cell sheet to the site of infarction and differentiated into car-diomyocytes and vasculature leading to improving cardiac contractile function and retarding LV remodelling. Thus, delivery of cardiac progenitor cells together with cardiac mesenchymal cells in a form of scaffold-free cell sheet is an effective approach for cardiac regeneration after MI.
Consistent with previous studies,5,11 here we showed that cardio-spheres are composed of multipotent precursors, which have the capacity to differentiate to cardiomyocytes and other cardiac cell types. When we fractioned cardiospheres based on c-Kit expression, we identified two subsets: Kitþ /KDR2/low/Nkx2.5þ and Kit2/KDRþ/ Nkx2.52(Supplementary material online, Figure S3d), which are likely reflecting cardiac and vascular progenitors.20
In the present study, delivery of cardiac progenitor cells as a cell sheet facilitates cell survival after transplantation. Necrotic cores, commonly observed in tissue engineered patches,23,24 are absent in cardiosphere sheets prior to transplantation (Figure 1B and C). Poor cell survival is caused by multiple processes such as: ischemia from the lack of vasculature and anoikis due to cell detachment from sub-strate.25 A possible mechanism of cell survival within the sheet is the induction of neo-vessels soon after transplantation due to the presence of endothelial cells within the sheet before transplantation (Figure 10). The cell sheet continued to grow in vivo (Figure 2B and C), suppressed cardiac wall thinning, and prevented LV remodelling at 21 days after transplantation (see Supplementary material online, Table S3). This maybe due to the induction of neovascularization (Figure 3), which may prevents ischemia-induced cell death (Figure 2G). Another likely mechanism of cell survival is that the cells within the scaffold-free sheet maintained cell-to-cell adhesion16 as shown by ICAM expression (Figure 1F). The cells also exhibit C43-positive junctions (Figure 10, see Supplementary material online, Figure S6), which may facilitate electromechanical coupling between the transplanted cells and the native myocardium.
We observed cell migration from the sheet to the infarcted myocardium (Figure 4A and B, E and F), which may be facilitated by the strong expression of MMP2 in the cell sheet (Figure 1F). Although, the mechanism of cardiac progenitor cell migration remains unclear, previous observations showed that SDF-1 is upregulated after MI and plays a role in bone-marrow and cardiac stem cell migration.26,27 Our data suggest that SDF-1-CXCR4 axis plays, at least in part, a role in cardiac progenitor cell migration from cell sheet to the infarcted myocardium. This conclusion is based on the following observations: (1) cell sheet expresses CXCR4 prior to transplantation (Figure 1F), (2) migrated cells are located in the vicinity of SDF-1 release (Figure 5A and B), and (3) about 20% of migrated cells expressed CXCR4. Note, not all the migrated cells expressed CXCR4 suggesting other mechanisms are involved in cell migration (Figure 5C).
Here we report that implanting cardiosphere-generated cell sheet onto infarcted myocardium not only improved vascularization but also promoted cardiogenesis within the infarcted area (Figure 6). A larger number of newly formed cardiomyocytes were found deep within the infarct compared with the cell sheet periphery. Notably the transplantation of the cell sheet resulted in a significant improvement of the cardiac contractile function after MI, as was shown by an increase of EF and decrease of LV end diastolic pressure (Table 1).
The beneficial effect of cell sheet is, in part, due to the presence of a large number of activated cardiac mesenchymal stromal cells (myofibroblasts) within the sheet. Myofibroblasts are known to provide a mechanical support for grafted cells, facilitating contraction28 and to induce neovascularization through the release of cytokines.17 In addition, mesenchymal cells are uniquely immunotolerant. In xenograft models unmatched mesenchymal cells transplanted to the heart of immunocompetent rats were shown to suppress host immune response29 presumably due to inhibition of T-cell activation.30 Consistently with previous study from our laboratory,31 here, we demonstrated host tolerance to the cell sheet 21 days after MI. Finally, phase II and III clinical trials are currently undergoing in which allogeneic MSCs are used to treat MI in patients (Osiris Therapeutic, Inc.).
In summary, our results show that cardiac progenitor cells can be delivered as a cell sheet, composed of a layer of cardiac stromal cells impregnated with cardiospheres. After transplantation, cells from the cell sheet migrated to the infarct, partially driven by SDF-1 gradient, and differentiated into cardiomyocytes and vasculature. Transplantation of cell sheet was associated with prevention of LV remodelling, reconstitution of cardiac mass, reversal of wall thinning, and significant improvement in cardiac contractile function after MI. Our data also suggest that strategies, which utilize undigested cells, intact cell–cell interactions, and combined cell types such as our scaffold-free cell sheet should be considered in designing effective cell therapy.

References

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Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):86–88.
Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–468.
Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006;113:1311–1325.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114: 763–776.
Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–12318.
Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433: 647–653.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.
Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci USA 2005;102:3766–3771.

 

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Reporter: Prabodh Kandala, PhD.

A transcription factor called Lyl-1 is necessary for production of the earliest cells that can become T-cells, critical cells born in the thymus that coordinate the immune response to cancer or infections, said a consortium of researchers led by those from Baylor College of Medicine in a report in the journal Nature Immunology.

These earliest progenitors (called early T lineage progenitor cells) are the first cells that can be identified as being on the road to becoming T-cells, said Dr. Margaret Goodell, director of the Stem Cells and Regenerative Medicine Center of Baylor College of Medicine. Without Lyl-1, only a few of these early T lineage progenitor cells get made.

“This finding gives us insight into the biology of these progenitor cells,” said Goodell, a professor of pediatrics at BCM and a member of the Center for Cell and Gene Therapy at BCM, Texas Children¹s Hospital and The Methodist Hospital.

Dr. Fabian Zohren, a post-doctoral student in Goodell¹s laboratory, found that mice lacking the gene for this factor had a T-cell deficiency and in particular, too few of these early progenitor cells.

“It showed that those early T lineage progenitor cells are really dependent on Lyl-1 for their generation,” said Goodell, who is also corresponding author of the report. “We think that Lyl-1 controls a program that allows survival and expansion of these critical progenitors.”

The finding may have particular import in understanding a form of leukemia known as T-cell acute lymphoblastic leukemia. The researchers found that the forms of the disease that have the worst prognosis are those in which the cancer cells resemble these early T lineage progenitor cells. These cells also have high levels of Lyl-1.

One possibility is the T-cell progenitors in patients with this type of T-cell leukemia continue to express Lyl-1, so continue to be programmed to expand. The excess Lyl-1 prevents the early T lineage progenitor cells from differentiating into active T-cells. Goodell said a recent grant from the Alex¹s Lemonade Stand Foundation will help test that hypothesis.

Abstract:

Thymopoiesis depends on the recruitment and expansion of bone marrow–derived progenitor populations; tight regulation of these processes is required for maintenance of the homeostasis of the T lineage. Lyl-1, a transcription factor that regulates hematopoietic progenitors, is expressed in thymocyte progenitors until T cell commitment. Here we demonstrate a requirement for Lyl-1 in lymphoid specification and the maintenance of early T lineage progenitors (ETPs). Lyl-1 deficiency resulted in profound defects in the generation of lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and ETPs. Lyl-1-deficient ETPs and thymocyte progenitors at the CD4CD8 double-negative 2 (DN2) stage showed more apoptosis, blocked differentiation and impaired population expansion. We identified Gfi1 as a critical transcriptional target of Lyl-1-mediated lymphopoiesis of T cells. Thus, Lyl-1 is a pivotal component of a transcriptional program that controls the lymphoid specification and maintenance of ETPs.

Ref:

http://www.sciencedaily.com/releases/2012/07/120708162320.htm

http://www.nature.com/ni/journal/vaop/ncurrent/full/ni.2365.html.

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