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Posts Tagged ‘Basic fibroblast growth factor’


Contributions to Cardiomyocyte Interactions and Signaling

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Introduction

This is Part II of the ongoing research in the Lee Laboratory, concerned with Richard T Lee’s dissection of the underlying problems that will lead to a successful resolution of myocardiocyte regeneration unhampered by toxicity, and having a suffuciently sustained effect for an evaluation and introduction to the clinic.  This would be a milestone in the treatment of heart failure, and an alternative to transplantation surgery.  This second presentation focuses on the basic science work underpinning the therapeutic investigations.  It is work that, if it was unsupported and did not occur because of insufficient funding, the Part I story could not be told.

Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF

J Yoshioka, RN Prince, H Huang, SB Perkins, FU Cruz, C MacGillivray, DA Lauffenburger, and RT Lee *
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; and Biological Engineering Division, MIT, Cambridge, MA
PNAS 2005; 302(30):10622-10627.  http://pnas.org/cgi/doi/10.1073/pnas.0501198102

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling. Within 3D tissues, cells must coordinate remodeling in response to stress or growth signals, and this communication may occur by direct contact or by secreted signaling molecules. Cardiac hypertrophy is a physiological response that enables the heart to adapt to an initial stress; however, hypertrophy can ultimately lead to the deterioration in cardiac function and an increase in cardiac arrhythmias. Although considerable progress has been made in elucidating the molecular pathogenesis of cardiac hypertrophy, the precise mechanisms guiding the hypertrophic process remain unknown. Recent evidence suggests that myocardial heparin-binding (HB)-epidermal growth factor participates in the hypertrophic response. In cardiomyocytes, hypertrophic stimuli markedly increase expression of the HB-EGF gene, suggesting that HB-EGF can act as an autocrine trophic factor that contributes to cellular growth. HB-EGF is first synthesized as a membrane-anchored form (proHB-EGF), and subsequent ectodo-main shedding at the cell surface releases the soluble form of HB-EGF. Soluble HB-EGF is a diffusible factor that can be captured by the receptors to activate the intracellular EGF receptor signaling cascade. Indeed, EGF receptor (EGFR) transactivation, triggered by shedding of HB-EGF from the cell surface, plays an important role in cardiac hypertrophy resulting from pressure overload in the aortic-banding model. EGFR activation can occur through autocrine and paracrine signaling. In autocrine signaling, a cell produces and responds to the same signaling molecules. Paracrine signaling molecules can target groups of distant cells or act as localized mediators affecting only cells in the immediate environment of the signaling cell. Thus, although locally produced HB-EGF may travel through the extra-cellular space, it may also be recaptured by the EGFR close to the point where it was released from the cell surface. The impact of spatially localized microenvironments of signaling could be extensive heterogeneous tissue remodeling, which can be particularly important in an electrically coupled tissue like myocardium. Interestingly, recent data suggest that EGF can regulate protea-some-dependent degradation of connexin43 (Cx43), a major trans-membrane gap junction protein, in liver epithelial cells, along with a rapid inhibition of cell–cell communication through gap junctions. One of the critical potential myocardial effects of HB-EGF could therefore be to increase degradation of Cx43 and reduce electrical stability of the heart. Reduced content of Cx43 is commonly observed in chronic heart diseases such as hypertrophy, myocardial infarction, and failure. Thus, we hypothesized that HB-EGF signals may operate in a spatially restricted local circuit in the extracellular space. We also hypothesized that HB-EGF secretion by a given cardiomyocyte could create a local remodeling microenvironment of decreased Cx43 within the myocardium. To explore whether HB-EGF signaling is highly spatially constrained, we took advantage of the nonuniform gene transfer to cardiac myocytes in vivo, normally considered a pitfall of gene therapy. We also performed computational modeling to predict HB-EGF dynamics and developed a 3D approach to measure cardiomyocyte hypertrophy.

Results

Autocrine HB-EGF and Cardiomyocyte Growth.

To assess the effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy, cells were infected with adenoviral vectors expressing GFP alone (Ad-GFP) or HB-EGF and GFP (Ad-HB-EGF). At this level of infection, 99% of cardiomyocytes were transduced. The incidence of apoptotic cell death (sub-G1 fraction) was not different between Ad-GFP cells, suggesting that expression of GFP by the adenoviral vector was not cardiotoxic in these conditions. Western analysis by using an anti-HB-EGF antibody confirmed successful gene transfer of HB-EGF in cardiomyocytes (18 ± 5-fold, n = 4, P < 0.01); HB-EGF appeared electrophoretically as several bands from 15 to 30 kDa (Fig. 1A). The strongest band corresponds to the soluble 20-kDa form of HB-EGF. To confirm that Ad-HB-EGF results in cellular hypertrophy, cell size and protein synthesis were measured. Ad-HB-EGF enlarged cardiomyocytes compared with Ad-GFP-infected cells by phase-contrast microscopy (24 ± 10% increase in cell surface area, n = 27, P < 0.05) and with flow cytometry analysis (26 ± 10% increase of Ad-GFP infected cells, P < 0.01, Fig. 1B). Overexpression of HB-EGF increased total protein synthesis in cardiomyocytes as measured by [3H]leucine uptake (34 ± 6% of Ad-GFP, n = 6, P < 0.01, Fig. 1C). Uninfected cells within the same dish (and thus sharing the same culture media) did not develop hypertrophy. Additionally, medium from cultures previously infected with Ad-HB-EGF for 48 h was collected and applied to adenovirus-free cultures. Conditioned medium from Ad-HB-EGF-infected cardiomyocytes failed to stimulate hypertrophy in naive cardiomyocytes (Fig. 1C), and there were no significant differences in cell size between noninfected cells from Ad-GFP and Ad-HB-EGF dishes. These results suggest that HB-EGF acts primarily as an autocrine growth factor in cardiomyocytes in vitro.
Because the dilution factor in culture media is important for autocrine/paracrine signaling, we determined the concentration of soluble HB-EGF in the conditioned medium and the effective concentration to stimulate cardiomyocyte growth. HB-EGF levels in the conditioned medium from Ad-HB-EGF dishes were 258 ± 73 pg/ml (n = 4), whereas HB-EGF levels from Ad-GFP dishes (n = 8) were below the limit of detection (6.7 pg/ml). The addition of 300 pg/ml of exogenous recombinant HB-EGF into fresh media failed to stimulate hypertrophy in cardiomyocytes as measured by [3H]leucine uptake (-12 ± 5.0% compared with control, n = 5,P = not significant), but 2,000 pg/ml of recombinant HB-EGF did result in a significant effect (+24 ± 5.5% compared with control, n = 6, P < 0.05). This comparison implies that the local concentration of autocrine ligand is substantially greater than that indicated by a bulk measurement of conditioned media, consistent with previous experimental and theoretical studies.

Fig. 1. Effects of gene transfer of HB-EGF on rat neonatal cardiomyocyte growth.

(A) Cells were infected with adenoviral vectors expressing GFP (Ad-GFP), or HB-EGF and GFP (Ad-HB-EGF). Western analysis showed the successful gene transfer of HB-EGF. (8) FACS analysis of 5,000 cardiomyocytes demonstrated that overexpression of HB-EGF produced a 26 ± 10% increase in cell size that was significantly greater than the overex-pression of GFP. Bar graphs with errors represent mean ± SEM from three independent experiments. **, P < 0.01 vs. Ad-HB-EGF-nonin-fected cells and Ad-GFP nonin-fected cells. , P < 0.05 vs. Ad-GFP infected cells. (C) Overexpression of HB-EGF resulted in a 34 ± 6% increase in [3H]leucine uptake compared with Ad-GFP (n = 6), whereas conditioned medium from Ad-HB-EGF cells caused an only insignificant increase. **, P < 0.05 vs. Ad-GFP control and conditioned medium Ad-GFP.

 Effects of HB-EGF on Cx43 Content in Cultured Cardiomyocytes

Because EGF can induce degradation of the gap junction protein Cx43 in other cells, we then determined whether Cx43 is regulated by HB-EGF in cardiomyocytes. Fig. 2A shows a representative immunoblot from three separate experiments in which Cx43 migrated as three major bands at 46, 43, and 41 kDa, as reported in ref. 16. Overexpression of HB-EGF decreased total Cx43 content (27 ± 11% compared with Ad-GFP, n = 4, P < 0.05) without affecting the intercellular adhesion protein, N-cadherin. The phosphorylation of ERK1/2, an intracellular signaling kinase downstream of EGFR transactivation, was augmented by HB-EGF (3.2 ± 1.0-fold compared with Ad-GFP, n = 4, P < 0.05). Northern analysis showed that HB-EGF did not reduce Cx43 gene expression, suggesting that HB-EGF decreases Cx43 by posttranslational modification (Fig. 2B). AG 1478 (10 iLM), a specific inhibitor of EGFR tyrosine kinase, abolished the effect of HB-EGF on Cx43 (Fig. 2C), indicating that the decrease in Cx43 content depends on EGFR transactivation by HB-EGF. The conditioned medium from Ad-HB-EGF-infected cells did not change expression of Cx43 in naive cells, even though ERK1/2 was slightly activated by the conditioned medium (Fig. 1D). These data are consistent with the hypertrophy data presented above, demonstrating that HB-EGF can act as a predominantly autocrine factor both in hypertrophy and in the reduction of Cx43 content in cardiomyocytes.

Computational Analysis Predicts HB-EGF Autocrine/Paracrine Signaling in Vivo.

Although these in vitro experiments showed HB-EGF as a predominantly autocrine cardiac growth factor, HB-EGF signaling in vivo takes place in a very different environment. Therefore, we sought to determine the extent that soluble HB-EGF may travel in the interstitial space of the myocardium with a simple 2D model of HB-EGF diffusion (Fig. 3A). An approximate geometric representation of myocytes in cross-section is a square (15 x 15 iLm), with each of the corners occupied by a capillary (diameter 5 iLm). The cell shape was chosen so that the extracellular matrix width (0.5 iLm), in which soluble HB-EGF is free to diffuse, was constant around all tissue features. This model geometry is based on a square array of capillaries; although a hexagonal pattern of capillary distribution is commonly accepted, the results are not expected to be substantially different with this simpler construction, because both have four capillaries surrounding each myocyte. The model represents a single central cell that is releasing HB-EGF at a constant rate, Rgen, (approximated from the HB-EGF concentration measurement in conditioned medium) into the extracellular space. HB-EGF then can diffuse throughout this space, or enter a capillary and leave the system. This system is governed by
  • the diffusion equation at steady state (DV2C = 0),
  • the boundary condition for the ligand producing cell (—DVC = Rgen),
  • the boundary condition for all other cells (—DVC = 0), and
  • the capillary boundary condition (DVC = h(C — Cblood)).

C denotes HB-EGF concentration, D is the diffusivity constant, h is the mass transfer coefficient, and Cblood is the concentration of HB-EGF in the blood, approximated to be zero.  The numerical solution in Fig. 3B illustrates that HB-EGF remained localized around the cell which produced it and did not diffuse farther because of the sink-like effect of the capillaries. The maximum concentration of soluble HB-EGF achieved is 0.27 nM, which is near the threshold level of HB-EGF measured to stimulate cardiomyocyte growth (2,000 pg/ml). Therefore, the central HB-EGF-producing cell only signals to its four adjacent neighbors where the HB-EGF concentration reaches this threshold. However, if the model geometry is altered to reflect a 50% and 150% increase in cross-sectional area in all cells because of hypertrophy, estimated from 1 and 4 weeks of transverse aortic constriction, the maximum concentration achieved increases slightly to 0.29 nM and 0.37 nM, respectively. As the cell width increases, HB-EGF must diffuse farther to reach a capillary, exposing adjacent cells to a higher concentration during hypertrophy. However, no additional cells are exposed to HB-EGF. 

Fig. 3. Computational modeling of HB-EGF diffusion in the myocardium.

Red areas represent capillaries, green represents the HB-EGF ligand producing cell, pink represents adjacent cells, and white is an extracellular matrix where HB-EGF is free to diffuse. (A) The model geometry where HB-EGF is generated by the ligand-producing cell at a constant rate, Rgen, and diffuses throughout the extracellular space or enters a capillary and leaves the system with a mass transfer coefficient, h. (B) Numerical solution of the steady-state HB-EGF concentration profile with Rgen = 10 cell-1s-1, D = 0.7 µm2/s, and h = 0.02 µm/s, where concentration is shown by the color scale and height depicted. The maximum concentration achieved with the stated parameters was 0.27 nM from a capillary. Myocyte length was assumed to be 100 µm.

The driving force that determined the extent to which HB-EGF traveled was the rate of HB-EGF transfer into the capillaries and the diffusivity of HB-EGF. The exact mechanism of macromolecule transport into capillaries is unknown; however, it is most likely through diffusion, transcytosis, or a combination of the two. In the case of diffusion, the mass transfer coefficient governing the flux of HB-EGF through the capillary wall is coupled to the diffusivity of HB-EGF, whereas the terms are uncoupled for the case of transcytosis. Therefore, this model assessed transcytosis as a conservative scenario for HB-EGF localization. Parameter perturbation with uncoupled diffusion and capillary mass transfer showed that HB-EGF remained localized around the origin of production and diffused only to immediate neighbors for mass transfer coefficients >0.002 µm/s. For values <0.002 µm/s, HB-EGF diffused distances more than two cells away from the origin. Although the actual mass transfer coefficient of ligands in the size range of HB-EGF is unknown, values for O2 (0.02 µm/s, 0.032 kDa) (19) and LDL (1.7 x 10-5 µm/s, 2,000–3,000 kDa) (20) have been reported, and we assumed HB-EGF is in the upper end of that range due to its small size. HB-EGF also binds to EGFRs, the extracellular matrix, and cell surface heparan sulfate proteoglycans. EGFR binding and internalization could serve to further localize HB-EGF. The number of extracellular binding sites does not affect the steady-state HB-EGF concentration profile if this binding is reversible. However, these binding sites could serve to localize HB-EGF as the cell begins to produce the ligand by slowing the travel of HB-EGF to the capillaries in the approach to the steady state, or as a source of HB-EGF as the cell slows or stops HB-EGF production. At a diffusivity of 0.7 µm2/s (21), HB-EGF traveled only one cell away, but traveled approximately five cells away at 51.8 µm2/s (22), with a peak concentration below the estimated threshold for stimulating.

Overexpression of HB-EGF Causes Hypertrophy on the Infected Cell and Its Immediate Neighbor in Vivo.

To explore whether HB-EGF signals operate in a spatially restricted local circuit in the in vivo myocardial extracellular space as predicted by computational modeling, adenoviral vectors were injected directly into the left ventricular free wall in 26 male mice (Ad-GFP, n = 12; Ad-HB-EGF, n = 14). Of the 26 mice, 5 (4 Ad-GFP and 1 Ad-HB-EGF) mice died after the surgery. Gene expression was confirmed as positive cellular fluorescence in the presence of GFP, allowing determination of which cells were infected at 7 days (Fig. 4A). Immunohis-tochemical staining revealed that HB-EGF was localized on the Ad-HB-EGF-infected cell membrane or in the extracellular space around the overexpressing cell (Fig. 4A). For comparison, remote cells were defined as noninfected cells far (15–20 cell dimensions) from the adenovirus-infected area and in the same field as infected cells. Conventional 2D cross-sectional analysis blinded to treatment group (Fig. 4B) showed that Ad-GFP-infected cells (n = 102) resulted in no cellular hypertrophy compared with noninfected, adjacent (n = 92), or remote (n = 97) cells (2D myocyte cross-sectional area, 250 ± 7 versus 251 ± 7 or 255 ± 6 µm2, respectively). These data suggest that expression of GFP in these conditions does not cause cellular hypertrophy. However, overexpression of HB-EGF caused hypertrophy in both Ad-HB-EGF-infected cells (a 41 ± 5% increase of Ad-GFP-infected cells, n = 119, P < 0.01) and noninfected adjacent cells (a 33 ± 5% increase of Ad-GFP-adjacent cells, n = 97, P < 0.01) compared with remote cells (n = 109). Because 2D analysis of cardiomyocyte hypertrophy can be influenced by the plane of sectioning, we then developed a 3D histology approach that allowed reconstruction of cardiomyocytes in situ (Fig. 4C). We performed an independent 3D histology analysis of cardiomyocytes to determine cell volumes, blinded to treatment group (Fig. 4B). The volumes of both HB-EGF-infected cells (n = 19, 42,700 ± 4,000 µm3) and their adjacent cells (n = 11, 33,500 ± 3,300 µm3) were significantly greater than volumes of remote cells (n = 13, 18,600 ± 1,700 µm3, P < 0.01 vs. HB-EGF-infected cells and P < 0.05 vs. HB-EGF-adjacent cells, Fig. 4D). In contrast, cells treated with Ad-GFP (n = 12) showed no hypertrophy in the Ad-GFP-adjacent (n = 10) or remote cells (n = 9). These data demonstrate that HB-EGF acts as both an autocrine and local paracrine growth factor within myocardium, as predicted by computational modeling.

Degradation of Cx43 Through Local Autocrine/Paracrine HB-EGF

To determine whether the spatially confined effect of HB-EGF reduces local myocardial Cx43 in vivo, Cx43 was assessed with immunohistochemistry and confocal fluorescence imaging. Cells infected with Ad-HB-EGF had significant decreases in Cx43 immunoreactive signal compared with Ad-GFP cells, consistent with the results of in vitro immunoblotting (Fig. 5A). Quantitative digital image analyses of Cx43 in a total of 22 fields in 6 Ad-HB-EGF hearts and 19 fields in 4 Ad-GFP hearts were analyzed (Fig. 5B). Although Ad-GFP-infected cells showed immunoreactive Cx43 at the appositional membrane, overexpression of HB-EGF increased Cx43 in intracellular vesicle-like components (Fig. 5C), with reduced gap junction plaques (percent Cx43 area per cell area, 52 ± 8% of Ad-GFP control, P < 0.01). These data suggest that reduced expression of Cx43 can be attributed to an increased rate of internalization and degradation in gap junction plaques in cardiomyocytes. Interestingly, HB-EGF secretion by a given cardiomyocyte caused a 37 ± 13% reduction of Cx43 content in its adjacent cells compared with GFP controls (P < 0.05). As degradation of Cx43 may accompany structural changes with marked rearrangement of intercellular connections.  In contrast to Cx43, there was no significant difference in total area occupied by N-cadherin immunoreactive signal in between Ad-GFP (n = 19) and Ad-HB-EGF hearts (1.8 ± 0.5-fold compared with Ad-GFP, n = 17, P = not significant), indicating that HB-EGF has a selective effect on Cx43. Taken together, these data show that HB-EGF leads to cardiomyocyte hypertrophy and degradation of Cx43 in the infected cell and its immediately adjacent neighbors because of autocrine/ paracrine signaling. It should be noted, however, that quantifying the Cx43 from immunostaining could be limited by a nonlinear relation between the amount of Cx43 present and the area of staining.

Fig. 4. Effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy in vivo.

(A) Adenoviral vectors (Ad-GFP or Ad-HB-EGF) were injected into the left ventricular free wall in mice. Myocytes were grouped as infected or noninfected on the basis of GFP fluorescence. Overex-pression of HB-EGF was confirmed by im-munohistochemistry. The presented image was pseudocolored with blue from that stained with Alexa Fluor 555 for the presence of HB-EGF. (Scale bars: 20 sm.) (B) 2D cross-sectional area of cardiomyo-cytes was measured in infected and non-infected cells in the same region of the same animal. Overexpression of HB-EGF caused cellular hypertrophy in both infected and adjacent cells. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01 vs. Ad-HB-EGF remote; and §, P < 0.01 vs. Ad-GFP adjacent cells. GFP (infected 102 cells, adjacent 92 cells, and remote 97 cells from 5 mice), and HB-EGF (infected 119 cells, adjacent 97 cells, and remote 109 cells from 7 mice). The 3D histology also revealed cellular hypertrophy in both Ad-HB-EGF-infected cell and its adjacent cell. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01; and *, P < 0.05 vs. Ad-HB-EGF remote cells. GFP (infected 12 cells, adjacent 10 cells, and remote 9 cells), and HB-EGF (infected 19 cells, adjacent 11 cells, and remote 13 cells). Statistical analysis was performed with one-way ANOVA. (C) Sample image of extracted myocytes in three dimensions.

Discussion

We have demonstrated in this study that HB-EGF secreted by cardiomyocytes leads to cellular growth and reduced expression of the principal ventricular gap junction protein Cx43 in a local autocrine/paracrine manner. Although proHB-EGF is biologically active as a juxtacrine growth factor that can signal to immediately neighboring cells in a nondiffusible mannerseveral studies have revealed the crucial role of metalloproteases in the enzymatic conversion of proHB-EGF to soluble HB-EGF, which binds to and activates the EGFR. Hypertrophic stimuli such as mechanical strain and G protein-coupled receptors agonists mediate cardiac hypertrophy through the shedding of membrane-bound proHB-EGF. Thus, an autocrine/paracrine loop, which requires the diffusible, soluble form of HB-EGF, is necessary for subsequent transactivation of the EGFR to produce the hypertrophic response.

To our knowledge, there have been no previous reports concerning the spatial extent of autocrine/paracrine ligand distribution and signaling in myocardial tissue. A theoretical analysis by Shvartsman et al. predicted, from computational modeling in an idealized cell culture environment, that autocrine ligands may remain highly localized, even within subcellular distances; this prediction has support from experimental data in the EGFR system. In contrast, a theoretical estimate by Francis and Palsson has suggested that cytokines might effectively communicate larger distances, approximated to be 200–300 m from the point of release. However, these studies have all focused on idealized cell culture systems, so our combined experimental and computational investigation here aimed at understanding both in vitro and in vivo situations offers insight.
Our computational model of diffusion in the extracellular space predicts that HB-EGF acts as both an autocrine and spatially restricted paracrine growth factor for neighboring cells. We studied the responses of the signaling cell and its immediate neighbors compared with more distant cells. For a paracrine signal to be delivered to its proper target, the secreted signaling molecules cannot diffuse too far; in vitro experiments, in fact, indicated that HB-EGF acts as a predominantly autocrine signal in cell culture, where diffusion into the medium is relatively unconstrained.
In contrast, in the extracellular space of the myocardium, HB-EGF is localized around the source of production because of tissue geometry, thereby acting in a local paracrine or autocrine manner only. Indeed, our results from in vivo gene transfer demonstrated that both the cell releasing soluble HB-EGF and its surrounding cells undergo hypertrophy. This localized conversation between neighboring cells may allow remodeling to be fine-tuned on a highly spatially restricted level within the myocardium and in other tissues.

Common genetic variation at the IL1RL1locus regulates IL-33/ST2 signaling

JE Ho, Wei-Yu Chen, Ming-Huei Chen, MG Larson, ElL McCabe, S Cheng, A Ghorbani, E Coglianese, V Emilsson, AD Johnson,….. CARDIoGRAM Consortium, CHARGE Inflammation Working Group, A Dehghan, C Lu, D Levy, C Newton-Cheh, CHARGE Heart Failure Working Group, …. JL Januzzi, RT Lee, and TJ Wang J Clin Invest Oct 2013; 123(10):4208-4218.  http://dx.doi.org/10.1172/JCI67119

Abstract and Introduction

The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framing­ham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide associ­ation study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway. Suppression of tumorigenicity 2 (ST2) is a member of the IL-1 receptor (IL-1R) family that plays a major role in immune and inflammatory responses. Alternative promoter activation and splicing produces both a membrane-bound protein (ST2L) and a soluble form (sST2). The transmembrane form of ST2 is selectively expressed on Th2- but not Th1-type T cells, and bind­ing of its ligand, IL-33, induces Th2 immune responses.  In contrast, the soluble form of ST2 acts as a decoy receptor by sequestering IL-33. The IL-33/ST2 pathway has important immunomodulatory effects. Clinically, the ST2/IL-33 signaling pathway participates in the pathophysiology of a number of inflammatory and immune diseases related to Th2 activation, including asthma, ulcera­tive colitis, and inflammatory arthritis. ST2 expression is also upregulated in cardiomyocytes in response to stress and appears to have cardioprotective effects in experimental studies. As a biomarker, circulating sST2 concentrations have been linked to worse prognosis in patients with heart failure, acute dyspnea, and acute coronary syndrome, and also predict mortality and incident cardiovascular events in individuals without existing cardiovascular disease. Both sST2 and its transmembrane form are encoded by IL-1R– like 1 (IL1RL1). Genetic variation in this pathway has been linked to a number of immune and inflammatory diseases. The contribution of IL1RL1 locus variants to interindividual variation in sST2 has not been investigated. The emergence of sST2 as an important predictor of cardiovascular risk and the important role outside of the ST2/IL-33 pathway in inflammatory diseases highlight the value of understanding genetic determinants of sST2. The fam­ily-based FHS cohort provides a unique opportunity to examine the heritability of sST2 and to identify specific variants involved using a genome-wide association study (GWAS). Thus, we per­formed a population-based study to examine genetic determinants of sST2 concentrations, coupled with experimental studies to elu­cidate the underlying molecular mechanisms.

Results

Clinical characteristics of the 2,991 FHS participants are presented in Supplemental Table 1 (supplemental material available online with this article; doi:10.1172/JCI67119DS1). The mean age of participants was 59 years, and 56% of participants were women. Soluble ST2 concentrations were higher in men compared with those in women (P < 0.001). Soluble ST2 concentrations were positively associated with age, systolic blood pressure, body-mass index, antihypertensive medication use, and diabetes mellitus (P < 0.05 for all). Together, these variables accounted for 14% of the variation in sST2 concentrations. The duration of hypertension or diabetes did not materially influence variation in sST2 concentra­tions. After additionally accounting for inflammatory conditions, clinical variables accounted for 14.8% of sST2 variation.

Heritability of sS72.

The age- and sex-adjusted heritability (h2) of sST2 was 0.45 (P = 5.3 x 10–16), suggesting that up to 45% of the vari­ation in sST2 not explained by clinical variables was attributable to genetic factors. Multivariable adjustment for clinical variables pre­viously shown to be associated with sST2 concentrations (21) did not attenuate the heritability estimate (adjusted h2 = 0.45, P = 8.2 x 10–16). To investigate the influence of shared environmental fac­tors, we examined the correlation of sST2 concentrations among 603 spousal pairs and found no significant correlation (r = 0.05, P = 0.25).

Genetic correlates of sS72.

We conducted a GWAS of circulating sST2 concentrations. Quantile-quantile, Manhattan, and regional linkage disequilibrium plots are shown in Supplemental Figures 1–3.  All genome-wide significant SNPs were located in a 400-kb linkage disequilibrium block that included IL1RL1 (the gene encoding ST2), IL1R1, IL1RL2, IL18R1, IL18RAP, and SLC9A4 (Figure 1). Results for 11 genome-wide significant “indepen­dent” SNPs, defined as pairwise r2 < 0.2, are shown in Table 1. In aggregate, these 11 “independent” genome-wide significant SNPs across the IL1RL1 locus accounted for 36% of heritability of sST2. In conditional analyses, 4 out of the 11 SNPs remained genome-wide significant, independent of each other (rs950880, rs13029918, rs1420103, and rs17639215), all within the IL1RL1 locus. The most significant SNP (rs950880, P = 7.1 x 10–94) accounted for 12% of the residual interindividual variability in circulating sST2 concentrations. Estimated mean sST2 concen­trations were 43% higher in major homozygotes (CC) compared with minor homozygotes (AA). Tree loci outside of the IL1RL1 locus had suggestive associations with sST2 (P < 1 x 10–6) and are displayed in Supplemental Table 3.

In silico association with expression SNPs.

The top 10 sST2 SNPs (among 11 listed in Table 1) were explored in collected gene expression databases. There were 5 genome-wide significant sST2 SNPs associated with gene expression across a variety of tissue types (Table 2). Specifically, rs13001325 was associated with IL1RL1 gene expression (the gene encoding both soluble and transmembrane ST2) in several subtypes of brain tissue (prefrontal cortex, P = 1.95 x 10–12; cerebellum, P = 1.54 x 10–5; visual cortex, P = 1.85 x 10–7). The CC genotype of rs13001325 was associated with a higher IL1RL1 gene expression level as well as a higher circulating sST2 concentration when compared with the TT genotype (Supplemental Figure 4). Other ST2 variants were significantly associated with IL18RAP (P = 8.50 x 10–41, blood) and IL18R1 gene expression (P = 2.99 x 10–12, prefrontal cortex).

In silico association with clinical phenotypes in published data

The G allele of rs1558648 was associated with lower sST2 concentra­tions in the FHS (0.88-fold change per G allele, P = 3.94 x 10–16) and higher all-cause mortality (hazard ratio [HR] 1.10 per G allele, 95% CI 1.03–1.16, P = 0.003) in the CHARGE consortium, which observed 8,444 deaths in 25,007 participants during an average fol­low-up of 10.6 years (22). The T allele of rs13019803 was associated with lower sST2 concentrations in the FHS (0.87-fold change per G allele, P = 5.95 x 10–20), higher mortality in the CHARGE consor­tium (HR 1.06 per C allele, 95% CI 1.01–1.12, P = 0.03), and higher risk of coronary artery disease (odds ratio 1.06, 95% CI 1.00–1.11, P = 0.035) in the CARDIoGRAM consortium, which included 22,233 individuals with coronary artery disease and 64,762 controls (23). In relating sST2 SNPs to other clinical phenotypes (including blood pressure, body-mass index, lipids, fasting glucose, natriuretic peptides, C-reactive protein, and echocardiographic traits) in pre­viously published studies, we found nominal associations with C-reactive protein for 2 SNPs (Supplemental Table 4).

Putative functional variants.

Using GeneCruiser, we examined nonsynonymous SNPs (nSNPs) (missense variants) that had at least suggestive association with sST2 (P < 1 x 10–4), includ­ing SNPs that served as proxies (r2 = 1.0) for nSNPs within the 1000 Genomes Pilot 1 data set (ref. 24 and Table 3). There were 6 missense variants located within the IL1RL1 gene, 5 of which had genome-wide significant associations with sST2 concen­trations, including rs6749114 (proxy for rs10192036, Q501K), rs4988956 (A433T), rs10204137 (Q501R), rs10192157 (T549I), rs10206753 (L551S), and rs1041973 (A78AE). Base substitutions and corresponding amino acid changes for these coding muta­tions are listed in Table 3. In combination, these 6 missense muta­tions accounted for 5% of estimated heritability, with an effect estimate of 0.23 (standard error [s.e.] 0.02, P = 2.4 x 10–20). When comparing major homozygotes with minor homozygotes, the esti­mated sST2 concentrations for these missense variants differed by 11% to 15% according to genotype (Supplemental Table 5). In conditional analyses, intracellular and extracellular variants appeared to be independently associated with sST2. For instance, in a model containing rs4988956 (A433T) and rs1041973 (A78E), both SNPs remained significantly associated with sST2 (P = 2.61 x 10–24 and P = 7.67 x 10–15, respectively). In total, missence variants added little to the proportion of sST2 variance explained by the 11 genome-wide significant nonmissense variants listed in Table 1. In relating these 6 missense variants to other clinical phenotypes in large consortia, we found an association with asthma for 4 out of the 6 variants (lowest P = 4.8 x 10–12 for rs10204137) (25).

Homology map of IL1RL1 missense variants and ST2 structure.

Of the 6 missense variants mapping to IL1RL1, 5 were within the cytoplas­mic Toll/IL-1R (TIR) domain of the transmembrane ST2 receptor (Figure 2A), and these intracellular variants are thus not part of the circulating sST2 protein. Of these cytoplasmic domain variants, A433T was located within the “box 2” region of sequence conserva­tion, described in the IL-1R1 TIR domain as important for IL-1 sig­naling . Q501R/K was within a conserved motif called “box 3,” but mutants of IL-1R1 in box 3 did not significantly affect IL-1 signaling in previous experiments (26). Both T549I and L551S were near the C terminus of the transmembrane ST2 receptor and were not predicted to alter signaling function based on previous exper­iments with the IL-1R . The A78E SNP was located within the extracellular domain of ST2 and is thus present in both the sST2 isoform and the transmembrane ST2 receptor. In models of the ST2/IL-33/IL-1RAcP complex derived from a crystal structure of the IL-1RII/IL-1β/IL-1RAcP complex (protein data bank ID 1T3G and 3O4O), A78E was predicted to be located on a surface loop within the first immunoglobulin-like domain (Figure 2B), distant from the putative IL-33 binding site or the site of interaction with IL-1RAcP. There were 2 rare extracellular variants that were not cap­tured in our GWAS due to low minor allele frequencies (A80E, MAF 0.008; A176T, MAF 0.002). Both were distant from the IL-33 bind­ing site on homology mapping and unlikely to affect IL-33 binding.

Functional effects of IL1RL1 missense variants on sST2 expression and promoter activity.

Since 5 of the IL1RL1 missense variants asso­ciated with sST2 levels mapped to the intracellular domain of ST2L and hence are not present on sST2 itself, we hypothesized that these missense variants exert effects via intracellular mecha­nisms downstream of ST2 transmembrane receptor signaling to regulate sST2 levels. To investigate the effect of IL1RL1 missense variants (identified by GWAS) on sST2 expression, stable cell lines expressing WT ST2L, IL1RL1 variants (A78E, A433T, T549I, Q501K, Q501R, and L551S), and a construct containing the 5 IL1RL1 intracellular domain variants (5-mut) were generated. Expression of ST2L mRNA and protein (detected in membrane fractions) was confirmed (Supplemental Figures 5 and 6). Eight different stable clones in each group were analyzed to reduce bias from clonal selection. Intracellular domain variants (A433T, T549I, Q501K, Q501R, L551S, and 5-mut), but not the extracellular domain variant (A78E), were associated with increased basal sST2 expression when compared with WT expression (P < 0.05 for all, Figure 3A). sST2 expression was highest in the 5-mut construct, suggesting that intracellular ST2L variants cooperatively regulate sST2 levels. This same pattern was consistent across different cell types (U937, Jurkat T, and A549 cells; Supplemental Figure 7). These findings suggest that intracellular domain variants of the transmembrane ST2 receptor may functionally regulate downstream signaling.   IL1RL1 transcription may occur via two alternative promoters (proximal vs. distal), which leads to differential expression of the soluble versus membrane-bound ST2 proteins. Similar to the sST2 protein expression results above, the intracellular domain variants, but not the extracellular domain variant, were associated with higher basal proximal promoter activity. Dis­tal promoter activity was also increased for most intracellular domain variants (Supplemental Figure 8).

IL1RL1 intracellular missense variants resulted in higher IL-33 pro­tein levels.

In addition to upregulation of sST2 protein levels, IL1RL1 intracellular missense variants caused increased basal IL-33 protein expression (Figure 3B), suggesting a possible autoregulatory loop whereby IL-33 signaling positively induces sST2 expression. IL-33 induced sST2 protein expression in cells expressing both WT and IL1RL1 missense variants. Interest­ingly, this effect was particularly pronounced in the A433T and Q501R variants (Supplemental Figure 9A).

Enhanced IL-33 responsiveness is mediated by IL-113 in A433T and Q501R variants.

Interaction among IL-33, sST2, and IL-113.

Inhibition of IL-113 by anti–IL-113 mAb reduced basal expression of sST2 (Supplemental Figure 11A). Blocking of IL-33 by sST2 did not reduce the induction of IL-113 levels by the IL1RL1 variants (Supplemental Figure 11B). Furthermore, inhibition of IL-113 by anti–IL-113 reduced the basal IL-33 levels. IL-33 itself upregulated sST2 levels, which in turn reduced IL-33 levels (Supplemental Figure 11C). Our results revealed that both IL-33 and IL-113 drive sST2 expression and that IL-113 acts as an upstream inducer of IL-33 and maintains IL-33 expression by intracellular IL1RL1 vari­ants (Supplemental Figure 11D). This suggests that IL1RL1 vari­ants upregulated sST2 mainly through IL-33 autoregulation and that the enhanced IL-33 responsiveness by A433T and Q501R was mediated by IL-113 upregulation.

IL1RL1 missense variants modulate ST2 signaling pathways

The effect of IL1RL1 missense variants on known ST2 downstream regulatory pathways, including NF-KB, AP-1/c-Jun, AKT, and STAT3 , was examined in the presence and absence of IL-33 (Figure 4 and Supplemental Figure 12). The IL1RL1 intracellular missense vari­ants (A433T, T549I, Q501K, Q501R, and L551S) were associated with higher basal phospho–NF-KB p65 and phospho–c-Jun levels (Figure 4, A and B). Consistent with enhanced IL-33 responsive­ness in A433T and Q501R cells, levels of IL-33–induced NF-KB and c-Jun phosphorylation were enhanced in these 2 variants (Figure 4, B and D). In contrast, A433T and Q501R variants showed lower basal phospho-AKT levels (Figure 4E). ……….. The majority of sST2 gene variants in our study were located within or near IL1RL1, the gene coding for both transmembrane ST2 and sST2. IL1RL1 resides within a linkage disequilibrium block of 400 kb on chromosome 2q12, a region that includes a number of other cytokines, including IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory protein (IL18RAP). Polymorphisms in this gene cluster have been associated previously with a num­ber of immune and inflammatory conditions, including asthma, celiac disease, and type 1 diabetes mellitus . Many of these variants were associated with sST2 concentrations in our analysis (Supplemental Table 6). The immune effects of ST2 are corroborated by experimental evidence: membrane-bound ST2 is selectively expressed on Th2- but not Th1-type T helper cells, and activation of the ST2/IL-33 axis elaborates Th2 responses. In general, the allergic phenotypes above are thought to be Th2-mediated processes, in contrast to atherosclerosis, which appears to be a Th1-driven process.

Fig 2  Models of ST2 illustrate IL1RL1 missense variant locations.

Figure 2 Models of ST2 illustrate IL1RL1 missense variant locations.

Models of the (A) intracellular TIR domain (ST2-TIR) and the (B) extracellular domain (ST2-ECD) of ST2 (protein data bank codes 3O4O and 1T3G, respectively). Domains of ST2 are shown in yellow, with identified mis-sense SNP positions represented as red spheres and labels. Note that positions 549 and 551 are near the C terminus of ST2, which is not defined in the crystal structure (protein data bank ID 1T3G, shown as dashed black line in A). Arrows point toward the transmembrane domain, which is also not observed in crystal structures.

Fig 3 IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Figure 3   IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Media from KU812 cells expressing WT and IL1RL1 missense variants were collected for ELISA analysis of (A) sST2, (B) IL-33, and (C) IL-113 levels. Horizontal bars indicate mean values, and symbols represent indi­vidual variants. *P < 0.05, **P < 0.01 vs. WT. (D) Effect of anti–IL-113 mAb on IL-33–induced sST2 expression. Dashed line indicates PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. *P < 0.05 vs. IL-33.

Fig 4  IL1RL1 missense variants modulated ST2 signaling pathways

Figure 4  IL1RL1 missense variants modulated ST2 signaling pathways. 

KU812 cells expressing WT or IL1RL1 variants were treated with PBS or IL-33. Levels of the following phosphorylated proteins were detected in cell lysates using ELISA: (A and B) phospho-NF-KB p65; (C and D) phospho-c-Jun activity; and (E and F) phospho-AKT. (A, C, and E) White bars represent basal levels, and (B, D, and F) gray bars represent relative fold increase (compared with PBS-treated group) after IL-33 treatment. *P < 0.05 vs. WT; **P < 0.01 vs. PBS-treated group. Dashed line in B, D, and F represents PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. Fig 5   IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

Figure 5  IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

(A) sST2 mRNA expression in KU812 cells after treatment with DMSO, IL-33, or IL-33 plus signal inhibitors (wortmannin, LY294002, rapamycin, PD98059, SP60125, BAY11-7082, or SR11302). (B) ST2L mRNA and (C) sST2 mRNA expression in KU812 cells treated with PBS (white columns), rapamycin (rapa), anti-ST2 mAb, IL-33, IL-33 plus anti-ST2, IL-33 plus rapamycin, IL-33 plus rapamycin plus anti-ST2 mAb, or rapamycin plus anti-ST2. (D) IL33 mRNA expression in KU812 cells after treatment with DMSO, signal inhibitors, IL-33 plus signal inhibitors, and IL-1n plus signal inhibitors. *P < 0.05 vs. PBS-treated group; #P < 0.05 vs. IL-33–treated group; &P < 0.05 vs. IL-1n–treated group. Error bars represent mean ± SEM from 2 independent experiments. (E) A schematic model illustrating the regulation of sST2 expression by IL1RL1 missense variants through enhanced induction of IL-33 via enhanced NF-KB and AP-1 signaling and enhanced IL-33 responsiveness via increasing ST2L expression.

Quantitating subcellular metabolism with multi-isotope imaging mass spectrometry

ML Steinhauser, A Bailey, SE Senyo, C Guillermier, TS Perlstein, AP Gould, RT Lee, and CP Lechene
Department of Medicine, Divisions of Cardiovascular Medicine & Genetics, Brigham and Women’s Hospital, Harvard Medical School & Harvard Stem Cell Institute Division of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, Mill Hill, London, UK National Resource for Imaging Mass Spectroscopy
Nature 2012;481(7382): 516–519.   http://dx. do.org/10.1038/nature10734

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research. MIMS combines ion microscopy with secondary ion mass spectrometry (SIMS), stable isotope reporters, and intensive computation (Supplemental Fig 1). MIMS allows imaging and measuring stable isotope labels in cell domains smaller than one micron cubed. We tested the potential of MIMS to quantitatively track DNA labeling with 15N-thymidine in vitro. In proliferating fibroblasts, we detected label incorporation within the nucleus by an increase in the 15N/14N ratio above natural ratio (Fig 1a). The labeling pattern resembled chromatin with either stable isotope-tagged thymidine or thymidine analogs (Fig 1b). We measured dose-dependent incorporation of 15N-thymidine over three orders of magnitude (Fig 1d, Supplemental Fig 2). We also tracked fibroblast division after a 24-hour label-free chase (Fig 1d,e, Supplemental Fig 3). Cells segregated into two populations, one indistinguishable from control cells suggesting no division, the other with halving of label, consistent with one division during chase. We found similar results by tracking cell division in vivo in the small intestine (Fig 1f,g, Supplemental Figs 4–6). We measured dose-dependent 15N-thymidine incorporation within nuclei of actively dividing crypt cells (Fig 1g, Supplemental Fig 4), down to a dose of 0.1µg/ g (Supplemental Fig 2). The cytoplasm was slightly above natural ratio, likely due to low level soluble 15N-thymidine or mitochondrial incorporation (Supplemental Fig 2). We measured halving of label with each division during label-free chase (Supplemental Fig 6). We then tested the “immortal strand hypothesis,” a concept that emerged from autoradiographic studies and that predicted long-term label retaining cells in the small intestinal crypt. It proposes that asymmetrically dividing stem cells also asymmetrically segregate DNA, such that older template strands are retained by daughter cells that will remain stem cells and newer strands are passed to daughters committed to differentiation (Supplemental Fig 7)5,6. Modern studies continue to argue both for or against the hypothesis, leading to the suggestion that definitive resolution of the debate will require a new experimental approach. Although prior evidence suggests a concentration of label-retaining cells in the +4 anatomic position, we searched for DNA label retention irrespective of anatomic position or molecular identity. We labeled mice with 15N-thymidine for the first 8 wks of life when intestinal stem cells are proposed to form. After a 4-wk chase, mice received bromodeoxyuridine (BrdU) for 24h prior to sacrifice to identify proliferating cells(Fig 2a, Supplemental Fig 8: Exp 1), specifically crypt base columnar (CBC) cells and transit amplifying cells (TA) (Supplemental Fig 9), which cycle at a rate of one and two times per 24h, respectively (Supplemental Fig 10). All crypt cell nuclei were highly labeled upon completion of 15N-thymidine; after a four-week chase, however, we found no label retention by non-Paneth crypt cells (Fig 2b–f; n=3 mice, 136 crypts analysed). 15N-labeling in BrdU/15N+ Paneth and mesenchymal cells was equivalent to that measured at pulse completion (Fig2b,c) suggesting quiescence during the chase (values above 15N/14N natural ratio: Paneth pulse=107.8 +/− 5.0% s.e.m. n=51 vs Paneth pulse-chase=96.3+/−2.8% s.e.m. n=218; mesenchymal pulse=92.0+/−5.0% s.e.m. n=89 vs mesenchymal pulse-chase=90.5+/ −2.2% s.e.m. n=543). The number of randomly selected crypt sections was sufficient to detect a frequency as low as one label-retaining stem cell per crypt irrespective of anatomic location within the crypt. Because each anatomic level contains approximately 16 circumferentially arrayed cells, a 2-dimensional analysis captures approximately 1/8th of the cells at each anatomic position (one on each side of the crypt; Supplemental Fig 9a). Therefore, assuming only 1 label-retaining stem cell per crypt we should have found 17 label-retaining cells in the 136 sampled crypts (1/8th of 136); we found 0 (binomial test p<0.0001). The significance of this result held after lowering the expected frequency of label-retaining cells by 25% to account for the development of new crypts, a process thought to continue into adulthood. In three additional experiments, using shorter labeling periods and including in utero development, we also found no label-retaining cells in the crypt other than Paneth cells (Supplemental Fig 8, Exps 2–4).

Fig 1 post-natal human DNA synthesis in the heart

In recent years, several protocols have been developed experimentally in an attempt to identify novel therapeutic interventions aiming at the reduction of infarct size and prevention of short and long term negative ventricular remodeling following ischemic myocardial injury. Three main strategies have been employed and a significant amount of work is being conducted to determine the most effective form of action for acute ischemic heart failure. The delivery of bone marrow progenitor cells (BMCs) has been highly controversial, but recent clinical data have shown improvement in ventricular performance and clinical outcome. These observations have not changed the nature of the debate concerning the efficacy of this cell category for the human disease and the mechanisms involved in the impact of BMCs on cardiac structure and function. Whether BMCs transdifferentiate and acquire the cardiomyocyte lineage has faced strong opposition and data in favor and against this possibility have been reported. However, this is the only cell class which has been introduced in the treatment of heart failure in patients and large clinical trials are in progress.
Human embryonic stem cells (ESCs) have repeatedly been utilized in animal models to restore the acutely infarcted myocardium, but limited cell engraftment, modest ability to generate vascular structures, teratoma formation and the apparent transient beneficial effects on cardiac hemodynamics have questioned the current feasibility of this approach clinically. Tremendous efforts are being performed to reduce the malignant tumorigenic potential of ESCs and promote their differentiation into cardiomyocytes with the expectation that these extremely powerful cells may be applied to human beings in the future. Additionally, the study of ESCs may provide unique understanding of the mechanisms of embryonic development that may lead to therapeutic interventions in utero and the correction of congenital malformations.
The recognition that a pool of primitive cells with the characteristics of stem cells resides in the myocardium and that these cells form myocytes, ECs and SMCs has provided a different perspective of the biology of the heart and mechanisms of cardiac homeostasis and tissue repair. Regeneration implies that dead cells are replaced by newly formed cells restoring the original structure of the organ. In adulthood, this process occurs during physiological cell turnover, in the absence of injury. However, myocardial damage interferes with recapitulation of cell turnover and restitutio ad integrum of the organ. Because of the inability of the adult heart to regenerate itself after infarction, previous studies have promoted tissue repair by injecting exogenously expanded CPCs in proximity of the necrotic myocardium or by activating resident CPCs through the delivery of growth factors known to induce cell migration and differentiation. These strategies have attenuated ventricular dilation and the impairment in cardiac function and in some cases have decreased animal mortality.

Although various subsets of CPCs have been used to reconstitute the infarcted myocardium and different degrees of muscle mass regeneration have been obtained, in all cases the newly formed cardiomyocytes possessed fetal-neonatal characteristics and failed to acquire the adult cell phenotype. In the current study, to enhance myocyte growth and differentiation, we have introduced cell therapy together with the delivery of self-assembly peptide nanofibers to provide a specific and prolonged local myocardial release of IGF-1. IGF-1 increases CPC growth and survival in vitro and in vivo and this effect resulted here in a major increase in the formation of cardiomyocytes and coronary vessels, decreasing infarct size and restoring partly cardiac performance. This therapeutic approach was superior to the administration of CPCs or NF-IGF-1 only. Combination therapy appeared to be additive; it promoted myocardial regeneration through the activation and differentiation of resident and exogenously delivered CPCs. Additionally, the strategy implemented here may be superior to the utilization of BMCs for cardiac repair. CPCs are destined to form myocytes, and vascular SMCs and ECs and, in contrast to BMCs, do not have to transdifferentiate to acquire cardiac cell lineages. Transdifferentiation involves chromatin reorganization with activation and silencing of transcription factors and epigenetic modifications.

Selected References

  1. Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest 2006;116:237–248. [PubMed: 16357943]
  2. Davis ME, Hsieh PC, Takahashi T, Song Q, Zhang S, Kamm RD, Grodzinsky AJ, Anversa P, Lee RT. Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci USA 2006;103:8155–8160. [PubMed: 16698918]
  3. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776. [PubMed: 14505575]
  4. Rota M, Padin-Iruegas ME, Misao Y, De Angelis A, Maestroni S, Ferreira-Martins J, Fiumana E, Rastaldo R, Arcarese ML, Mitchell TS, Boni A, Bolli R, Urbanek K, Hosoda T, Anversa P, Leri A, Kajstura J. Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function. Circ Res 2008;103:107–116. [PubMed: 18556576]

Cardiac anatomy.

Figure 2.  Cardiac anatomy.

(A and B) Cardiac weights and infarct size. R and L correspond, respectively, to the number of myocytes remaining and lost after infarction. (C–G) LV dimensions. Sham-operated: SO. *Indicates P<0.05 vs SO; **vs untreated infarcts (UN); †vs infarcts treated with CPCs; ‡vs infarcts treated with NF-IGF-1.

Ventricular function

Figure 3.  Ventricular function.

Combination therapy (CPC-NF-IGF-1) attenuated the most the negative impact of myocardial infarction on cardiac performance. See Figure 2 for symbols.

Endothelial Cells Promote Cardiac Myocyte Survival and Spatial Reorganization: Implications for Cardiac Regeneration

Daria A. Narmoneva, Rada Vukmirovic, Michael E. Davis, Roger D. Kamm,  and Richard T. Lee
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, and the Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA
Circulation. 2004 August 24; 110(8): 962–968.        http://dx.doi.org/10.1161/01.CIR.0000140667.37070.07

Background

Endothelial-cardiac myocyte (CM) interactions play a key role in regulating cardiac function, but the role of these interactions in CM survival is unknown. This study tested the hypothesis that endothelial cells (ECs) promote CM survival and enhance spatial organization in a 3-dimensional configuration.

Methods and Results

Microvascular ECs and neonatal CMs were seeded on peptide hydrogels in 1 of 3 experimental configurations:

  1. CMs alone,
  2. CMs mixed with ECs (coculture), or
  3. CMs seeded on preformed EC networks (prevascularized).

Capillary-like networks formed by ECs promoted marked CM reorganization along the EC structures, in contrast to limited organization of CMs cultured alone. The presence of ECs markedly inhibited CM apoptosis and necrosis at all time points. In addition, CMs on preformed EC networks resulted in significantly less CM apoptosis and necrosis compared with simultaneous EC-CM seeding (P<0.01, ANOVA). Furthermore, ECs promoted synchronized contraction of CMs as well as connexin 43 expression.

Conclusions

These results provide direct evidence for a novel role of endothelium in survival and organization of nearby CMs. Successful strategies for cardiac regeneration may therefore depend on establishing functional CM-endothelium interactions.

Keywords:  endothelium; cardiomyopathy; heart failure; tissue

Introduction

Recent studies suggest that the mammalian heart possesses some ability to regenerate itself through several potential mechanisms, including generation of new cardiomyocytes (CMs) from extracardiac progenitors, CM proliferation, or fusion with stem cells with subsequent hybrid cell division. These mechanisms are insufficient to regenerate adequate heart tissue in humans, although some vertebrates can regenerate large volumes of injured myocardium.
Several approaches in cell transplantation and cardiac tissue engineering have been investigated as potential treatments to enhance cardiac function after myocardial injury. Implantation of skeletal muscle cells, bone marrow cells, embryonic stem cell-derived CMs, and myoblasts can enhance cardiac function. Cell-seeded grafts have been used instead of isolated cells for in vitro cardiac tissue growth or in vivo transplantation. These grafts can develop a high degree of myocyte spatial organization, differentiation, and spontaneous and coordinated contractions. On implantation in vivo, cardiac grafts can integrate into the host tissue and neovascularization can develop. However, the presence of scar tissue and the death of cells in the graft can limit the amount of new myocardium formed, most likely due to ischemia. Therefore, creating a favorable environment to promote survival of transplanted cells and differentiation of progenitor cells remains one of the most important steps in regeneration of heart tissue.
One of the key factors for myocardial regeneration is revascularization of damaged tissue. In the normal heart, there is a capillary next to almost every CM, and endothelial cells (ECs) outnumber cardiomyocytes by ≈3:1. Developmental biology experiments reveal that myocardial cell maturation and function depend on the presence of endocardial endothelium at an early stage. Experiments with inactivation or overexpression of vascular endothelial growth factor (VEGF) demonstrated that at later stages, either an excess or a deficit in blood vessel formation results in lethality due to cardiac dysfunction. Both endocardium and myocardial capillaries have been shown to modulate cardiac performance, rhythmicity, and growth. In addition, a recent study showed the critical importance of CM-derived VEGF in paracrine regulation of cardiac morphogenesis. These findings and others highlight the significance of interactions between CMs and endothelium for normal cardiac function. However, little is known about the specific mechanisms for these interactions, as well as the role of a complex, 3-dimensional organization of myocytes, ECs, and fibroblasts in the maintenance of healthy cardiac muscle.
The critical relation of CMs and the microvasculature suggests that successful cardiac regeneration will require a strategy that promotes survival of both ECs and CMs. The present study explored the hypothesis that ECs (both as preexisting capillary-like structures and mixed with myocytes at the time of seeding) promote myocyte survival and enhance spatial reorganization in a 3-dimensional configuration. The results demonstrate that CM interactions with ECs markedly decrease myocyte death and show that endothelium may be important not only for the delivery of blood and oxygen but also for the formation and maintenance of myocardial structure.

Methods

  • Three-Dimensional Culture
  • Immunohistochemistry and Cell Death Assays
  • Evaluation of Contractile Areas

Results

  • EC-CM Interactions Affect Myocyte Reorganization
  • ECs Improve Survival of CMs
  • Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions
  • ECs Promote Cx43 Expression

EC-CM Interactions Affect Myocyte Reorganization

To explore interactions between CMs and ECs in 3-dimensional culture, we used peptide hydrogels, a tissue engineering scaffold. Cells seeded on the surface of the hydrogel attach and then migrate into the hydrogel. When CMs alone were used, cells attached on day 1 and then formed small clusters of cells at days 3 and 7 (Figure 1). In contrast, when CMs were seeded together with ECs, cells formed interconnected linear networks, as commonly seen with ECs in 3-dimensional culture environments, with increasing spatial organization from day 1 to day 7 (Figure 1).

Figure 1.  ECs promote CM reorganization. 

When CMs were cultured alone (left column), they aggregated into sparse clusters. When CMs were cultured with ECs (center), cells organized into capillary-like networks. There was no difference in morphological appearance between coculture or prevascularized cultures (not shown) and ECs alone (right column). Bar=100 μm. Abbreviations are as defined in text.

To establish whether preformed endothelial networks enhanced the organization of myocytes, we also seeded ECs 1 day before myocytes were added. These ECs formed similar interconnected networks in the absence of myocytes; preforming the vascular network did not lead to significant differences in morphology (data not shown). Furthermore, to exclude the possibility that the increasing cell density of added ECs caused the spatial organization, we also performed control experiments with varying numbers and combinations of cells; there was no effect of doubling or halving cell numbers, indicating that the spatial organization effect was specifically due to ECs. To establish that both myocytes and ECs were forming networks together, we performed immunofluorescence studies with specific antibodies, as well as analysis of cross sections of CM-EC cocultures, whereby cells were labeled with CellTracker dyes before seeding. Immunofluorescent staining demonstrated that >95% of CMs were present within these networks, suggesting that CMs preferentially migrate to or survive better near ECs (Figure 2).

Figure 2.  CMs appear on outside of endothelial networks.

CMs appear on outside of endothelial networks. High-magnification, double-immunofluorescence image of structures formed in EC-CM coculture at day 7 demonstrating CMs (sarcomeric actinin, red) spread on top of ECs (von Willebrand factor, green) with no myocytes present outside structure. Bar=100 μm. Abbreviations are as defined in text.

The analysis of cross sections demonstrated the presence of what appeared to be EC-derived, tubelike structures (Figure 3), with myocytes spread on the outer part of the capillary wall. Along with the capillary-like structures, clusters of intermingled cells (both myocytes and ECs) not containing the lumen were also observed (not shown). However, when the lumen was present, ECs were always on the inner side and myocytes on the outer side of the structure.

Figure 3.  ECs form tubelike structures with myocytes spreading on outer wall.

Cross section of paraffin-embedded sample of 3-day coculture of myocytes (red) and ECs (green) incubated in CellTracker dye before seeding on hydrogel. Bar=50 μm. Abbreviations are as defined in text.

In CM-fibroblast cocultures, cells rapidly (within 24 hours) formed large clusters consisting of cells of both types (not shown). At later time points, fibroblast proliferation resulted in their migration outside the clusters and spreading on the hydrogel without any pattern. However, in contrast to EC-CM cocultures, CMs remained in the clusters and demonstrated only limited spreading. Immunofluorescent staining revealed that there was no orientation of myocytes relative to the fibroblasts in the clusters. In cultures with EC-conditioned medium, myocyte morphology and spatial organization remained similar to those of myocyte controls.

ECs Improve Survival of CMs

To test the hypothesis that ECs promote CM survival, we assessed apoptosis and necrosis in the 3-dimensional cultures. Quantitative analyses of CMs positive for TUNEL and necrosis staining demonstrated significantly decreased myocyte apoptosis and necrosis when cultured with ECs, compared with CM-only cultures (Figure 4, P<0.01). This effect was observed at all 3 time points, although the decreased necrosis was most pronounced at day 1. In addition, CMs seeded on the preformed EC networks had a lower rate of apoptosis at day 1 relative to same-time seeding cultures (P<0.05, post hoc test), suggesting that early EC-CM interactions provided by the presence of well-attached and prearranged ECs may further promote CM survival. In contrast to the ECs, cardiac fibroblasts did not affect myocyte survival (P>0.05, Figure 4), with ratios for myocyte apoptosis and necrosis in the myocyte-fibroblast cocultures being similar to those for myocyte-only controls. However, addition of EC-conditioned medium resulted in a significant decrease in apoptosis and necrosis ratios of myocytes (P<0.01). Interestingly, the effect of conditioned medium on myocyte necrosis was similar in magnitude to the effect of ECs, whereas myocyte apoptosis ratios in the conditioned-medium group were only partially decreased compared with those in the presence of ECs. These results suggest that the prosurvival effect of ECs on CMs may not only be merely due to the local interactions between myocytes and ECs during myocyte attachment but may also involve direct signaling between myocytes and ECs.

Figure 4.  ECs prolong survival of CMs

Top, dual immunostaining of CMs and EC-myocyte prevascularized groups at day 3 in culture, with TUNEL-positive cells in red; green indicates sarcomeric actinin; blue, DAPI. Bottom, presence of ECs decreased CM apoptosis and necrosis, both in coculture conditions and when cultures were prevascularized by seeding with ECs 1 day before CMs (mean±SD, P<0.01). EC-conditioned medium decreased myocyte apoptosis and necrosis (P<0.01), whereas fibroblasts did not have any effect (P>0.05). *Different from myocytes alone; **different from EC-myocyte coculture and pre-vascularized. Bar=100 μm. Abbreviations are as defined in text.

Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions

In the prevascularized group with preformed vascular structures, synchronized, spontaneous contractions of large areas (Figure 5, top panels) were detected as early as days 2 to 3after seeding, in contrast to the coculture group, wherein such contractions were observed on days 6 to 7. In CM-only cultures, beating of separate cells and small cell clusters was also detected at days 2 to 3, similar to that in the prevascularized group. However, the average area of synchronized beating at day 3 in the myocyte-only group (3.5±0.5×102 μm2) was nearly 3 orders of magnitude smaller than the synchronously contracting area in the prevascularized group (4.3±2.5×105 μm2, mean±SD, n=5). These data suggest that ECs promote synchronized CM contraction, particularly when vascular networks are already formed.

Figure 5.  ECs promote large-scale, synchronized contraction of CMs.

Left, phase-contrast video of beating areas in CM-only and prevascularized groups (day 3). Right, motion analysis of video showing regions of synchronized contractions (connected areas in purple are contracting synchronously) and nonmoving areas in blue. Bars=100 μm. Abbreviations are as defined in text.

ECs Promote Cx43 Expression

Staining for Cx43 showed striking differences in the distribution pattern of this gap junction protein between EC-CM cocultures and CMs cultured alone. In myocyte-only cultures, Cx43 expression was barely detectable at day 1 (not shown); at days 3 and 7, Cx43 expression was sparse throughout the cell clusters (Figure 6). In the presence of ECs (in both coculture and prevascularized groups), Cx43 staining was evident at day 1, both between ECs and distributed among CMs. As early as day 3 in culture, patches of localized junction-like Cx43, in addition to diffuse staining, were observed for myocytes in the coculture group (Figure 6). In the prevascularized group at day 3, wherein spontaneous contractions were already observed, more junction-like patches of Cx43 were observed compared with the coculture group, indicating electrical connections between myocytes (Figure 6). In addition to junctions between myocytes, there was also evidence of Cx43 localized at the interface between ECs and myocytes (Figure 6) detected in both the coculture group (at day 7) and the preculture group (as early as day 3). When myocytes and myocyte-EC coculture groups were cultured for 3 days with or without addition of 100 ng/mL of neutralizing anti-mouse VEGF antibody (R&D Systems), we observed no differences in either apoptosis or Cx43 staining between VEGF antibody-containing cultures and controls.

Figure 6. ECs promote Cx43 expression

Cultures at 3 days immunostained for Cx43 (red) and anti-sarcomeric actinin (green); nuclei are stained with DAPI (blue). For CMs alone (left), Cx43 staining is diffuse and sparse, with no evidence of gap junctions; for coculture (center), both diffuse (yellow arrow) and patchlike (thin, white arrow) Cx43 staining is observed; for prevascularized (right), increased patchlike staining indicates presence of gap junctions. Thick arrow-heads indicate junctions between myocytes and ECs. Bar=50 μm. Abbreviations are as defined in text.

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Endothelial-Cardiomyocyte Interactions in Cardiac Development and Repair: Implications for Cardiac Regeneration

Patrick C.H. Hsieh, Michael E. Davis, Laura K. Lisowski, and Richard T. Lee

Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Annu Rev Physiol.    PMC 2009 September 30

The ongoing molecular conversation between endothelial cells and cardiomyocytes is highly relevant to the recent excitement in promoting cardiac regeneration. The ultimate goal of myocardial regeneration is to rebuild a functional tissue that closely resembles mature myocardium, not just to improve systolic function transiently. Thus, regenerating myocardium will require rebuilding the vascular network along with the cardiomyocyte architecture. Here we review evidence demonstrating crucial molecular interactions between endothelial cells and cardiomyocytes. We first discuss endothelial-cardiomyocyte interactions during embryonic cardiogenesis, followed with morphological and functional characteristics of endothelial-cardiomyocyte interactions in mature myocardium. Finally, we consider strategies exploiting endothelial-cardiomyocyte interplay for cardiac regeneration.

Signaling from Cardiomyocytes to Endothelial Cells

The examples of neuregulin-1, NF1, and PDGF-B demonstrate that signals from endothelial cells regulate the formation of primary myocardium. Similarly, signaling from myocardial cells to endothelial cells is also required for cardiac development. Two examples of myocardial-to-endothelial signaling are vascular endothelial growth factor (VEGF)-A and angiopoietin-1.

VASCULAR ENDOTHELIAL GROWTH FACTOR-A

VEGF-A is a key regulator of angiogenesis during embryogenesis. In mice, a mutation in VEGF-A causes endocardial detachment from an underdeveloped myocardium. A mutation in VEGF receptor-2 (or Flk-1) also results in failure of the endocardium and myocardium to develop (18). Furthermore, cardiomyocyte-specific deletion of VEGF-A results in defects in vasculogenesis/angiogenesis and a thinned ventricular wall, further confirming reciprocal signaling from the myocardial cell to the endothelial cell during cardiac development. Interestingly, this cardiomyocyte-selective VEGF-A-deletion mouse has underdeveloped myocardial microvasculature but preserved coronary artery structure, implying a different signaling mechanism for vasculogenesis/angiogenesis in the myocardium and in the epicardial coronary arteries.
Cardiomyocyte-derived VEGF-A also inhibits cardiac endocardial-to-mesenchymal transformation. This process is essential in the formation of the cardiac cushions and requires delicate control of VEGF-A concentration. A minimal amount of VEGF initiates endocardial-to-mesenchymal transformation, whereas higher doses of VEGF-A terminate this transformation. Interestingly, this cardiomyocyte-derived VEGF-A signaling for endocardial-to-mesenchymal transformation may be controlled by an endothelial-derived feedback mechanism through the calcineurin/NFAT pathway (24), demonstrating the importance of endothelial-cardiomyocyte interactions for cardiac morphogenesis.

ANGIOPOIETIN-1

Another mechanism of cardiomyocyte control of endothelial cells during cardiac development is the angiopoietin-Tie-2 system. Both angiopoietin-1 and angiopoietin-2 may bind to Tie-2 receptors in a competitive manner, but with opposite effects: Angiopoietin-1 activates the Tie-2 receptor and prevents vascular edema, whereas angiopoietin-2 blocks Tie-2 phosphorylation and increases vascular permeability. During angiogenesis/vasculogenesis, angiopoietin-1 is produced primarily by pericytes, and Tie-2 receptors are expressed on endothelial cells. Angiopoietin-1 regulates the stabilization and maturation of neovasculature; genetic deletion of angiopoietin-1 or Tie-2 causes a defect in early vasculogenesis/angiogenesis and is lethal.
Cardiac endocardium is one of the earliest vascular components (along with the dorsal aorta and yolk sac vessels) and the adult heart can be regarded as a fully vascularized organ, angiopoietin-Tie-2 signaling may also be required for early cardiac development. Indeed, mice with mutations in Tie-2 have underdeveloped endocardium and myocardium. These Tie-2 knockout mice display defects in the endocardium but have normal vascular morphology at E10.5, suggesting that the endocardial defect is the fundamental cause of death. In addition, a recent study showed that overexpression, and not deletion, of angiopoietin-1 from cardiomyocytes caused embryonic death between E12.5-15.5 due to cardiac hemorrhage. The mice had defects in the endocardium and myocardium and lack of coronary arteries, suggesting that, as with VEGF-A, a delicate control of angiopoietin-1 concentration is critical for early heart development.

ENDOTHELIAL-CARDIOMYOCYTE INTERACTIONS IN NORMAL CARDIAC FUNCTION

Cardiac Endothelial Cells Regulate Cardiomyocyte Contraction

The vascular endothelium senses the shear stress of flowing blood and regulates vascular smooth muscle contraction. It is therefore not surprising that cardiac endothelial cells—the endocardial endothelial cells as well as the endothelial cells of intramyocardial capillaries— regulate the contractile state of cardiomyocytes. Autocrine and paracrine signaling molecules released or activated by cardiac endothelial cells are responsible for this contractile response (Figure 2).

NITRIC OXIDE

Three different nitric oxide synthase isoenzymes synthesize nitric oxide (NO) from L-arginine. The neuronal and endothelial NO synthases (nNOS and eNOS, respectively) are expressed in normal physiological conditions, whereas the inducible NO synthase is induced by stress or cytokines. Like NO in the vessel, which causes relaxation of vascular smooth muscle, NO in the heart affects the onset of ventricular relaxation, which allows for a precise optimization of pump function beat by beat. Although NO is principally a paracrine effector secreted by cardiac endothelial cells, cardiomyocytes also express both nNOS and eNOS. Endothelial expression of eNOS exceeds that in cardiomyocytes by greater than 4:1. Cardiomyocyte autocrine eNOS signaling can regulate β-adrenergic and muscarinic control of contractile state.
Barouch et al. demonstrated that cardiomyocyte nNOS and eNOS may have opposing effects on cardiac structure and function. Using mice with nNOS or eNOS deficiency, they found that nNOS and eNOS have not only different localization in cardiomyocytes but also opposite effects on cardiomyocyte contractility; eNOS localizes to caveolae and inhibits L-type Ca2+ channels, leading to negative inotropy, whereas nNOS is targeted to the sarcoplasmic reticulum and facilitates Ca2+ release and thus positive inotropy (31). These results demonstrate that spatial confinement of different NO synthase isoforms contribute independently to the maintenance of cardiomyocyte structure and phenotype.
As indicated above, mutation of neuregulin or either of two of its cognate receptors, erbB2 and erbB4, causes embryonic death during mid-embryogenesis due to aborted development of myocardial trabeculation . Neuregulin also appears to play a role in fully developed myocardium. In adult mice, cardiomyocyte-specific deletion of erbB2 leads to dilated cardiomyopathy. Neuregulin from endothelial cells may induce a negative inotropic effect in isolated rabbit papillary muscles. This suggests that, along with NO, the neuregulin signaling pathway acts as an endothelial-derived regulator of cardiac inotropism.  In fact, the negative inotropic effect of neuregulin may require NO synthase because L-NMMA, an inhibitor of NO synthase, significantly attenuates the negative inotropy of neuregulin.

Studies to date indicate that cardiac regeneration in mammals may be feasible, but the response is inadequate to preserve myocardial function after a substantial injury. Thus, understanding how normal myocardial structure can be regenerated in adult hearts is essential. It is clear that endothelial cells play a role in cardiac morphogenesis and most likely also in survival and function of mature cardiomyocytes. Initial attempts to promote angiogenesis in myocardium were based on the premise that persistent ischemia could be alleviated. However, it is also possible that endothelial-cardiomyocyte interactions are essential in normal cardiomyocyte function and for protection from injury. Understanding the molecular and cellular mechanisms controlling these cell-cell interactions will not only enhance our understanding of the establishment of vascular network in the heart but also allow the development of new targeted therapies for cardiac regeneration by improving cardiomyocyte survival and maturation.

Endothelial-cardiomyocyte assembly

Figure 1.  Endothelial-cardiomyocyte assembly in adult mouse myocardium.
Normal adult mouse myocardium is stained with intravital perfusion techniques to demonstrate cardiomyocyte (outlined in red) and capillary (green; stained with isolectin-fluorescein) assembly. Nuclei are blue (Hoechst). Original magnification: 600X

Endothelial dysfunction

Intramyocardial Fibroblast – Myocyte Communication

Rahul Kakkar, M.D. and Richard T. Lee, M.D.
From the Cardiology Division, Massachusetts General Hospital and the Cardiovascular Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA
Circ Res. 2010 January 8; 106(1): 47–57.    http://dx.doi.org/10.1161/CIRCRESAHA.109.207456

Cardiac fibroblasts have received relatively little attention compared to their more famous neighbors, the cardiomyocytes. Cardiac fibroblasts are often regarded as the “spotters”, nonchalantly watching the cardiomyocytes do the real weight-lifting, and waiting for a catastrophe that requires their actions. However, emerging data now reveal the fibroblast as not only a critical player in the response to injury, but also as an active participant in normal cardiac function.
Interest in cardiac fibroblasts has grown with the recognition that cardiac fibrosis is a prominent contributor to diverse forms of myocardial disease. In the early 1990’s, identification of angiotensin receptors on the surface of cardiac fibroblasts linked the renin-angiotensin-aldosterone system directly with pathologic myocardial and matrix extracellular remodeling.  Fibroblasts were also revealed as a major source of not only extracellular matrix, but the proteases that regulate and organize matrix. New research has uncovered paracrine and well as direct cell-to-cell interactions between fibroblasts and their cardiomyocyte neighbors, and cardiac fibroblasts appear to be dynamic participants in ventricular physiology and pathophysiology.
This review will focus on several aspects of fibroblast-myocyte communication, including mechanisms of paracrine communication.  Ongoing efforts at regeneration of cardiac tissue focus primarily on increasing the number of cardiomyocytes in damaged myocardium. Although getting cardiomyocytes into myocardium is an important goal, understanding intercellular paracrine communication between different cell types, including endothelial cells but also fibroblasts, may prove crucial to regenerating stable myocardium that responds to physiological conditions appropriately.

An area of active research in cardiovascular therapeutics is the attempt to engineer, ex vivo, functional myocardial tissue that may be engrafted onto areas of injured ventricle. Recent data suggests that the inclusion of cardiac fibroblasts in three-dimensional cultures greatly enhances the stability and growth of the nascent myocardium. Cardiac fibroblasts when included in polymer scaffolds seeded with myocytes and endothelial cells have the ability to promote and stabilize vascular structures. Naito and colleagues constructed three dimensional cultures of neonatal rat cell isolates on collagen type I and Matrigel (a basement membrane protein mixture), and isolates of a mixed cell population versus a myocyte-enriched population were compared. The mixed population cultures, which contained a higher fraction of cardiac fibroblasts than the myocyte-enriched cultures, displayed improved contractile force generation and greater inotropic response despite an equivalent overall cell number. Greater vascularity was also seen in the mixed-pool cultures.(160) Building on this, Nichol and colleagues demonstrated that in a self-assembling nanopeptide scaffold, embedded rat neonatal cardiomyocytes exhibit greater cellular alignment and reduced apoptosis when cardiac fibroblasts were included in the initial culture. A similar result was noted when polymer scaffolds were pre-treated with cardiac fibroblasts before myocyte seeding, suggesting a persistent paracrine effect. These data reinforce the concept that engineering functional myocardium, either in situ or ex vivo will require attention to the nature of cell-cell interactions, including fibroblasts.

To date, a broad initial sketch of cardiac fibroblast-myocyte interactions has been drawn. Future studies in this field will better describe these interactions. How do multiple paracrine factors interact to produce a cohesive and coordinated communication scheme? What are the changes in coordinated bidirectional signaling that during development promotes myocyte progenitor proliferation but have different roles in the adult? Might fibroblasts actually be required for improved cardiac repair and regeneration?
Recent studies have begun to apply genetic and cellular fate-mapping techniques to document the origins of cardiac fibroblasts, the dynamic nature of their population, and how that population may be in flux during time of injury or pressure overload. It is crucial to define on a more specific molecular basis the origins and fates of cardiac fibroblasts. Do fibroblasts that have been resident within the ventricle since development fundamentally differ from those that arise from endothelial transition or that infiltrate from the bone marrow during adulthood? Do fibroblasts with these different origins behave differently or take on different roles in the face of ventricular strain or injury?

Our understanding of the nature of the cardiac fibroblast is evolving from the concept of the fibroblast as a bystander that causes unwanted fibrosis to the picture of a more complex role of fibroblasts in the healthy as well as diseased heart. The pathways used by cardiac fibroblasts to communicate with their neighboring myocytes are only partially described, but the data to date indicate that these pathways will be important for cardiac repair and regeneration.

. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Figure 2. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Under biomechanical overload, cardiac fibroblasts and myocytes respond to an altered environment via multiple mechanisms including integrin-extracellular matrix interactions and renin-angiotensin-aldosterone axis activation. Cardiac fibroblasts increase synthesis of matrix proteins and secrete a variety of paracrine factors that can stimulate myocyte hypertrophy. Cardiac myocytes similarly respond by secreting a conglomerate of factors. Hormones such as TGFβ1, FGF-2, and the IL-6 family members LIF and CT-1 have all been implicated in this bidirectional fibroblast-myocyte hormonal crosstalk.

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Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES


Drug Eluting Stents: On MIT‘s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES

Author: Larry H Bernstein, MD, FACP

and 

Curator: Aviva Lev-Ari, PhD, RN
http://PharmaceuticalIntelligence.com/2013/04/25/Contributions
-to-vascular-biology/

This is the first of a three part series on the evolution of vascular biology and the studies of the effects of biomaterials in vascular reconstruction and on drug delivery, which has embraced a collaboration of cardiologists at Harvard Medical School , Affiliated Hospitals, and MIT,
requiring cardiovascular scientists at the PhD and MD level, physicists, and computational biologists working in concert, and
an exploration of the depth of the contributions by a distinguished physician, scientist, and thinker.

The first part – Vascular Biology and Disease – will cover the advances in the research on

  • vascular biology,
  • signaling pathways,
  • drug diffusion across the endothelium and
  • the interactions with the underlying muscularis (media),
  • with additional considerations for type 2 diabetes mellitus.

The second part – Stents and Drug Delivery – will cover the

  • purposes,
  • properties and
  • evolution of stent technology with
  • the acquired knowledge of the pharmacodynamics of drug interactions and drug distribution.

The third part – Problems and Promise of Biomaterials Technology – will cover the shortcomings of the cardiovascular devices, and opportunities for improvement

Vascular Biology and Cardiovascular Disease

Early work on endothelial injury and drug release principles

The insertion of a catheter for the administration of heparin is not an innocuous procedure. Heparin is infused to block coagulation, lowering the risk of a dangerous

  • clot formation and
  • dissemination.

It was shown experimentally that the continuous infusion of heparin

  • suppresses smooth muscle proliferation after endothelial injury. It may lead to
  • hemorrhage as a primary effect.

The anticoagulant property of heparin was removed by chemical modification without loss of the anti-proliferative effect.

In this study, MIT researches placed ethylene-vinyl acetate copolymer matrices containing standard and modified heparin adjacent to rat carotid arteries at the time of balloon deendothelialization.

Matrix delivery of both heparin compounds effectively diminished this proliferation in comparison to controls without producing systemic anticoagulation or side effects.

This mode of therapy appeared more effective than administering the agents by either

  • intravenous pumps or
  • heparin/polymer matrices placed in a subcutaneous site distant from the injured carotid artery

This indicated that the site of placement at the site of injury is a factor in the microenvironment, and is a preference for avoiding restenosis after angioplasty and other interventions.

This raised the question of why the proliferation of vascular muscle occurs in the first place.
 Edelman, Nugent and Karnovsky  (1) showed that the proliferation required first the denudation of vascular surface endothelium. This exposed the underlayer to the effect of basic fibroblast growth factor, which stimulates mitogenesis of the exposed cell, explained by the endothelium as a barrier from circulating bFGF.

To answer this question, they compared the effect of

  • 125I-labelled bFGF intravenously given with perivascular controlled bFGF release.
  • Polymeric controlled release devices delivered bFGF to the extravascular space without transendothelial transport. 
Deposition within the blood vessel wall was rapidly distributed circumferentially and was substantially greater than that observed following intravenous injection.

The amount of bFGF deposited in arteries adjacent to the release devices was 40 times that deposited in similar arteries in animals who received a single intravenous bolus of bFGF.

The presence of intimal hyperplasia increased deposition of perivascularly released bFGF 2.4-fold but decreased the deposition of intravenously injected bFGF by 67%.

  • bFGF was 5- to 30-fold more abundant in solid organs after intravenous injection than it was following perivascular release, and
  • bFGF deposition was greatest in the kidney, liver, and spleen and was substantially lower in the heart and lung.

This result indicated that vascular deposition of bFGF is independent of endothelium, and

  • bFGF delivery is effectively perivascular. (2)

Drug activity studies have to be done in well controlled and representative conditions.
 Edelsman’s Lab researchers studied the

  • dose response of injured arteries to exogenous heparin in vivo by providing steady and predictable arterial levels of drug.
  • Controlled-release devices were fabricated to direct heparin uniformly and at a steady rate to the adventitial surface of balloon-injured rat carotid arteries.

Researchers predicted the distribution of heparin throughout the arterial wall using computational simulations and correlated these concentrations with the biologic response of the tissues.

Researchers determined from this process that an in vivo arterial concentration of 0.3 mg/ml of heparin is required to maximallyinhibit intimal hyperplasia after injury.

This estimation of the required tissue concentration of a drug is

  • independent of the route of administration and
  • applies to all forms of drug release.

In this way the Team was able to

  • evaluate the potential of  widely disparate forms of drug release and, to finally
  • create some rigorous criteria by which to guide the development of particular delivery strategies for local diseases. (3)

Chiefly, the following three effects:

(1) Effect of controlled adventitial heparin delivery on smooth muscle cell proliferation following endothelial injury. ER Edelman, DH Adams, and MJ Karnovsky. PNAS May 1990; 87: 3773-3777.


(2) Perivascular and intravenous administration of basic fibroblast growth factor: Vascular and solid organ deposition. ER Edelman, MA Nugent, and MJ Karnovsky. PNAS Feb 1993; 90: 1513-1517.


(3) Tissue concentration of heparin, not administered dose, correlates with the biological response of injured arteries in vivo. MA Lovich and ER Edelman. PNAS Sep 1999; 96: 11111–11116.

Vascular Injury and Repair

Perlecan is a heparin-sulfate proteoglycan that might be critical for regulation of vascular repair by inhibiting the binding and mitogenic activity of basic fibroblast growth factor-2 (bFGF-2) in vascular smooth muscle cells .

The Team generated

  • Clones of endothelial cells expressing an antisense vector targeting domain III of perlecan. The transfected cells produced significantly less perlecan than parent cells, and they had reduced bFGF in vascular smooth muscle cells.
  • Endothelial cells were seeded onto three-dimensional polymeric matrices and implanted adjacent to porcine carotid arteries subjected to deep injury.
  • The parent endothelial cells prevented thrombosis, but perlecan deficient cells were ineffective.

The ability of endothelial cells to inhibit intimal hyperplasia, however, was only in part suppressed by perlecan. The differential regulation by perlecan of these aspects of vascular repair may clarify why control of clinical clot formation does not lead to full control of intimal hyperplasia.

The use of genetically modified tissue engineered cells provides a new approach for dissecting the role of specific factors within the blood vessel wall.(1) Successful implementation of local arterial drug delivery requires transmural distribution of drug. The physicochemical properties of the applied compound govern its transport and tissue binding.

  • Hydrophilic compounds are cleared rapidly.
  • Hydrophobic drugs bind to fixed tissue elements, potentially prolonging tissue residence and biological effect.

Local vascular drug delivery provides

  • elevated concentrations of drug in the target tissue while
  • minimizing systemic side effects.

To better characterize local pharmacokinetics the Team examined the arterial transport of locally applied dextran and dextran derivatives in vivo.

Using a two-compartment pharmacokinetic model to correct

  • The measured transmural flux of these compounds for systemic
  • Redistribution and elimination as delivered from a photo-polymerizable hydrogel.
  • The diffusivities and the transendothelial permeabilities were strongly dependent on molecular weight and charge
  • For neutral dextrans, the diffusive resistance increased with molecular weightapproximately 4.1-fold between the molecular weights of 10 and 282 kDa.
  • Endothelial resistance increased 28-fold over the same molecular weight range.
  • The effective medial diffusive resistance was unaffected by cationic charge as such molecules moved identically to neutral compounds, but increased approximately 40% when dextrans were negatively charged.

Transendothelial resistance was 20-fold lower for the cationic dextrans, and 11-fold higher for the anionic dextrans, when both were compared to neutral counterparts.

These results suggest that, while

  • low molecular weight drugs will rapidly traverse the arterial wall with the endothelium posing a minimal barrier,
  • the reverse is true for high molecular weight agents.

The deposition and distribution of locally released vascular therapeutic compounds might be predicted based upon chemical properties, such as molecular weight and charge. (2)

Paclitaxel is hydrophobic and has therapeutic potential against proliferative vascular disease.
 The favorable preclinical data with this compound may, in part, result from preferential tissue binding.
 The complexity of Paclitaxel pharmacokinetics required in-depth investigation if this drug is to reach its full clinical potential in proliferative vascular diseases.

Equilibrium distribution of Paclitaxel reveals partitioning above and beyond perfusate concentration and a spatial gradient of drug across the arterial wall.

The effective diffusivity (Deff) was estimated from the Paclitaxel distribution data to

  • facilitate comparison of transport of Paclitaxel through arterial parenchyma with that of other vasoactive agents and to
  • characterize the disparity between endovascular and perivascular application of drug.

This transport parameter described the motion of drug in tissues given an applied concentration gradient and includes, in addition to diffusion,

  • the impact of steric hindrance within the arterial interstitium;
  • nonspecific binding to arterial elements; and, in the preparation used here,
  • convective effects from the applied transmural pressure gradient.

At all times, the effective diffusivity for endovascular delivery exceeded that of perivascular delivery. The arterial transport of Paclitaxel was quantified through application ex vivo and measurement of the subsequent transmural distribution.

  • Arterial Paclitaxel deposition at equilibrium varied across the arterial wall.
  • Permeation into the wall increased with time, from 15 minutes to 4 hours, and
  • varied with the origin of delivery.

In contrast to hydrophilic compounds, the concentration in tissue exceeded the applied concentration and the rate of transport was markedly slower. Furthermore, endovascular and perivascular Paclitaxel application led to differences in deposition across the blood vessel wall.

This leads to a conclusion that Paclitaxel interacts with arterial tissue elements  as it moves under the forces of

  • diffusion and
  • convection and
  • can establish substantial partitioning and spatial gradients across the tissue. (3)

Endovascular drug-eluting stents have changed the practice of  cardiovascular vascularization, and yet it is unclear how they so dramatically reduce restenosis

We don’t know how to distinguish between the different formulations available.
 Researchers are now questioning whether individual properties of different drugs beyond lipid avidity effect arterial transport and distribution.

In bovine internal carotid segments, tissue-loading profiles for

  • Hydrophobic Paclitaxel and Rapamycin are indistinguishable, reaching load steady state after 2 days.
  • Hydrophilic dextran reaches equilibrium in hours.

Paclitaxel and Rapamycin bind to the artery at 30–40 times bulk concentration, and bind to specific tissue elements.

Transmural drug distribution profiles are markedly different for the two compounds.

  • Rapamycin binds specifically to FKBP12 binding protein and it distributes evenly through the artery,
  • Paclitaxel binds specifically to microtubules, and remains primarily in the subintimal space.

The binding of Rapamycin and Paclitaxel to specific intracellular proteins plays an essential role in

  • determining arterial transport and distribution and in
  • distinguishing one compound from another.

These results offer further insight into the

  • mechanism of local drug delivery and the
  • specific use of existing drug-eluting stent formulations. (4)

The Role of Amyloid beta (A) in Creation of Vascular Toxic Plaque

Amyloid beta (A) is a peptide family produced and deposited in neurons and endothelial cells (EC).
It is found at subnanomolar concentrations in the plasma of healthy individuals.
 Simple conformational changes produce a form of A-beta , A-beta 42, which creates toxic plaque in the brains of Alzheimer’s patients.

Oxidative stress induced blood brain barrier degeneration has been proposed as a key factor for A-beta 42 toxicity.

This cannot account for lack of injury from the same peptide in healthy tissues.
Researchers hypothesized that cell state mediates A-beta’s effect.
 They examined the viability in the presence of A-beta secreted from transfected
Chinese hamster ovary cells (CHO) of

  • aortic Endothelial Cells (EC),
  • vascular smooth muscle cells (SMC) and
  • epithelial cells (EPI) in different states

A-beta was more toxic to all cell types when they were subconfluent.
 Subconfluent EC sprouted and SMC and EPI were inhibited by A-beta.
Confluent EC were virtually resistant to A-beta and suppressed A-beta production by A-beta +CHO.

Products of subconfluent EC overcame this resistant state, stimulating the production and toxicity of A-beta 42. Confluent EC overgrew >35% beyond their quiescent state in the presence of A-beta conditioned in media from subconfluent EC.

These findings imply that A-beta 42 may well be even more cytotoxic to cells in injured or growth states and potentially explain the variable and potent effects of this protein.

One may now need to consider tissue and cell state in addition to local concentration of and exposure duration to A-beta.

The specific interactions of A-beta and EC in a state-dependent fashion may help understand further the common and divergent forms of vascular and cerebral toxicity of A-beta and the spectrum of AD. (5)

(1) Perlecan is required to inhibit thrombosis after deep vascular injury and contributes
to endothelial cell-mediated inhibition of intimal hyperplasia. MA Nugent, HM Nugent,
RV Iozzoi, K Sanchack, and ER Edelman. PNAS Jun 2000; 97(12): 6722-6727


(2) Correlation of transarterial transport of various dextrans with their physicochemical properties.
O Elmalak, MA Lovich, E Edelman. Biomaterials 2000; 21: 2263-2272


(3) Arterial Paclitaxel Distribution and Deposition. CJ Creel, MA Lovich, ER Edelman. Circ Res. 2000;86:879-884


(4) Specific binding to intracellular proteins determines arterial transport properties for rapamycin and Paclitaxel.
AD Levin, N Vukmirovic, Chao-Wei Hwang, and ER Edelman. PNAS Jun 2004; 101(25): 9463–9467.
www.pnas.org/cgi/doi/10.1073/pnas.0400918101

(5) Amyloid beta toxicity dependent upon endothelial cell state. M Balcells, JS Wallins, ER Edelman.
Neuroscience Letters 441 (2008) 319–322

Endothelial Damage as an Inflammatory State

Autoimmunity may drive vascular disease through anti-endothelial cell (EC) antibodies. This raises a question about whether an increased morbidity of cardiovascular diseases in concert with systemic illnesses may involve these antibodies.

Matrix-embedded ECs act as powerful regulators of vascular repair accompanied by significant reduction in expected systemic and local inflammation.

The Lab researchers compared the immune response against free and matrix-embedded ECs in naive mice and mice with heightened EC immune reactivity. Mice were presensitized to EC with repeated subcutaneous injections of saline-suspended porcine EC (PAE) (5*10^5 cells).

On day 42, both naive mice (controls) and mice with heightened EC immune reactivity received 5*10^5 matrix-embedded or free PAEs. Circulating PAE-specific antibodies and effector T-cells were analyzed 90 days after implantation for –

  • PAE-specific antibody-titers,
  • frequency of CD4+-effector cells, and
  • xenoreactive splenocytes

These were 2- to 4-fold lower (P<0.0001) when naıve mice were injected with matrix-embedded instead of saline-suspended PAEs.

Though basal levels of circulating antibodies were significantly elevated after serial PAE injections (2210+341 mean fluorescence intensity, day 42) and almost doubled again 90 days after injection of a fourth set of free PAEs, antibody levels declined by half in recipients of matrix-embedded PAEs at day 42 (P<0.0001), as did levels of CD4+-effector cells and xenoreactive splenocytes.

A significant immune response to implantation of free PAE is elicited in naıve mice, that is even more pronounced in mice with pre-developed anti-endothelial immunity.

Matrix-embedding protects xenogeneic ECs against immune reaction in naive mice and in mice with heightened immune reactivity.

Matrix-embedded EC might offer a promising approach for treatment of advanced cardiovascular disease. (1)

Researchers examined the molecular mechanisms through which

mechanical force and hypertension modulate

endothelial cell regulation of vascular homeostasis.

Exposure to mechanical strain increased the paracrine inhibition of vascular smooth muscle cells (VSMCs) by endothelial cells.

Mechanical strain stimulated the production by endothelial cells of perlecan and heparan-sulfate glycosaminoglycans. By inhibiting the expression of perlecan with an antisense vector researchers demonstrated that perlecan was essential to the strain-mediated effects on endothelial cell growth control.

Mechanical regulation of perlecan expression in endothelial cells was

  • governed by a mechano-transduction pathway
  • requiring transforming growth factor (TGF-β) signaling and
  • intracellular signaling through the ERK pathway.

Immunohistochemical staining of the aortae of spontaneously hypertensive rats
demonstrated strong correlations between

  • endothelial TGF-β,
  • phosphorylated signaling intermediates, and
  • arterial thickening.

Studies on ex vivo arteries exposed to varying levels of pressure demonstrated that

ERK and TGF-beta signaling were required for pressure-induced upregulation of endothelial HSPG.

The Team’s findings suggest a novel feedback control mechanism in which

  • net arterial remodeling to hemodynamic forces is controlled by a dynamic interplay between growth stimulatory signals from vSMCs and
  • growth inhibitory signals from endothelial cells. (2)

Heparan-sulfate proteoglycans (HSPGs) are potent regulators of vascular remodeling and repair.
 The major enzyme capable of degrading HSPGs is heparanase, which led us to examine
the role of heparanase in controlling

  • arterial structure,
  • mechanics, and
  • remodeling.

In vitro studies suggested heparanase expression in endothelial cells serves as a negative regulator of endothelial inhibition of vascular smooth muscle cell (vSMC) proliferation.

ECs inhibit vSMC proliferation through the interplay between

  • growth stimulatory signals from vSMCs and
  • growth inhibitory signals from ECs.

This would be expected if ECs had HSPGs that are degraded by heparanase.
Arterial structure and remodeling to injury is modified by heparanase expression.
Transgenic mice overexpressing heparanase had

  • increased arterial thickness,
  • cellular density, and
  • mechanical compliance.

Endovascular stenting studies in Zucker rats demonstrated increased heparanase expression in the neointima of obese, hyperlipidemic rats in comparison to lean rats.

The extent of heparanase expression within the neointima strongly correlated with the neointimal thickness following injury. To test the effects of heparanase overexpression on arterial repair, researchers developed a novel murine model of stent injury using small diameter self-expanding stents.

Using this model, researchers found that increased

  • neointimal formation and
  • macrophage recruitment occurs in transgenic mice overexpressing heparanase.
  • Taken together, these results support a role for heparanase in the regulation of arterial structure, mechanics, and repair. (3)

The first host–donor reaction in transplantation occurs at the blood–tissue interface.
When the primary component of the implant (donor) is the endothelial cells, it incites an immunologic reaction. Injections of free endothelial cell implants elicit a profound major histocompatibility complex (MHC) II dominated immune response.

Endothelial cells embedded within three-dimensional matrices behave like quiescent endothelial cells.

Perivascular implants of such embedded ECs cells are the most potent inhibitor of intimal hyperplasia and thrombosis following controlled vascular injury, but without any immune reactivity.

Allo- and even exenogenic endothelial cells evoke no significant humoral or
cellular immune response in immune-competent hosts when embedded within matrices.
 Moreover,  endothelial implants are immune-modulatory, reducing the extent of the memory response to previous free cell implants.

Attenuated immunogenicity results in muted activation of adaptive and innate immune cells. These findings point toward a pivotal role of matrix–cell-interconnectivity for

  • the cellular immune phenotype and might therefore assist in the design  of
  • extracellular matrix components for successful tissue engineering. (4)

Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, researchers sought to understand if

  • cytokine-induced NF-κB activity and
  • downstream effects depend on substrate adherence of endothelial cells (EC).

The team compared the upstream

  • phosphorylation cascade,
  • activation of NF-ĸβ, and
  • expression/secretion

of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC).

Adhesion of natural killer (NK) cells was quantified in vitro and in vivo.

  • NF-κβ subunit p65 nuclear levels were significantly lower and
  • p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS.

Despite similar surface expression of TNF-α receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1.

Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 ± 420 vs. 1,735 ± 135 cpm; p < 0.0002).

Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix.

Matrix embedding enables control of EC substratum interaction.

This in turn regulates chemokine and surface molecule expression and secretion, in particular – of those compounds within NF-κβ pathways,

  • chemoattraction of NK cells,
  • local inflammation, and
  • tissue repair. (5)

Monocyte recruitment and interaction with the endothelium is imperative to vascular recovery.

Tie2 plays a key role in endothelial health and vascular remodeling.
Researchers studied monocyte-mediated Tie2/angiopoietin signaling following interaction of primary monocytes with endothelial cells and its role in endothelial cell survival.

The direct interaction of primary monocytes with subconfluent endothelial cells

resulted in transient secretion of angiopoietin-1 from monocytes and

the activation of endothelial Tie2. This effect was abolished by preactivation of monocytes with tumor necrosis factor-α (TNFα).

Although primary monocytes contained high levels of

  • both angiopoietin 1 and 2,
  • endothelial cells contained primarily angiopoietin 2.

Seeding of monocytes on serum-starved endothelial cells reduced caspase-3 activity by 46+5.1%, and 52+5.8% after TNFα treatment, and it decreased single-stranded DNA levels by 41+4.2% and 40+ 3.5%, respectively.

This protective effect of monocytes on endothelial cells was reversed by Tie2 silencing with specific short interfering RNA.

The antiapoptotic effect of monocytes was further supported by the

  • activation of cell survival signaling pathways involving phosphatidylinositol 3-kinase,
  • STAT3, and
  • AKT.

Monocytes and endothelial cells form a unique Tie2/angiopoietin-1 signaling system that affects endothelial cell survival and may play critical a role in vascular remodeling and homeostasis. (6)

(1) Cell–Matrix Contact Prevents Recognition and Damage of Endothelial Cells in States of Heightened Immunity.
H Methe, ER Edelman. Circulation. 2006;114[suppl I]:I-233–I-238.
http://www.circulationaha.org/DOI/10.1161/CIRCULATIONAHA.105.000687

(2) Endothelial Cells Provide Feedback Control for Vascular Remodeling Through a Mechanosensitive Autocrine
TGFβ Signaling Pathway. AB Baker, DS Ettenson, M Jonas, MA Nugent, RV Iozzo, ER Edelman.
Circ. Res. 2008;103;289-297   http://dx.doi.org/10.1161/CIRCRESAHA.108.179465http://circres.ahajournals.org/cgi/content/full/103/3/289

(3) Heparanase Alters Arterial Structure, Mechanics, and Repair Following Endovascular Stenting in Mice.
AB Baker, A Groothuis, M Jonas, DS Ettenson…ER Edelman.   Circ. Res. 2009;104;380-387;
http://dx.doi.org/10.1161/CIRCRESAHA.108.180695  http://circres.ahajournals.org/cgi/content/full/104/3/380

(4) The effect of three-dimensional matrix-embedding of endothelial cells on the humoral and cellular immune response.
H Methe, S Hess, ER Edelman. Seminars in Immunology 20 (2008) 117–122. http://dx.doi.org/10.1016/j.smim.2007.12.005

(5) NF-kB Activity in Endothelial Cells Is Modulated by Cell Substratum Inter-actions and Influences Chemokine-Mediated
Adhesion of Natural Killer Cells.  S Hess, H Methe, Jong-Oh Kim, ER Edelman.
Cell Transplantation 2009; 18: 261–273


(6) Primary Monocytes Regulate Endothelial Cell Survival Through Secretion of Angiopoietin-1 and Activation of Endothelial Tie2.
SY Schubert, A Benarroch, J Monter-Solans and ER Edelman. Arterioscler Thromb Vasc Biol 2011;31;870-875
http://dx.doi.org/10.1161/ATVBAHA.110.218255

Neointimal Formation, Shear Stress, and Remodelling with Reference to Diabetes

Innate immunity is of major importance in vascular repair. The present study evaluated whether

  • systemic and transient depletion of monocytes and macrophages with
  • liposome-encapsulated bisphosphonates inhibits experimental in-stent neointimal formation.

The Experiment

Rabbits fed on a hypercholesterolemic diet underwent bilateral iliac artery balloon denudation and stent deployment.

Liposomal alendronate (3 or 6 mg/kg) was given concurrently with stenting.

  • Monocyte counts were reduced by 90% 24 to 48 hours aftera single injection of liposomal alendronate, returning to basal levels at 6 days.

This treatment significantly reduced

  • intimal area at 28 days, from 3.88+0.93 to 2.08+0.58 and 2.16 +0.62 mm2.
  • Lumen area was increased from 2.87+0.44 to 3.57­+0.65 and 3.45+0.58 mm2, and
  • arterial stenosis was reduced from 58 11% to 37 8% and 38 7% in controls, in rabbits treated with 3 mg/kg, and with 6 mg/kg, respectively (mean+SD, n=8 rabbits/group, P< 0.01 for all 3 parameters).

No drug-related adverse effects were observed.
Reduction in neointimal formation was associated with

  • reduced arterial macrophage infiltration and proliferation at 6 days and with an
  • equal reduction in intimal macrophage and smooth muscle cell content at 28 days after injury.

Conversely, drug regimens ineffective in reducing monocyte levels did not inhibit neointimal formation.
Researchers have shown that a

  • single liposomal bisphosphonates injection concurrent with injury reduces in-stent neointimal formation and
  • arterial stenosis in hypercholesterolemic rabbits, accompanied by systemic transient depletion of monocytes and macrophages. (1)

Diabetes and insulin resistance are associated with increased disease risk and poor outcomes from cardiovascular interventions.

Even drug-eluting stents exhibit reduced efficacy in patients with diabetes.
Researchers reported the first study of vascular response to stent injury in insulin-resistant and diabetic animal models.

Endovascular stents were expanded in the aortae of

  • obese insulin-resistant and
  • type 2 diabetic Zucker rats,
  • in streptozotocin-induced type 1 diabetic Sprague-Dawley rats, and
  • in matched controls.

Insulin-resistant rats developed thicker neointima (0.46+0.08 versus 0.37+0.06 mm2, P 0.05), with  decreased lumen area (2.95+0.26 versus 3.29+0.15 mm2, P 0.03) 14 days after stenting compared with controls, but without increased vascular inflammation (tissue macrophages).

Insulin-resistant and diabetic rat vessels did exhibit markedly altered signaling pathway activation 1 and 2 weeks after stenting, with up to a 98% increase in p-ERK (anti-phospho ERK) and a 54% reduction in p-Akt (anti-phospho Akt) stained cells. Western blotting confirmed a profound effect of insulin resistance and diabetes on Akt and ERK signaling in stented segments. p-ERK/p-Akt ratio in stented segments uniquely correlated with neointimal response (R2 = 0.888, P< 0.04) , but not in lean controls.

Transfemoral aortic stenting in rats provides insight into vascular responses in insulin resistance and diabetes.

Shifts in ERK and Akt signaling related to insulin resistance may reflect altered tissue repair in diabetes accompanied by a

  • shift in metabolic : proliferative balance.

These findings may help explain the increased vascular morbidity in diabetes and suggest specific therapies for patients with insulin resistance and diabetes. (2)

Researchers investigated the role of Valsartan (V) alone or in combination with Simvastatin (S) on coronary atherosclerosis and vascular remodeling, and tested the hypothesis that V or V/S attenuate the pro-inflammatory effect of low endothelial shear stress (ESS).

Twenty-four diabetic, hyperlipidemic swine were allocated into Early (n = 12) and Late (n=12) groups.
Diabetic swine in each group were treated with Placebo (n=4), V (n = 4) and V/S (n = 4) and  followed for 8 weeks in the Early group and 30 weeks in the Late group.

Blood pressure, serum cholesterol and glucose were similar across the treatment subgroups.
ESS was calculated in plaque-free subsegments of interest (n = 109) in the Late group at week 23.
Coronary arteries of this group were harvested at week 30, and the subsegments of interest were identified, and analyzed histopathologically.

Intravascular geometrically correct 3-dimensional reconstruction of the coronary arteries of 12 swine was performed 23 weeks after initiation of diabetes mellitus and a hyperlipidemic diet. Local endothelial shear stress was calculated

  • in plaque-free subsegments of interest (n=142) with computational fluid dynamics, and
  • the coronary arteries (n=31) were harvested and the same subsegments were identified at 30 weeks.

V alone or with S

  • reduced the severity of inflammation in high-risk plaques.
Both regimens attenuated the severity of enzymatic degradation of the arterial wall, reducing the severity of expansive remodeling.
  • attenuated the pro-inflammatory effect of low ESS.
V alone or with S
  • exerts a beneficial effect of reducing and stabilizing high-risk plaque characteristics independent of a blood pressure- and lipid-lowering effect. (3)

This study tested the hypothesis that low endothelial shear stress  augments the

  • expression of matrix-degrading proteases, promoting the
  • formation of thin-capped atheromata.

Researchers assessed the messenger RNA and protein expression, and elastolytic activity of selected elastases and their endogenous inhibitors.

Subsegments with low endothelial shear stress at week 23 showed

  • reduced endothelial coverage,
  • enhanced lipid accumulation, and
  • intense infiltration of activated inflammatory cells at week 30.

These lesions showed increased expression of messenger RNAs encoding

  • matrix metalloproteinase-2, -9, and -12, and cathepsins K and S
  • relative to their endogenous inhibitors and
  • increased elastolytic activity.

Expression of these enzymes correlated positively with the severity of internal elastic lamina fragmentation.

Thin-capped atheromata in regions with

  • lower preceding endothelial shear stress had
  • reduced endothelial coverage,
  • intense lipid and inflammatory cell accumulation,
  • enhanced messenger RNA expression and
  • elastolytic activity of MMPs and cathepsins with
  • severe internal elastic lamina fragmentation.

Low endothelial shear stress induces endothelial discontinuity and

  • accumulation of activated inflammatory cells, thereby
  • augmenting the expression and activity of elastases in the intima and
  • shifting the balance with their inhibitors toward matrix breakdown.

Team’s results provide new insight into the mechanisms of regional formation of plaques with thin fibrous caps. (4)

Elevated CRP levels predict increased incidence of cardiovascular events and poor outcomes following interventions. There is the suggestion that CRP is also a mediator of vascular injury.

Transgenic mice carrying the human CRP gene (CRPtg) are predisposed to arterial thrombosis post-injury.

Researchers examined whether CRP similarly modulates the proliferative and hyperplastic phases of vascular repair in CRPtg when thrombosis is controlled with daily aspirin and heparin at the time of trans-femoral arterial wire-injury.

Complete thrombotic arterial occlusion at 28 days was comparable for wild-type and CRPtg mice (14 and 19%, respectively). Neointimal area at 28d was 2.5 fold lower in CRPtg (4190±3134 m2, n = 12) compared to wild-types (10,157±8890 m2, n = 11, p < 0.05).

Likewise, neointimal/media area ratio was 1.10±0.87 in wild-types and 0.45±0.24 in CRPtg (p < 0.05).

  • Seven days post-injury, cellular proliferation and apoptotic cell number in the intima were both less pronounced in CRPtg than wild-type.
  • No differences were seen in leukocyte infiltration or endothelial coverage.
CRPtg mice had significantly reduced p38 MAPK signaling pathway activation following injury.

The pro-thrombotic phenotype of CRPtg mice was suppressed by aspirin/heparin, revealing CRP’s influence on neointimal growth after trans-femoral arterial wire-injury.

  • Signaling pathway activation,
  • cellular proliferation, and
  • neointimal formation

were all reduced in CRPtg following vascular injury.
 Increasingly the Team was aware of CRP multipotent effects.
 Once considered only a risk factor, and recently a harmful agent, CRP is a far more complex regulator of vascular biology. (5)

(1) Liposomal Alendronate Inhibits Systemic Innate Immunity and Reduces In-Stent Neointimal
Hyperplasia in Rabbits. HD Danenberg, G Golomb, A Groothuis, J Gao…, ER Edelman.
Circulation. 2003;108:2798-2804


(2) Vascular Neointimal Formation and Signaling Pathway Activation in Response to Stent Injury
in Insulin-Resistant and Diabetic Animals. M Jonas, ER Edelman, A Groothuis, AB Baker, P Seifert, C Rogers.
Circ. Res. 2005;97;725-733.        http://dx.doi.org/10.1161/01.RES.0000183730.52908.C6
http://circres.ahajournals.org/cgi/content/full/97/7/725

(3) Attenuation of inflammation and expansive remodeling by Valsartan alone or in combination with
Simvastatin in high-risk coronary atherosclerotic plaques. YS Chatzizisis, M Jonas, R Beigel, AU Coskun…
ER Edelman, CL Feldman, PH Stone.  Atherosclerosis 203 (2009) 387–394


(4) Augmented Expression and Activity of Extracellular Matrix-Degrading Enzymes in Regions of Low
Endothelial Shear Stress Colocalize With Coronary Atheromata With Thin Fibrous Caps in Pigs.
YS Chatzizisis, AB Baker, GK Sukhova,…P Libby, CL Feldman, ER Edelman, PH Stone
Circulation 2011;123;621-630     http://dx.doi.org/10.1161/CIRCULATIONAHA.110.970038
http://circ.ahajournals.org/cgi/content/full/123/6/621


(5) Neointimal formation is reduced after arterial injury in human crp transgenic mice
HD Danenberg, E Grad, RV Swaminathan, Z Chenc,…ER Edelman
Atherosclerosis 201 (2008) 85–91

A Rattle Bag of Science and the Art of Translation

Science Translational Medicine – A rattle bag of science and the art of translation
E. R. Edelman, G. A. FitzGerald.
Sci.Transl. Med. 3, 104ed3 (2011). http://dx.doi.org/10.1126/scitranslmed.3002131

Elazer R. Edelman is the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology at MIT,
Professor of Medicine at Harvard Medical School, a coronary care unit cardiologist at the Brigham and Women’s
Hospital, and Director of the Harvard-MIT Biomedical Engineering Center. E-mail: ere@mit.edu

Garret A. FitzGerald is the McNeil Professor in Translational Medicine and Therapeutics, Chair of the Department of
Pharmacology, and Director of the Institute for Translational Medicine & Therapeutics, University of Pennsylvania.
E-mail: garret@upenn.edu

In 2011, the American Association for the Advancement of Science (AAAS)  founded Science Translational Medicine (STM)
to disseminate interdisciplinary science integrating basic and clinical research that defines and fosters new therapeutics, devices, and diagnostics.

Conceived and nourished under the creative vision of Elias Zerhouni and Katrina Kelner, the journal has attracted widespread attention.
Now, as we assume the mantle of co-chief scientific advisors, we look back on the journal’s early accomplishments, restate our mission, and make clear the kinds of manuscripts we seek and accept for publication.

STM’s mission, as articulated by Elias and Katrina, was to

“promote human health by providing a forum for communication and cross-fertilization among basic, translational, and clinical research practitioners and trainees from all relevant established and emerging disciplines.”

This statement remains relevant and accurate today.
 With this mission on our masthead, STM now receives ~25 manuscripts (full-length research articles) per week and publishes ~10% of them. Roughly half of the submissions are deemed inappropriate for the journal and are returned without review within 8 to 10 days of receipt.

Of those papers that undergo full peer review,

decisions to reject are made within 48 days and

the mean time to acceptance (including the revision period) is 125 days.

There is now an average wait of only 24 days between acceptance and publication.

Defining TRANSLATIONAL Medicine

In accord with the journal’s broad readership, the ideal manuscript meets five criteria: It
(i) reports a discovery of translational relevance with high-impact potential;
(ii) has a conceptual focus with interdisciplinary appeal;
(iii) elucidates a biological mechanism;
(iv) is innovative and novel; and
(v) is presented in clear, broadly accessible language.
 STM seeks to publish research that describes

  • how innovative concepts drive the creative biomedical science
  • that ultimately improves the quality of people’s lives—

This is the broadest of our journal’s criteria but is the one that sets us apart as well.
Translational relevance does not require demonstration of benefit in humans but does require the evident potential to advance clinical medicine, thus impacting the direction of our culture and the welfare of our communities. Conceptual focus and mechanistic emphasis discriminate our papers from those that contain observational descriptions of technical findings for which value is restricted to a specific discipline.

However, innovation and novelty may apply to a fundamental scientific discovery or to the nature of its application and relevance to the translational process. Criteria enable the journal to consider versatile technological advances that apply new and creative thinking but may not necessarily offer fresh insights into biological mechanisms. Finally, while the subsequent additional efforts of the STM editorial staff are not to be discounted, the clarity of writing and coherence of argument presented within a submitted manuscript are likely to facilitate its progress through the challenge of peer review.

On Causes – Hippocrates, Aristotle, Robert Koch, and the Dread Pirate Roberts

Elazer R. Edelman
Circulation 2001;104:2509-2512

The idea of risk factors for vascular disease has evolved

  • from a dichotomous to continuous hazard analysis and
  • from the consideration of a few factors to
  • mechanistic investigation of many interrelated risks.

However, confusion still abounds regarding issues of association and causation. Originally, the simple presence of

  • tobacco abuse, hypertension, and/or hypercholesterolemia were tallied, and
  • the cumulative score was predictive of subsequent coronary artery disease.

Since then, dose responses have been defined for these and other factors and it has been suggested that almost 300 factors place patients at risk; these factors include elevations in plasma homocysteine.
 Recent studies shed interesting light on the mechanism of this potentially causal relationship, which was first noted in 1969.

Aside from putative effects on vessel wall dynamics, there is now direct evidence that homocysteine is atherogenic. Twenty-fold increases in plasma homocysteine achieved by dietary manipulation of apoE–/– mice increased aortic root lesion size 2-fold and produced a prolonged chronic inflammatory mural response accompanied by elevations in vascular cell adhesion molecule-1 (VCAM) and tumor necrosis factor-a (TNF-a).

In long term followup, homocysteine levels elevated by

  • dietary supplementation with methionine or homocysteine
  • promoted lesion size and plaque fibrosis in these
  • atherosclerosis-prone mice early in life, but without influencing ultimate plaque burden as the animals aged.

A number of mechanisms were proposed by which homocysteine achieved this effect, including

  • promotion of inflammation,
  • regulation of lipoprotein metabolism, and
  • modification of critical biochemical pathways and
  • metabolites including nitric oxide (NO).

See p 2569
In the present issue of Circulation,

Stühlinger et al 7 advance these mechanistic insights one critical step further by defining homocysteine’s effects at an enzymatic level.

The group led by Lentz published an association between levels of the

  • endogenous inhibitor of Nirtic Oxide synthase,
  • asymmetric dimethyl arginine (ADMA), and
  • homocysteine in cultured endothelial cells and in the serum of cynomolgus monkeys.

Such an association is interesting because the L-arginine–NO synthase pathway seems to be a critical component in the full range of endothelial cell biology and vascular dysfunction.

Stühlinger et al 7  now show that increased cultured endothelial cell elaboration of ADMA by homocysteine and its precursor L-methionine is associated with a dose-dependent impairment of the activity of endothelial dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades ADMA. Homocysteine directly inhibited DDAH activity in a cell-free system by targeting a critical sulfhydryl group on this enzyme.

Thus, one could envision that the balance of cardiovascular health and disease could well be determined by the ability of an intact Nirtic Oxide synthase system to overcome environmental, dietary, and even genetic factors.

In patients with altered enzymatic defense systems,

  • elevated homocysteine,
  • oxidized lipoproteins,
  • inflammation, and other
  • vasotoxins

may dominate even the most potent defense mechanisms.
These studies raise a number of issues.
Do we need to add to our list of established cardiovascular risk factors to accommodate new findings and associations?
Is there a final common pathway for all risk factors or perhaps even a unified factor theory into which all potential risks can be grouped?
And, as always, should we consider Nirtic Oxide at the core of this universality?
Finally, should we change our focus altogether and speak not of risk factors but of

  • genetic predisposition,
  • extent of biochemical aberration, and
  • degree of physical damage?

Some would view these remarkable success stories and the repeated association of hyperhomocyst(e)inemia with coronary, cerebral, and peripheral vascular disease and simply advocate for increased folic acid intake for all.

Indeed, this intervention of negligible cost and

  • insignificant side effect is already partially in place;
  • many foods are fortified with folate to prevent congenital neural tube defects.

This reader considers the seminal work by Vernon Young and Yves Ingenbleek on the relationship between

  • S8 and regions distant from lava flows in Asia and Indian subcontinents,
  • where they have determined hyperhomocysteinemia and the consequence associated with:
  • veganism (not voluntary)
  • impaired methyl donor reactions and transsulfuration pathways (not corrected by B12, folate)
  • loss of lean body mass due to the constant relationship of S:N (insufficient from plant sources)

What happens, when we fail to continue to pursue causality,

  • the linkage of biological significance or scientific plausibility with
  • epidemiologically or statistically significant association?

In medicine, risk becomes the likelihood that people without a disease will acquire the disease through contact with factors thought to increase disease risk.

All of these risk factors are then, by nature, imprecise and nonspecific.
 They are stochastic measures of what will happen to normal people who fall into particular measures of these parameters.

The daring may be willing to accept these risks, citing friend and foe who live well beyond or for far lesser times than anticipated by risk alone. Such concerns may well become moot if we can simultaneously identify patients at risk

  • by linking phenotype with genotype,
  • gene expression with protein elaboration, and
  • environmental exposures with the biochemical consequences and
  • direct anatomic aberrations they induce.

This kind of characterization may well replace a family history of arterial disease as a rough estimate of

  • genotype,
  • serum cholesterol as an indirect measure of the health of lipoprotein metabolism,
  • serum glucose as a crude determinant of the ravages of diabetes mellitus,
  • blood pressure measurement as a marker of long-standing endogenous exposure to altered flow, and
  • tobacco abuse as a maker of long-standing exposure to exogenous toxins.

Rather than identifying patients on the basis of their serum cholesterol, we will have a direct measure of their

  • LDL receptor number,
  • internalization rate,
  • macrophage content in the blood vessel wall,
  • metalloproteinase activity, etc.
  • insulin receptor metabolism,
  • oxidative state, and
  • glycated burden.
  • Serum glucose will similarly give way to these tests

Evaluating a new way to open clogged arteries: Computational model offers insight into mechanisms of drug-coated balloons.

A new study from MIT analyzes the potential usefulness of a new treatment that combines the benefits of angioplasty balloons and drug-releasing stents, but may pose fewer risks. With this new approach, a balloon is inflated in the artery for only a brief period, during which it releases a drug that prevents cells from accumulating and clogging the arteries over time.
While approved for limited use in Europe, these drug-coated balloons are still in development in the United States and have not received FDA approval. The MIT study, which models the behavior of the balloons, should help scientists optimize their performance and aid regulators in evaluating their effectiveness and safety.
“Until now, people who evaluate such technology could not distinguish hype from promise,” says Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology and senior author of the paper describing the study, which appeared online recently in the journal Circulation.
Lead author of the paper is Vijaya Kolachalama, a former MIT postdoc who is now a principal member of the technical staff at the Charles Stark Draper Laboratory.
Edelman’s lab is investigating a possible alternative to the current treatments: drug-coated balloons. “We’re trying to understand how and when this therapy could work and identify the conditions in which it may not,” Kolachalama says. “It has its merits; it has some disadvantages.”

Modeling drug release

The drug-coated balloons are delivered by a catheter and inflated at the narrowed artery for about 30 seconds, sometimes longer. During that time, the balloon coating, containing a drug such as Zotarolimus, is released from the balloon. The properties of the coating allow the drug to be absorbed in the body’s tissues. Once the drug is released, the balloon is removed.
In their new study, Kolachalama, Edelman and colleagues set out to rigorously characterize the properties of the drug-coated balloons. After performing experiments in tissue grown in the lab and in pigs, they developed a computer model that explains the dynamics of drug release and distribution. They found that factors such as the size of the balloon, the duration of delivery time, and the composition of the drug coating all influence how long the drug stays at the injury site and how effectively it clears the arteries.
One significant finding is that when the drug is released, some of it sticks to the lining of the blood vessels. Over time, that drug is slowly released back into the tissue, which explains why the drug’s effects last much longer than the initial 30-second release period.
“This is the first time we can explain the reasons why drug-coated balloons can work,” Kolachalama says. “The study also offers areas where people can consider thinking about optimizing drug transfer and delivery.”

http://circ.ahajournals.org/content/127/20/2047.short  
http://www.mit.edu/people/vbk/Circulation_2013.pdf 
http://www.sciencedaily.com/…13/05/130521121513.ht…    
Circulation, 2013; 127 (20): 2047 – 2055
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.002051;

 

Conclusion

MIT’s Edelman’s Lab conducted the pioneering work in Vascular biology, animal models of drug eluting stents and was at the forefront of Empirical Molecular Cardiology in its studies in vascular physiology, biology and biomaterials for medical devices.

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