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Posts Tagged ‘cardiac muscle repair’


Regulation of mesenchymal cell generation

 

Curator: Larry H. Bernstein, MD, FCAP

from Butyrov

LPBI

 

 

Controlling Mesenchymal Stem Cell Activity With Microparticles Loaded With Small Molecules

M. Butyrov      Beyond the Dish

Mesenchymal stem cells are the subject of many clinical trials and show a potent ability to down-regulate unwanted immune responses and quell inflammation. A genuine challenge with mesenchymal stem cells (MSCs) is controlling the genes they express and the proteins they secrete.

A new publication details the strategy of one enterprising laboratory to control MSC function. Work by Jeffery Karp from the Harvard Stem Cell Institute and Maneesha Inamdar from the Institute for Stem Cell Biology and Regenerative Medicine in Bangalore, India and their colleagues had use microparticles that are loaded with small molecules and are readily taken up by cultures MSCs.

In this paper, which appeared in Stem Cell Reports (DOI:http://dx.doi.org/10.1016/j.stemcr.2016.05.003), human MSCs were stimulated with a small signaling protein called Tumor Necrosis Factor-alpha (TNF-alpha). TNF-alpha makes MSCs “angry” and they pour out pro-inflammatory molecules upon stimulation with TNF-alpha. However, to these TNF-alpha-stimulated, MSC, Karp and others added tiny microparticles loaded with a small molecule called TPCA-1. TPCA-1 inhibits the NF-κB signaling pathway, which is one of the major signal transduction pathways involved in inflammation.

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Delivery of these TPCA-1-containing microparticles thinned-out the production of pro-inflammatory molecules by these TNF-alpha-treated MSCs for at least 6 days. When the culture medium from TPCA-1-loaded MSCs was given to different cell types, the molecules secreted by these cells reduced the recruitment of white blood cells called monocytes. This is indicative of the anti-inflammatory nature of TPCA-1-treated MSCs. The culture medium from these cells also prevented the differentiation of human cardiac fibroblasts into collagen-making cells called “myofibroblasts.” Myofibroblasts lay down the collagen that produces the heart scar after a heart attack. This is a further indication of the anti-inflammatory nature of the molecules made by these TPCA-1-treated MSCs.

These results are important because it shows that MSC activities can be manipulated without gene therapy. It is possible that such non-gene therapy-based approached can be used to fine-tune MSC activity and the types of molecules secreted by implanted MSCs. Furthermore, given the effect of these cells on monocytes and cardiac fibroblasts, perhaps microparticle-treated MSCs can prevent the adverse remodeling that occurs in the heart after a heart attack.

 

Controlled Inhibition of the Mesenchymal Stromal Cell Pro-inflammatory Secretome via Microparticle Engineering

Sudhir H. Ranganath, Zhixiang Tong, Oren Levy, Keir Martyn, Jeffrey M. Karpcorrespondence, Maneesha S. Inamdar
Stem Cell Reports June 2016; Volume 6 (Issue 6): 926–939    http://dx.doi.org/10.1016/j.stemcr.2016.05.003
Mesenchymal stromal cells (MSCs) are promising therapeutic candidates given their potent immunomodulatory and anti-inflammatory secretome. However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. To address this, we used a microparticle-based engineering approach to non-genetically modulate pro-inflammatory pathways in human MSCs (hMSCs) under simulated inflammatory conditions. Here we show that microparticles loaded with TPCA-1, a smallmolecule NF-kB inhibitor, when delivered to hMSCs can attenuate secretion of pro-inflammatory factors for at least 6 days in vitro. Conditioned medium (CM) derived from TPCA-1-loaded hMSCs also showed reduced ability to attract human monocytes and prevented differentiation of human cardiac fibroblasts to myofibroblasts, compared with CM from untreated or TPCA-1-preconditioned hMSCs. Thus, we provide a broadly applicable bioengineering solution to facilitate intracellular sustained release of agents that modulate signaling. We propose that this approach could be harnessed to improve control over MSC secretome post-transplantation, especially to prevent adverse remodeling post-myocardial infarction.
Mesenchymal stromal cells (MSCs; also known as bone marrow stromal cells and earlier known as mesenchymal stem cells) are being explored as therapeutics in over 550 clinical trials registered with the US Food and Drug Administration (www.FDA.gov) for the treatment of a wide range of diseases (Ankrum et al., 2014c). Their immuneevasive properties (Ankrum et al., 2014c) and safe transplant record, allowing allogeneic administration without an immunosuppressive regimen, positions MSCs as an appealing candidate for a potential off-the-shelf product. One of the primary mechanisms exploited in MSC therapeutics is a secretome-based paracrine effect as evidenced in many pre-clinical studies (Ranganath et al., 2012). However, controlling the MSC secretome post-transplantation is considered a major challenge that hinders their clinical efficacy. For instance, upon transplantation, MSCs are subjected to a complex inflammatory milieu (soluble mediators and immune cells) in most injury settings. MSCs not only secrete anti-inflammatory factors, but also produce pro-inflammatory factors that may compromise their therapeutic efficacy. Table S1 lists a few in vitro and in vivo conditions that demonstrate the complex microenvironment under which MSCs switch between anti-inflammatory and pro-inflammatory phenotypes.
Levels of pro- or anti-inflammatory cytokines are not always predictive of the response, possibly due to the dynamic cytokine combinations (and concentrations) present in the cell microenvironment (Table S1). For example, relatively low inflammatory stimulus (<20 ng/ml tumor necrosis factor alpha [TNF-a] alone or along with interferon-g [IFN-g]) can polarize MSCs toward pro-inflammatory effects (Bernardo and Fibbe, 2013) resulting in increased inflammation characterized by T cell proliferation and transplant rejection. Conversely, exposure to high levels of the inflammatory cytokine TNF-a has been shown in certain studies to result in MSC-mediated anti-inflammatory effects via secretion of potent mediators such as TSG6, PGE2, STC-1, IL-1Ra, and sTNFR1 as demonstrated in multiple inflammation-associated disease models (Prockop and Oh, 2012; Ylostalo et al., 2012). These effects are mediated via molecular pathways such as NF-kB, PI3K, Akt, and JAK-STAT (Ranganath et al., 2012). However, it is not clear that low and high levels of TNF-a always exert the same effect on anti- versus pro-inflammatory MSC secretome. NF-kB is a central regulator of the anti-inflammatory secretome response in monolayer (Yagi et al., 2010), spheroid MSCs (Bartosh et al., 2013; Ylostalo et al., 2012) and TNFa-mediated (20 ng/ml for 120 min) apoptosis (Peng et al., 2011). Given that NF-kB can promote secretion of proinflammatory components in the MSC secretome (Lee et al., 2010), we hypothesized that NF-kB inhibition via small molecules in MSC subjected to a representative in- flammatory stimulus (10 ng/ml TNF-a) would inhibit their pro-inflammatory responses.
Adverse remodeling or cardiac fibrosis due to differentiation of cardiac fibroblasts (CF) into cardiac myofibroblasts (CMF) with pro-inflammatory phenotype and collagen deposition is the leading cause for heart failure. The secretome from exogenous MSCs has anti-fibrotic and angiogenic effects that can reduce scar formation (Preda et al., 2014) and improve ejection fraction when administered early or prior to adverse remodeling (Preda et al., 2014; Tang et al., 2010; Williams et al., 2013). Unfortunately in many cases, due to poor prognosis, MSCs may not be administered in time to prevent adverse remodeling to inhibit CF differentiation to CMF (Virag and Murry, 2003) or to prevent myocardial expression of TNF-a (Bozkurt et al., 1998; Mann, 2001). Also, when administered following an adverse remodeling event including CMF differentiation, MSCs may assume a pro-inflammatory phenotype and secretome (Naftali-Shani et al., 2013) under TNF-a (typically 5 pg/mg of total protein in myocardial infarction (MI) rat myocardium which is not significantly higher than 1–3 pg/mg protein in control rat myocardium) (Moro et al., 2007) resulting in impaired heart function. Hence, if the pro-inflammatory response of hMSCs can be suppressed it may maximize efficacy.
While the MSC phenotype can be controlled under regulated conditions in vitro, the in vivo response of MSCs post-transplantation is poorly controlled as it is dictated by highly dynamic and complex host microenvironments (Discher et al., 2009; Rodrigues et al., 2010). Factors including MSC tissue source (Melief et al., 2013; NaftaliShani et al., 2013), donor and batch-to-batch variability with respect to cytokine secretion and response to inflammatory stimuli (Zhukareva et al., 2010), gender (Crisostomo et al., 2007), and age (Liang et al., 2013) also affect the response of MSCs. Thus, it is important to develop approaches to control the MSC secretome post-transplantation regardless of their source or expansion conditions. We hypothesized that engineering MSCs to induce a specific secretome profile under a simulated host microenvironment may maximize their therapeutic utility. Previously, the MSC secretome has been regulated via genetic engineering (Gnecchi et al., 2006; Wang et al., 2009) or cytokine/small-molecule preconditioning approaches (Crisostomo et al., 2008; Mias et al., 2008). Genetically engineered human MSCs (hMSCs) pose challenging long-term regulatory hurdles given that the potential tumorigenicity has not been well characterized, and while preconditioning hMSCs with cytokines/small molecules may be safer, the phenotype-altering effects are transient. …
While TPCA-1 pretreatment of hMSCs before TNF-a stimulation showed a significant reduction in CMF numbers, this was greatly enhanced when TPCA-1 was available intracellularly via the microparticles. CM from TNF + TPCAmP-hMSC likely prevented differentiation of CF to CMF due to the continued intracellular inhibition of IKK-mediated NF-kB activation, thus preventing the release of an hMSC pro-inflammatory secretome. Surprisingly, the CMF number was reduced by over 2-fold, suggesting that TPCA-1 may activate hMSC pathways that revert the CMF phenotype. We cannot rule out the possibility that other contributors in the hMSC secretome that were not profiled might also be contributing, and their action is facilitated by intracellular TPCA-1. For instance, in an in vitro 3D model of cardiac fibrosis under hypoxic conditions, reversal of CMF to the CF phenotype was shown to be due to reduced MSC TGF-b levels (Galie and Stegemann, 2014). Our assay revealed a similar trend in terms of collagen production from CF. CF treated with CM from control-hMSCs, or TPCApre + TNF-hMSCs or mP-hMSCs secreted elevated levels of collagen into the media suggesting that only inhibited levels of pro-inflammatory mediators could not be implicated in the reduction in collagen secretion. The reduced number of a-SMA+ CMF in CM from TNF + TPCAmP-hMSCs possibly contributed to the lower secretion level of collagen in the media (Figure 5B). Upon dedifferentiation or lowered a-SMA expression, it is possible that CMFs lose collagen secretion ability. In regions of MI, CF switch to the myofibroblast phenotype due to stress from the infarct scar (Tomasek et al., 2002). High expression of a-SMA typical in such CMF (Teunissen et al., 2007) has been implicated for remodeling due to their high contractility (Santiago et al., 2010). In addition, the collagen secretion capacity of CMF is very high (Petrov et al., 2002). Overall, attenuation in the number of collagen-secreting a-SMA+ CMF could be beneficial in preventing pathological remodeling or irreversible scar formation and allowing cardiac regeneration.
Here we have demonstrated that the pro-inflammatory hMSC secretome could be inhibited using a microparticle engineering approach delivering an intracellular NF-kB inhibitor, TPCA-1. It is however important to note that the MSC secretome composition may change depending on the level of TNF-a encountered in vivo following transplantation. Nevertheless, a similar approach could be beneficial in inflammatory disease settings such as chronic inflammation and macrophage-mediated atherosclerosis. The approach of microparticle engineering of an exogenous cell population by modulating a central regulatory pathway, may find application in other cell types and pathways and could provide an attractive strategy for harnessing any cell secretome for therapy. This approach could also be potentially employed to modulate the composition of extracellular vesicles (exosomes) for therapy.
Stem Cell Reports j Vol. 6 j 926–939 j June 14, 2016
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Resident-cell-based Therapy in Human Ischaemic Heart Disease: Evolution in the PROMISE of Thymosin beta4 for Cardiac Repair

Curator: Aviva Lev-Ari, PhD, RN

2012 – STATE OF PHARMACEUTICAL RESEARCH

Scientists Report that Process of Converting Non-Beating Heart Cells into Functional, Beating Heart Cells is Enhanced Using Thymosin Beta 4 in Conjunction with Gene Therapy

ROCKVILLE, Md.–(BUSINESS WIRE)–Apr. 18, 2012– Regenerx Biopharmaceuticals, Inc. (OTC Bulletin Board: RGRX) (“the Company” or “RegeneRx”) announced today that scientists at the Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, have published new animal data in the current issue of Nature showing that the process of converting non-beating heart cells (which normally form scar tissue after a heart attack), into functional, beating heart muscle cells can be enhanced using Thymosin beta 4 (Tβ4). Delivery of Tβ4, in conjunction with GMT (an acronym for three genes that normally guide embryonic heart development), into the damaged region resulted in reduction of scar area and improvement in cardiac function compared to GMT or Tβ4 alone. Within a month, non-beating cells that normally form scar tissue transformed into beating heart-muscle cells. Within three months, the hearts were beating even stronger and pumping more blood.

“Our experiments in mice are a proof of concept that we can reprogram non-beating cells directly into fully functional, beating heart cells – offering an innovative and less invasive way to restore heart function after a heart attack,” stated Dr. Deepak Srivastava, who directs cardiovascular and stem cell research at Gladstone and a member of RegeneRx’s scientific advisory board.

“These findings could have a significant impact on heart-failure patients whose damaged hearts make it difficult for them to engage in normal activities like walking up a flight of stairs,” said Dr. Li Qian, PhD, who is a postdoctoral scholar at Gladstone and a member of Dr. Srivastava’s research team. “This research may result in a much needed alternative to heart transplants for which donors are extremely limited. And because we are reprogramming cells directly in the heart, we eliminate the need to surgically implant cells that were created in a petri dish,” he further commented.

According to the Institute news release, “The results have broad human health implications” and are a “medical breakthrough [that] holds promise for millions with heart failure.”

The results are described in the latest issue of Nature, available online today.

About RegeneRx Biopharmaceuticals, Inc. (www.regenerx.com)

RegeneRx is focused on the development of a novel therapeutic peptide, Thymosin beta 4, or Tβ4, for tissue and organ protection, repair and regeneration. RegeneRx currently has three drug candidates in Phase 2 clinical development and has an extensive worldwide patent portfolio covering its products.

2012 –  STATE OF SCIENCE: MOLECULAR CELL CARDIOLOGY
Recent research by Zhou BHonor LBMa QOh JHLin RZMelero-Martin JMvon Gise AZhou PHu THe LWu KHZhang HZhang YPu WT of the Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China concluded that Thymosin beta 4 treatment after myocardial infarction does not reprogram epicardial cells into cardiomyocytes J Mol Cell Cardiol. 2012 Jan;52(1):43-7. Epub 2011 Aug 26.
Kispert A. commented:“No muscle for a damaged heart: thymosin beta 4 treatment after myocardial infarction does not induce myocardial differentiation of epicardial cells.” http://www.ncbi.nlm.nih.gov/pubmed?term=Kispert%20A.
2012 – 2003 MILESTONES IN THE EVOLUTION OF THE PROMISE OF THYMOSIN beta4 FOR CARDIAC REPAIR

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal–relative to cell transplantation–would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury, indicating that  an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Shrivastava SSrivastava DOlson ENDiMaio JMBock-Marquette I. of Department of Cardiovascular and Thoracic Surgery, University of Texas, Southwestern Medical Center, Dallas, Texas, USA in Ann N Y Acad Sci. 2010 Apr;1194:87-96. asserted that Tbeta4 to be the first known molecule able to initiate simultaneous myocardial and vascular regeneration.

Another a study by Smart NRisebro CAClark JEEhler EMiquerol LRossdeutsch AMarber MSRiley PR, of the Molecular Medicine Unit, UCL Institute of Child Health, London, UK. concluded that Thymosin beta4 facilitates epicardial neovascularization of the injured adult heart, Ann N Y Acad Sci. 2010 Apr;1194:97-104

Additional research on De novo cardiomyocytes from within the activated adult heart after injury by Smart NBollini SDubé KNVieira JMZhou BDavidson SYellon DRiegler JPrice ANLythgoe MFPu WTRiley PR. Molecular Medicine Unit, UCL Institute of Child Health, London,Nature. 2011 Jun 8;474(7353):640-4 . They demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. They reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm’s tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury.

 In search for new strategies to repair and/or regenerate the myocardium after ischemia and infarction in order to prevent maladaptive remodeling and cardiac dysfunction, Cavasin MA, of the Hypertension and Vascular Research Division, Henry Ford Health System, Detroit, Michigan, in Therapeutic potential of thymosin-beta4 and its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) in cardiac healing after infarction, Am J Cardiovasc Drugs. 2006;6(5):305-11 compiles and analyzes the available experimental data regarding the potential therapeutic effects of thymosin-beta4 and its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) in cardiac healing after myocardial infarction (MI) as well as discussing the possible mechanisms involved. More recently it has been shown that thymosin-beta4 facilitates cardiac repair after infarction by promoting cell migration and myocyte survival. Additionally, the tetrapeptide Ac-SDKP was reported to reduce left ventricular fibrosis in hypertensive rats, reverse fibrosis and inflammation in rats with MI, and stimulate both in vitro and in vivo angiogenesis. Ac-SDKP also reduced cardiac rupture rate in mice post-MI. Some of the effects of Ac-SDKP, such as the enhancement of angiogenesis and the decrease in inflammation and collagenase activity, are similar to those described for thymosin-beta4. Thus, it is possible that Ac-SDKP could be mediating some of the beneficial effects of its precursor. Although the experimental evidence is very promising, there are no data available from a clinical trial supporting the use of thymosin-beta(4) or Ac-SDKP as means of healing the myocardium after MI in patients.
EXPLORATIONS with THYMOSIN beta4 FOR INDUCING ADULT EPICARDIAL PROGENETOR MOBILIZATION AND NEOVASCULARIZATION
 
Smart et al. (2007) implicate Thymosine beta4 (Tb4) with the following functions: (a) Tb4 in regulating all three key stages of cardiac vessel development: coronary vasculogenesis, angiogenesis and arteriogenesis – collateral growth; (b) identify the adult epicardium as a potential source of vascular progenitors which, when stimulated by Tb4, migrate and differentiate into smooth muscle and endothelial cells; (c) the ability of Tb4 to promote coronary vascularization both during development and in the adult, enhances cardiomyocyte survival and contributes significantly towards Tb4-induced cardioprotection.

Nature, 2007, 445, 177-182.

The reaction in the scientific community to these investigative results was most favorable.

“These results are very exciting because most humans suffering from ischemic cardiac events, either acutely or chronically, do not develop the collateral vessel growth necessary to preserve and restore heart tissue. If, in humans, we see the same effects as seen in mice, TB4 would be the first drug to prevent loss of (heart) muscle cells and restore blood flow in this manner and provide a new and much needed treatment modality for these patients,”

commented Deepak Srivastava, M.D., Professor and Director, Gladstone Institute of Cardiovascular Disease, University of California San Francisco, CA. Dr. Srivastava and his colleagues published the first paper on TB4’s effects on myocardial infarction in Nature in November 2004.

http://phx.corporate-ir.net/phoenix.zhtml?c=144396&p=irol-newsArticle&ID=932573&highlight=

 Dr. Paul Riley, Institute of Child Health, University College London, Research Lead on the Smart et al. (2007) article in Nature, said: “In 2006, through (British Heart Foundation) BHF-funded work, we discovered that a protein called Thymosin beta4 could mobilise dormant cells from the epicardium to form new blood vessels in the heart. This is a major step towards finding a DIY repair mechanism to repair injury following heart attack.”

http://www.ich.ucl.ac.uk/pressoffice/pressrelease_00498

 “To investigate whether Thymosin beta 4 could have a therapeutic effect on damaged adult hearts, my research team took cells from the outermost layer of adult mice and grew them in the lab. We found that, when treated with the protein, these adult cells have as much potential as embryonic cells to create healthy heart tissue. This suggests that the protein could have a therapeutic use,” explained lead researcher, Dr. Paul Riley. Furthermore, “current treatments for a damaged heart are limited by the ability of the adult tissue to respond. By using this protein to guide progenitor cells from the outer layer of the heart, to form new blood vessels and nourish tissue, it could be possible to better repair damaged adult hearts.”

“Our research has shown that blood vessel regeneration is still possible in the adult heart. In the future, if we can figure out how to direct the progenitor cells using Thymosin beta 4, there could be potential for therapy based on the patients’ own heart cells”, Dr. Riley explained. He said that this process has the added benefit in that the cells are already located in the right place – within the heart itself.

“All these cells need is the appropriate instructions to guide them towards new blood vessel formation that will help in the repair of muscle damage following a heart attack”, Dr. Riley added.

http://www.irishhealth.com/clin/cholesterol/newsstory.php?id=10581

Professor Jeremy Pearson, BHF associate medical director, said: “These results are important and exciting.” By identifying for the first time a molecule that can cause cells in the adult heart to form new blood vessels, Dr. Riley’s group have taken a large step towards practical therapy to encourage damaged hearts to repair themselves, a goal that researchers are urgently aiming for.” Here, we target pharmaco-therapy for their discovery.

Professor Colin Blakemore, MRC chief executive, said: ”Finding out how this protein helps to heal the heart offers enormous potential in fighting heart disease, which kills more than 105,000 people in the UK every year.”

http://news.bbc.co.uk/2/hi/health/6143286.stm

Philip et al., (2003) reported that Thymosin beta4 is angiogenic and can promote endothelial cell migration and adhesion, tubule formation, aortic ring sprouting, and angiogenesis. It also accelerates wound healing and reduces inflammation when applied in dermal wound-healing assays. Using naturally occurring Thymosin beta4, proteolytic fragments, and synthetic peptides, they found that a seven amino acid actin binding motif of Thymosin beta4 was essential for its angiogenic activity. Migration assays with human umbilical vein endothelial cells and vessel sprouting assays using chick aortic arches showed that Thymosin beta4 and the actin-binding motif of the peptide display near-identical activity at ~50 nM, whereas peptides lacking any portion of the actin motif were inactive. Furthermore, adhesion to Thymosin beta4 was blocked by this seven amino acid peptide demonstrating it as the major Thymosin beta4 cell binding site on the molecule. The adhesion and sprouting activity of Thymosin beta4 was inhibited with the addition of 5-50 nM soluble actin. These results demonstrate that the actin binding motif of Thymosin beta4 is an essential site for its angiogenic activity. FASEB Journal,2003, published on line 9/18/2003. Retrieved 3/1/2007, FASEB Journal,2007.

Smart et al. (2007) describe the mechanism by which Thymosin beta4 stimulates coronary vessel development which in this regard involves Thymosin beta4 directly promoting Epicardium-Derived Cells (EPDC) migration from the epicardium via its previously known function of actin binding, filament assembly and lamellipodia formation. Thymosin beta4 is presented in their Nature article as a single factor that can potentially couple myocardial and coronary vascular regeneration in failing mouse hearts. A major shortcoming of current angiogenic therapy in response to myocardial ischaemia in humans is that the outcome may be limited to capillary growth without concomitant collateral support of arterioles. Smart et al. (2007) findings that, in mice, Thymosin beta4 can promote vessel formation and collateral growth not only during development but also critically from adult epicardium, suggest Thymosin beta4 has considerable therapeutic potential in humans. They revealed the mechanism by which Thymosin beta4 may act to promote cardiomyocyte survival following acute myocardial damage in mice and identify the biopeptide AcSDKP as a small molecule that potentially offers further protection following cardiac injury. Nature, 2007, 445, 177-182.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) stimulates endothelial cell differentiation from adult epicardium

The first report demonstrating the ability of AcSDKP to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo was reported by Liu, et al., (2003). A novel biologic function of AcSDKP, is its function as a mediator of angiogenesis, as measured by its ability to modulate endothelial cell function in vitro and angiogenesis in vivo. The tetrapeptide acetyl-Ser-Asp-Lys-Pro (AcSDKP), purified from bone marrow and constitutively synthesized in vivo, belongs to the family of negative regulators of hematopoiesis. It protects the stem cell compartment from the toxicity of anticancer drugs and irradiation and consequently contributes to a reduction in marrow failure. AcSDKP at nanomolar concentrations stimulates in vitro endothelial cell migration and differentiation into capillary-like structures on Matrigel as well as enhances the secretion of an active form of matrix metalloproteinase-1 (MMP-1). In vivo, AcSDKP promotes a significant angiogenic response in the chicken embryo chorioallantoic membrane (CAM) and in the abdominal muscle of the rat. Moreover, it induces the formation of blood vessels in Matrigel plugs implanted subcutaneously in the rat (Liu, et al., 2003). Blood,2003, 101 (8), 3014-3020

Wang et al., (2004) reported three findings, that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) (a) stimulated endothelial cell proliferation and migration and tube formation in a dose-dependent manner, (b) enhanced corneal neovascularization, and (c) increased myocardial capillary density. Endothelial cell proliferation and angiogenesis stimulated by Ac-SDKP could be beneficial in cardiovascular diseases such as hypertension and MI. Furthermore, they wrote, because Ac-SDKP is mainly cleaved by ACE, it may partially mediate the cardioprotective effect of ACE inhibitors. .” Am J Physiol Heart Circ Physiol., 2004, 287, H2099-H2105.

Fleming (2006) reports that recent evidence suggests that some of the beneficial effects of ACE inhibitors on cardiovascular function and homeostasis can be attributed to novel mechanisms. These include the accumulation of the ACE substrate N-acetyl-seryl-aspartyl-lysyl-proline, which blocks collagen deposition in the injured heart, as well as the activation of an ACE signaling cascade that involves the activation of the kinase CK2 and the c-Jun N-terminal kinase in endothelial cells and leads to changes in gene expression. Circulation Research,2006, 98, 887.

Waeckel et al. (2006) report the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lees-Pro (AcSDKP) in a model of surgically induced hind limb ischemia. AcSDKP stimulated MCP-1 mRNA and protein levels in cultured endothelial cells and ischemic tissue. AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway. Arteriosclerosis, Thrombosis, and Vascular Biology,2006, 26, 773

Smart et al. (2007) in Nature, identified the biopeptide AcSDKP as a small molecule that potentially offers further protection following cardiac injury. Their research on AcSDKP, continue the work of Fleming (2006), Liu et al. (2003), Waeckel at al. (2006), Wang et al. (2004) and references 19 to 25 in Smart (2007). In their paper they report that scope exists for a non actin-mediated vasculo-, angio- and arteriogenic function for Thymosin beta4 by virtue of its endoproteinase activity to produce the pro-angiogenic tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP). They therefore quantified AcSDKP levels in their mutant knockdown hearts by competitive enzyme immunoassay on extracted myocardium, and found they were decreased to 62% and 60%, respectively, of that of controls.

They concluded that (a) this is robust evidence for a peptide and precursor peptide relationship between Thymosin beta4 and AcSDKP in a physiological setting. They investigated whether AcSDKP could rescue any of the vasculogenesis defects observed in the Thymosin beta4 mutant hearts, and (b) stated that their results support their interpretation of the primary phenotype in Thymosin beta4 mutants. AcSDKP lacks actin binding function, rendering it unable to stimulate filamentous actin assembly and lamellipodia-based cell migration and consequently unable to rescue the Epicardium-Derived Cells (EPDC) defect.

However, in the adult, consistent with reported cardioprotective effects of AcSDKP (refs 23–25 in Smart et al. (2007)), they observed a significant upregulation in levels of both endogenous Thymosin beta4 and AcSDKP in response to ischaemia after 1 day and 1 week. They reported that addition of AcSDKP to adult epicardial explants resulted in a striking increase in differentiated (Flk1-positive) endothelial cells. Although unable to promote epicardial outgrowth beyond control levels, AcSDKP brought about rapid differentiation of any emerging EPDCs. The differentiated cells were almost exclusively endothelial, with only very few smooth muscle cells observed in AcSDKP-treated cultures.  They stated  that it suggests that cleavage of AcSDKP from Thymosin beta4 exclusively promotes EPDC endothelial cell differentiation, and may underlie a compound vasculogenic effect of Thymosin beta4 aside from simply promoting EPDC migration into overlying myocardium as an instructive cue for differentiation.

Lastly, they concluded that crucial to the further understanding of this two-step function will be the identification of the respective receptors for Thymosin beta4 and AcSDKP. Receptors for Thymosin beta4 and AcSDKP will promote research activity for drug discovery.

Leading drug developer of synthetic Thymosin beta4 is RegeneRx Biopharmaceuticals, Inc. They are developing Thymosin beta4, a 43 amino acid peptide as a potential therapeutic target in part, under an exclusive world-wide license from the National Institutes of Health. RegeneRx holds nearly 60 world-wide patents and patent applications related to dermal, ocular, and internal wounds and tissue repair, cardiac and neurological injuries, septic shock and several consumer product areas. RegeneRx is currently sponsoring three Phase 2 chronic dermal wound healing clinical trials and has additionally targeted ophthalmic and cardiac trials in 2007 as part of its ongoing clinical development program. J.J. Finkelstein is RegeneRx’s president and chief executive officer.

http://phx.corporate-ir.net/phoenix.zhtml?c=144396&p=irol-newsArticle&ID=953115&highlight=

Scientists Find Heart Stem Cells

by Constance Holden on 2 July 2009, 12:00 AM in http://news.sciencemag.org/sciencenow/2009/07/02-01.html

Scientists have identified a cardiac stem cell that gives rise to all of the major cell types in the human heart. The find opens the way to using patients’ own cells to heal their damaged hearts.

The cells in question express a protein, called Islet 1, which is present in the early stages of fetal heart formation. In recent years, scientists have identified the cells in embryonic mouse hearts. And now, a team in the laboratory of Kenneth Chien, director of the Cardiovascular Research Center at Massachusetts General Hospital in Boston, has found the same cell type in human fetal hearts.

Once the group pinpointed the cells, it took the next important step: generating new cardiac stem cells from human embryonic stem cells. Using fluorescent tags to identify the ones containing Islet 1, the researchers obtained a purified population. They then proved that the Islet 1 cells are what Chien calls “master stem cells” by showing that single cells could be made to grow into any of the heart’s major cell types: heart muscle (cardiomyocytes), smooth muscle, and blood vessel lining (endothelium). The team reports its work today in Nature.

Chien cautions that these primordial stem cells, which are found only in fetuses, could not be used for therapy because they could develop into undesired cell types. Instead, he says, researchers need to isolate “intermediate” cells that are already heading for a particular fate. In the meantime, the primordial cells could be used for disease modeling and drug screening. They may also help shed light on congenital heart malformations. In the fetal heart, Islet 1 cells are clustered in areas that are “hot spots” for congenital heart defects, says Chien: “Congenital heart disease may be a stem cell disease.”

Ultimately, researchers may be able to use the cells to grow human “heart parts” such as strips of heart muscle or a valve on scaffolds that could be inserted into patients, Chien says.

“The findings are very important if they can be reproduced,” says cardiologist Richard Schatz of Scripps Clinic in San Diego, California. But Eduardo Marbán, director of the Cedars-Sinai Heart Institute in Los Angeles, says he’s doubtful that the identification of Islet 1 cells will hasten new therapies. Marbán is currently heading a trial that involves removing a tiny chunk of heart tissue from a patient, cultivating cells from it, and reinjecting them into the patient’s heart. He says Islet 1 cells “do appear to be important in development” but that “normal heart tissue can and does form in the complete absence” of the protein.

Chien has a different view, saying that there’s little or no evidence that scientists can obtain stem cells by “grinding up hearts and culturing cells from them.” It’s important to identify authentic progenitor cells, he says, in order to identify cells that will help repair damaged hearts.

http://news.softpedia.com/newsImage/Stem-Cells-for-the-Hear-Found-2.jpg/

http://news.sciencemag.org/sciencenow/2009/07/02-01.html

http://www.readcube.com/articles/10.1038/nature08191

Pluripotent stem cell-based heart regeneration: from the developmental and immunological perspectives.

Kathy O KO LuiLei L BuRonald A RA LiCamie W CW Chan
Birth Defects Res A Clin Mol Teratol 96(1):98-108 (2012), PMID 22457181

Heart diseases such as myocardial infarction cause massive loss of cardiomyocytes, but the human heart lacks the innate ability to regenerate. In the adult mammalian heart, a resident progenitor cell population, termed epicardial progenitors, has been identified and reported to stay quiescent under uninjured conditions; however, myocardial infarction induces their proliferation and de novo differentiation into cardiac cells. It is conceivable to develop novel therapeutic approaches for myocardial repair by targeting such expandable sources of cardiac progenitors, thereby giving rise to new muscle and vasculatures. Human pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can self-renew and differentiate into the three major cell types of the heart, namely cardiomyocytes, smooth muscle, and endothelial cells. In this review, we describe our current knowledge of the therapeutic potential and challenges associated with the use of pluripotent stem cell and progenitor biology in cell therapy. An emphasis is placed on the contribution of paracrine factors in the growth of myocardium and neovascularization as well as the role of immunogenicity in cell survival and engraftment.

Multipotent Embryonic Isl1^+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Alessandra A MorettiLeslie L CaronAtsushi A NakanoJason T JT LamAlexandra A Bernshausen,Yinhong Y ChenYibing Y QyangLei L BuMika M SasakiSilvia S Martin-PuigYunfu Y SunSylvia M SM EvansKarl-Ludwig KL LaugwitzKenneth R KR Chien
Cell 127(6):15 (2006), PMID 17123592

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1^+ precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1^+ cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1^+/Nkx2.5^+/flk1^+ defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1^+ cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

http://pubget.com/paper/17123592/Multipotent_Embryonic_Isl1___Progenitor_Cells_Lead_to_Cardiac__Smooth_Muscle__and_Endothelial_Cell_Diversification

 

 

 

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