Angiogenesis is the normal physiological process by which new blood vessels and capillary beds sprout from preexisting vessels, resulting in the creation or expansion of a vascular network within a region of tissue. The construction and maintenance of architecture of blood vessels functions primarily to provide the hosting tissue, and those cells involved in its structure, with a means for importing those nutrients required for survival and maintenance, and removing unnecessary waste. Consequently, the angiogenic process is a fundamental component of embryonic growth and development, tissue repair and wound healing, the resolution of inflammatory responses, and the onset of neoplasia. The expansion of a vascular network is a relatively fragile process governed by a delicate balance between stimulatory and inhibitory factors, and is, therefore, highly susceptible to instances of disruptive interference at several levels. Occurrences of angiogenic perversion can result in pathological angiogenesis, which is characterized by the abnormally rapid and uncontrolled proliferation of blood vessels. Pathological angiogenesis is critical to the transitioning of a tumor to malignancy, and a contributing factor to a multitude of other diseases, including ischemic chronic wounds, cardiovascular disease, diabetic retinopathy, rheumatoid arthritis, macular degeneration, and psoriasis. Due to its involvement in such an array of diseases, the ability to manipulate angiogenesis, through both natural and synthetic inhibitors and activators, represents a promising prospect for the prevention and treatment of diseases characterized by abnormal vascularization (Ref. 1).

Regeneration, the process of regrowth of damaged tissues or organs in response to injury, is a property that  varies  among  organisms.  Organ  and  tissue  regeneration  promotes  survival,  longevity  and  optimal health (Ref. 2 & 3). In humans, many tissues, such as skin, liver, blood, and intestinal mucosa, routinely and efficiently undergoing regeneration.  One of  the least  regenerative organs  in the  human body  is the heart. The self-regenerative capacity of the heart is insufficient to make up for the cardiac muscle loss after an ischemic injury. This limitation has triggered interest in different stem cell-based therapies aimed at repairing failing human hearts. Heart disease is the leading cause of death in developed countries. Myocardial infarction (MI) and normal aging lead to loss of viable cardiomyocytes, which may lead to heart failure. This cardiomyocyte loss is generally considered irreversible. The goal of cardiovascular regeneration is repairing and restoring damaged heart tissues through innovative methods, including stem cell and gene therapy (Ref. 4). The process of cardiovascular regeneration enables reprogramming of cells that were terminally differentiated, which may provide an opportunity for developing  therapeutic  interventions (Ref. 5). Stem cell-based therapies are a potential alternative therapy for myocardial regeneration in patients  with ischemic heart disease. Many different types of stem cells, such as crude bone marrow mononuclear cells, mesenchymal stem cells, adult stem cells from adipose or cardiac tissue, and embryonic stem cells, have been used in clinical trials. The selection of a suitable cell type is the most important  criteria  for its successful application (Ref. 6 & 7).

Adult stem cells, especially mesenchymal stem cells, secrete a variety of growth factors, extracellular matrix (ECM) molecules, paracrine cytokines, and chemokines that are believed to play a major role in cardiac repair (Ref. 8). Under hypoxic conditions, stem cells can release growth factors and cytokines such as transforming growth factor (TGF)-b, interleukin (IL)-6, vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2 or basic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), angiopoietin (Ang)-1, stromal cell-derived factor (SDF)-1, matrix metalloproteinase (MMP)-9, and tumor necrosis factor (TNF)-α, among others. These secreted cytokines and their relative signaling pathways, which represent key mechanisms for heart regeneration, may serve as a promising future therapeutic strategy for myocardial infarction patients. TGF-β, IL-6 family members, including IL-6, Oncostatin-M, cardiotrophin-1 (CT-1), and leukemia inhibitory factor (LIF), as well as several chemokines are key players in cardiomyocyte regeneration. TGF-β contributes to cardiac repair and renewing cardiac function after MI by reducing inflammation, promoting myofibroblasts and ECM placement, Ischemia-reperfusion injury and myocardial infarction induce IL-6 production by cardiac myocytes (Ref. 8 & 9). On the other hand, IL-6 family members protect myocytes against oxidative stress by inducing an anti-apoptotic program. Another cytokine, Granulocyte Colony-Stimulating Factor (G-CSF), is believed to be involved in myocardium regeneration, myocardial fibrosis reduction, healing acceleration and cardiomyocyte protection. Cardiac repair following cytokine therapy depends on a number of variables, and further research is required to accurately determine the true therapeutic potential of such therapy. Chemokine superfamily members are rapidly upregulated in the infarcted myocardium and may modulate infarct angiogenesis and fibrous tissue deposition. Upregulated CXCL8 may induce neutrophil infiltration. Other chemokines, such as the CC chemokines Monocyte Chemoattractant Protein (MCP)-1, Macrophage Inflammatory Protein (MIP)-1α, and CCL4 may regulate monocyte and lymphocyte recruitment. Stromal-Cell Derived Factor-1 (SDF-1α) is the most extensively studied chemokine for cardiogenesis.

Growth factors promote myocardial repair and improved cardiac function. The discovery of growth factor involvement in cardiac regeneration mechanisms, including angiogenesis, anti-apoptosis, cardiomyocyte proliferation, CSCs chemotaxis, ECM remodeling and others, has generated increasedinterest in cardiovascular medicine. EGF, due to its angiogenic property, is seen as a promising molecule for promoting neovascularization in the infarcted heart. VEGF also mediates eNOS phosphorylation and helps in the regulation of angioblast and embryonic endothelial cell (EC) proliferation. VEGF is required for effective cardiomyocyte differentiation of human induced pluripotent stem cells (iPSCs). Several others anti-apoptotic
growth factors, such as fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF), induce mitigation of the ischemic injury in the cardiac tissues. Erythropoietin (EPO), a glycoprotein hormone, protects the myocardium from ischemic injury and promotes cardiac remodeling. Hypoxic ischemic cardiomyocytes contain EPO receptors that are potential targets for EPO treatment. Thus, the paracrine secretion of cytokines, chemokines, and growth factors increases cardiac recovery and tissue regeneration (Ref. 9 & 10).

Besides cytokines and growth factors, small molecules may also play an important role in cardiac regenerative therapy. A variety of cells, such as cardiac fibroblasts, PSCs, and cardiomyocytes, can serve as a cell source for cardiac repair and regenerative medicine. Small molecules can be used to improve or enable cell reprogramming towards pluripotency. Treatment of multipotent cells with small molecules may also activate repair mechanisms, opening new avenues to regenerative medicine. Small molecules may be used for stem cell differentiation to cardiomyocytes, which is an importance source for replacement therapy. Among small molecules, ascorbic acid has been identified to increase cardiogenic differentiation of embryonic stem cells (ESCs). Other cardiogenic small molecules include cardiogenols, isoxazolyl-serine-based agonists of peroxisome proliferator-activated receptors (PPARs), verapamil, SB203580, sulfonylhydrazones, and cinchona alkaloid derivatives. AG1478, BMS-189453, Diazoxide, 1-EBIO, Retinoic acid (RA), and Purmorphamine are some other small molecules that enhances graft integration, cardiac differentiation and heart regeneration. Retinoic acid enhances cardiomyogenesis and ventricular cardiomyocyte development in mouse ESCs. Noggin, dorsomorphin or other BMP antagonists also promote cardiomyogenesis in mouse ESCs during developmental stages. Cardiomyogenesis can also be promoted by modulating calcium signaling pathways by using small molecules. Cyclosporine, a calcineurin inhibitor, as well as verapamil, an L-type calcium channel blocker, have been identified as cardiomyogenesis promoters. Small molecules targeting BMPs, TGF-  , and Wnt, enable the efficient cardiac differentiation of PSCs in humans. Cardiogenic small molecubles discovered in phenotypic screening assays are of immense use and investigating their cellular targets could potentially shed more light on the process of cardiac differentiation. Small molecules could replace cardiac reprogramming transcription factors and can serve as an initial step towards cardiac cell induction and enhancement of reprogramming for in vitro applications. Cardiogenic small molecules have proven to be important keys for determining novel drug targets and may serve as promising therapeutics for the treatment of ischemic cardiomyopathy (Ref. 11, 12 & 13).

Figure: Three approaches for differentiating hPSCs into cardiomyocytes, divided into six steps: pluripotent culture, pre-differentiation culture, differentiation format, treatment with mesoderm induction factors, treatment with cardiac specification factors, and treatment with cardiac differentiation factors. (A)Methods using suspension of embryoid bodies (EBs) in StemPro34; (B) Methods using forced aggregation of EBs; (C), Methods using monolayer differentiation.
Abbreviations: KSR, Knockout Serum Replacement; FGF-2, fibroblast growth factor 2; StemPro34, proprietary medium from Invitrogen; BMP-4, bone morphogenic protein 4; VEGF-A, vascular endothelial growth factor A; DKK-1, dickkopf homolog 1; SB431542, TGF-β/Activin/NODAL signaling inhibitor (ALK4,5,7); dorsomorphin, BMP signaling inhibitor (ALK2,3,6); IWR-1, WNT signaling inhibitor; MEF CM, mouse embryonic fibroblast conditioned hESC medium; IMDM/F12+PVA, IMDM/F12-based media supplemented with polyvinyl alcohol; RPMI, Roswell Park Memorial Institute 1640 basal medium; FBS, fetal bovine serum; DMEM, basal media; RPMI+PVA, RPMI-based media supplemented with polyvinyl alcohol; RPMI-INS, RPMI-based media without insulin; B27, media supplement; NOGGIN, BMP signaling inhibitor; RAi, retinoic acid signaling inhibitor; LI-APEL, low insulin, Albucult, polyvinyl alcohol, essential lipids media; SCF, stem cell factor (KITLG); LI-BEL, low insulin, bovine serum albumin, essential lipids media; IWP-4, WNT signaling inhibitor (Ref. 14).

All the above methods have utilized PSCs that were differentiated into cardiomyocytes. Since obtaining enough PSCs for practical applications in regenerative medicine could present a real challenge, iPSCs were viewed as a promising solution. However, currently, reprogramming efficiency is very low and there are safety issues that might limit the use of iPSCs. Transdifferentiation, which is the direct conversion of one mature somatic cell into another mature somatic cell, offers another option for obtaining cardiomyocyte-like cells by using forced expression of transcription factors and microRNAs, yet, these genetic manipulations can raise safety issues as well. The discovery that iPSCs can be obtained by chemical induction led to the discovery that it is possible to transdifferentiate induced cardiomyocyte like cells (iCMs) from fibroblasts by using a defined chemical cocktail comprised of CHIR 99021, E
616452, Forskolin, Valproic Acid, Tranylcypromine, TTNPB, L-Ascorbic Acid, Rolipram and PD 0325901, together with LIF, NRG-1 and G-CSF (Ref. 25).

An important aspect of cardiovascular diseases and cardiovascular regeneration includes the identification of biomarkers. Cardiac biomarkers are released into the blood when the heart is damaged or stressed. They can potentially be used to detect a wide range of cardiac conditions like acute coronary syndrome (ACS) and cardiac ischemia. Traditional cardiac biomarkers include glucose level, lipid profile, and hormonal biomarkers.

Physiological cardiac biomarkers are based on serum lipid, triglyceride to HDLp ratio, LDL cholesterol level, Sphingolipids, Omega-3 Index, Lipophorin-cholesterol ratio and ST2 level. Other cardiac biomarkers, such as imaging, anatomical, immunohistochemical, therapeutic and genetic biomarkers may be important in risk prediction and morbidity status. The most important markers in cardiovascular disease include molecular markers, both surface and internal markers. Cardiac troponins (cTn), including Troponin I (cTnI) and Cardiac Troponin-T (cTnT), and natriuretic peptides, are the most prominent molecular biomarkers used in clinical cardiology, especially in cases of ACS. High levels of cTn1 are generally associated with increased risk of heart failure (Ref. 26 & 27).

Transcription factors may also function as cardiac biomarkers. During cardiogenesis, cardiac progenitors expressing the transcription factors Nkx2.5 and Isl1 may give rise to myocytes. Nkx2.5, or cardiac homeobox protein, is a marker of cardiomyocyte differentiation and is necessary for proper development of the ventricular myocardial lineage. Isl1, a LIM homeodomain transcription factor, is a pan-cardiac progenitor marker, and is expressed in cardiac progenitor fields during early development. Nkx2.5 is present at high levels in embryonic differentiated cardiomyocytes and is expressed throughout cardiac development while Isl1, which is downregulated in myocardial differentiation, is mostly restricted to a progenitor cell state in the heart. Nkx2.5 acts in combination with both MEF2C and Hand2, also considered core cardiac transcription factors, to control ventricular identity. Tbx5, another transcription factor, is predominantly expressed in first heart field (FHF) precursors and was down regulated in the early stages of embryoid body (EB) differentiation of Nkx2.5OE ESCs. GATA-4, 5, and 6 are expressed in the heart and regulate developmental processes, including differentiation and migration of cardiomyoctes. GATA-4 is one of the first transcription factors expressed in cardiac cells and is important in transcriptional regulation during cardiac development (Ref. 26 & 27).

Additional gene markers for cardiac mesoderm and cardiomyocytes include Brachyury, atrial natriuretic factor, mesoderm posterior factor 1, myosin light chain 2 atrial and ventricular transcripts, and α-myosin heavy chain (MHC-α). NT-proBNP and sST2 are two promising biomarkers for identifying patients with little potential to benefit from Implantable Cardioverter Defibrillator (ICD) therapy. Other cardiac-specific structural genes identified include ion channel proteins (MYH7, MYH6, MLC-2A) and ionic channels (CACNA1C, CACNA1D, hERG, HCN-2). Many cell membrane proteins, such as potassium voltage- gated channel subfamily A member 6 (KCNA6), and N-cadherin, a calcium-dependent transmembrane adhesion protein, are also important markers in cardiovascular diseases. Cell surface markers are generally used to verify heart muscle cells. Other common cardiac cell membrane markers include β1- and β2-adrenergic receptors, Connexin 43, and Popeye domain containing 2 (POPDC2), which has a high frequency of expression along with MHC-α. Recently, miRNAs have also been identified as regulators of cardiovascular diseases and may provide new insights into disease mechanisms. Many miRNAs have been implicated incardiovascular disease as viable biomarkers and drug targets (Ref. 28).

Ischemic cardiomyopathy, myocardial infarction and congestive heart failure have become major clinical issues due to their role in increasing morbidity and mortality. Cardiac regenerative therapy, including drugs, growth factors and numerous pharmacological and device therapies have improved adverse cardiac remodeling and mortality in heart failure. However, few of these strategies have found success in clinical trials. A number of issues will need to be addressed for the advancement of regenerative medicine as a field. Stem cell-based therapies using multipotent and pluripotent stem cells could potentially achieve the elusive goal of true cardiac regeneration. Until now, both clinical as well as preclinical studies have utilized simple delivery methods for regenerative therapeutics. Recently, the focus has shifted to advanced delivery concepts such as the use of biomaterial carriers, multimodal therapeutic strategies, nanoparticulate encapsulation, and minimally invasive delivery systems. These potential strategies may lead to a whole new world of cardiac regeneration in the future (Ref. 29 & 30).

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