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.
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 S, Srivastava D, Olson EN, DiMaio JM, Bock-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 N, Risebro CA, Clark JE, Ehler E, Miquerol L, Rossdeutsch A, Marber MS, Riley 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 N, Bollini S, Dubé KN, Vieira JM, Zhou B, Davidson S, Yellon D, Riegler J, Price AN, Lythgoe MF, Pu WT, Riley 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.
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.
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.”
“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.
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.”
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.
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.
Pluripotent stem cell-based heart regeneration: from the developmental and immunological perspectives.
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 Moretti, Leslie L Caron, Atsushi A Nakano, Jason T JT Lam, Alexandra A Bernshausen,Yinhong Y Chen, Yibing Y Qyang, Lei L Bu, Mika M Sasaki, Silvia S Martin-Puig, Yunfu Y Sun, Sylvia M SM Evans, Karl-Ludwig KL Laugwitz, Kenneth 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.