Intervention for Cardiac Repair: A Clinical Perspective

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REVIEW ARTICLE

Intervention for Cardiac Repair: A Clinical Perspective Esther Hui Na Tan, Zhe Yuan Tay and Boon Seng Soh* Institute of Molecular and Cell Biology - Stem Cell, Regenerative Medicine And Ageing Singapore, Singapore; National University of Singapore - Department of Biological Sciences Singapore, Singapore

ARTICLE HISTORY Received: September 9, 2016 Revised: November 28, 2016 Accepted: January 6, 2017 DOI: 10.2174/1574888X12666170113094 234

Abstract: Cardiovascular disease remains the leading cause of death worldwide. Damage to the heart resulting from cardiovascular disease leads to gradual loss of function and reduced quality of life. Cardiac injury is particularly debilitating, more so than injury to any other organ, given our current inability to either generate new and functional cardiac tissue or to mimic the actions of the heart using external devices. Advances in the field of stem cells and genetics have paved the way for the development of a variety of novel therapies. A number of these therapies have shown great promise in regenerating cardiac tissue in non-human disease models and some have progressed towards clinical trials. Given the rapid progress and emergence of novel targets for therapy, it is perhaps timely that we assess the practicality of these techniques and their potential for translation to bedside. Hence, this review aims to outline the major therapies in development and to provide insight into the feasibility of the respective techniques with the hope that research can be steered towards developing therapies with greater potential of being employed at the bedside.

INTRODUCTION Ischemic heart disease is the main cause of death worldwide, accounting for 8.1 million or 14.8% of deaths in 2013 [1]. With the higher incidence of cardiac risks factors such as obesity, hypertension, and diabetes in addition to the increasing number of aging population, the future burden of heart disease is expected to rise. Unlike organisms such as the newt, which possesses the ability to regenerate the damaged heart [2, 3], the human heart lacks the ability to replace lost cardiomyocytes after myocardial injury. As such, current research focuses on the ability to regenerate the damaged heart and restore its function after an infarct. Evolution of stem cell research and genetics has led to the development of a myriad of strategies aimed at restoring heart functions (Fig. 1). In this review, we detail the latest treatments and therapies that are being developed, and discuss on the feasibility of applying these approaches in the clinic. A list of uncommon acronyms or abbreviations with their respective full names has been provided in Table (1). MOLECULAR BASED INTERVENTION Direct Reprogramming of Cardiac Fibroblasts into Cardiomyocytes Myocardial scarring often occurs as a result of wound healing after cardiac injury such as myocardial infarction. *Address correspondence to this author at the Institute of Molecular and Cell Biology - Stem Cell, Regenerative Medicine And Ageing Singapore, Singapore; National University of Singapore - Department of Biological Sciences Singapore, Singapore; E-mail: [email protected] 1574-888X/17 $58.00+.00

Fibroblasts are often regarded as the main culprits in scar tissue formation, which adversely affect the cardiac function eventually leading to heart failure. It is also known that cardiac fibroblasts form less than 20% of nonmyocytes in the heart, making them the second largest cell population of the nonmyocytes in the heart after endothelial cells [4]. All these, coupled with recent advances in the conversion of fibroblasts to cardiomyocytes via direct lineage programming, suggest that the cardiac fibroblast is an important target in cardiac repair. At present, there is no single transcription factor that is able to transdifferentiate fibroblasts into cardiomyocytes; unlike the ability of MyoD in skeletal muscle reprogramming [5]. Instead, cardiac reprogramming requires the combination of transcription factors; Gata4, Mef2c and Tbx5 (GMT) [6]. With the addition of heart and neural crest derivatives expressed protein 2 (Hand2), Song et al. further reported the improvement of cardiac reprogramming efficiency by 4-fold as compared to a similar study done by Ieda et al. using adult and neonatal tail tip fibroblasts [6, 7]. While GMT or GHMT can successfully transdifferentiate nonmyocyte cells into induced cardiomyocyte-like cells (iCMs) in the mouse model [6-10], they are insufficient to transdifferentiate human fibroblasts into human iCMs. In 2013, Wada et al. reported that the addition of MESP1 and myocardin to GMT (GMTMM) could transdifferentitate human cardiac and dermal fibroblasts into human iCMs that exhibited calcium oscillations. Synchronous contractions were observed only after co-culturing with murine cardiomyocytes [11]. While Nam et al. reasoned that myocardin is © 2017 Bentham Science Publishers

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Fig. (1). Schematic summarizing various interventions for cardiac repair and the possible clinical outcome. Table 1.

List of uncommon acronyms or abbreviations with their respective full names.

Acronym/ Abbreviation

Full Name

AAV

Adeno-associated virus

ALCADIA

Autologous Human Cardiac-derived Stem Cell To Treat Ischemic cArdiomyocpathy

ALLSTAR

ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration

CADUCEUS

CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction

CAREMI

Cardio Repair European Multidisciplinary Initiative

CDC

Cardiosphere derived CPCs

CPCs

Cardiac progenitor cells

ECM

Extracellular matrix

EPCs

Endothelial progenitor cell

EPDCs

Epicardium derived cells

ESCs

Embryonic stem cells

hPSCs

Human pluripotent stem cells

Hystem-C™

Hyaluronan-based hydrogel cross-linked using thiol-reactive poly(ethylene glycol) diacrylate

iCMs

Induced cardiomyocyte-like cells

LVEF

Left ventricular ejection fraction

MI

Myocardial infarction

MLR

Mixed lymphocyte reactions

MPs

Microparticles

PEAD

Poly(ethylene argininylaspartate diglyceride)

PLGA

Poly(lactic-co-glycolic acid)

SIS-ECM

Small intestine submucosa extracellular matrix

SPIONs

Superparamagnetic iron oxide nanoparticles

SA

Sarcomeric -actinin

Intervention for Cardiac Repair

essential for human cardiac reprogramming and GHMT can inhibit the activation of smooth muscle gene program of myocardin, Fu et al. on the other hand stated otherwise [1012]. They reported that only 5 factors (GMT, MESP1 and estrogen related receptor gamma (ESRRG) are critical for the generation of human iCMs, and that the addition of myocardin and Zinc Finger Protein, FOG Family Member 2 (ZFPM2) only function to further enhance cardiac reprogramming efficiency, rhythmic calcium transients, action potential and sarcomere development [10]. It is possible that the importance of myocardin in human iCMs largely depends on the combination of cocktail used for cardiac reprogramming. In addition, transforming growth factor beta (TGF-) signaling pathway was shown to affect the efficiency of iCMs programming and play a role in the early stages of cardiac reprogramming [10, 13]. While Fu et al. claimed that TGF-1 doubled the human iCMs generated by the 5 factors programming, Zhao et al. reported that TGF-1 reduced the efficiency of beating iCMs by 100 fold in GHMT2m (GHMT plus miR-1 and miR-133) treated mouse embryonic fibroblast (MEF) iCMs [10, 13]. In a latter study, Zhao et al. revealed that the reprogramming efficiency of iCMs improved significantly even with the suppression of other profibrotic signaling such as the Rho-associated kinase pathway [10, 13]. Similarly, Ifkovits et al. reported a five-fold increase in reprogramming efficiency in both the MEF and the adult mouse cardiac fibroblast using TGF- inhibitor SB431542 [14]. Functional human iCMs with sarcomere-like structures and spontaneous contractility can be formed from adult human cardiac fibroblasts [12] with the introduction of GATA4, HAND2, TBX5, myocardin, and the microRNAs miR-1 and miR-133 (which are described in more detail in the next section). Nevertheless, human iCMs that were derived from adult human fibroblasts and human foreskin fibroblasts did not show spontaneous contraction. This indicates that cardiac fibroblasts are more readily transdifferentiated into human iCMs as compared to fibroblasts of noncardiac origin, which may suggest that the epigenetic memory of parent cell influences the differentiation potential of a cell [15-17]. In addition, both miR-1 and miR-133 are believed to be crucial in cardiac reprogramming efficiency and formation of sarcomere [12, 18]. In vivo direct reprogramming of fibroblasts into cardiomyocytes is a promising new avenue of research that can endogenously substitute lost or damaged cardiomyocytes without the need for cell transplantation. This approach has several advantages such as reducing the risk of tumorigenesis since embryonic stem cells (ESCs) derived cardiomyocytes are not used, as well as avoiding complications arising from cellular rejection. There are, however, also challenges in ensuring that the iCMs formed can regenerate into the specific mature cardiomyocyte sub-types required at each particular site and their ability to interact and function with neighboring cells. Clinically, this approach is not viable at the moment as the cardiac reprogramming efficiency to transdifferentiate human fibroblast into human iCMs is low.

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Role of miRNAs in Cardiomyocyte Survival and Proliferation MicroRNAs (miRNAs) are small noncoding RNAs that induce RNA silencing and regulate gene expression at the post-transcriptional level. Due to their capacity to target multiple genes, miRNAs are more useful and potent as compared to other therapeutic agents that target a single gene. Here, we will summarize some of the miRNAs that have been reported to be critical in cardiac repair as they regulate cardiomyocyte survival and proliferation. It is known that cardiomyocytes die by apoptosis or necrosis in response to ischemia. Aurora et al. demonstrated that miR-214 was upregulated during cardiac stress and has a protective function against excessive Ca2+ uptake and Ca2+overload-induced-cell-death. miR-214 directly inhibits the sodium/calcium exchanger 1 (NCX1) and effectors of Ca2+ overload signaling pathways such as calcium/calmodulindependent protein kinase type ll subunit (CaMKII), Cyclophilin D (CypD), and Bcl-2-like protein (BIM). Mice with miR-214 deletions were found to have poorer heart function and were more prone to cell death after ischemic reperfusion injury [19]. In addition, overexpression of miR-214 protects cardiomyocytes against H2O2-induced apoptosis via phosphatase and tensin homolog (PTEN) and inhibits fibrosis though TGF-1 suppression and matrix metalloproteinase-1/ tissue inhibitors of metalloproteinases-1 (MMP-1/TIMP-1) regulation [20, 21]. Clinically, while Lu et al. reported that patients with coronary heart disease have significantly decreased miR-214 levels as compared to healthy control [22], elevated miR-214 levels are not necessarily good. Overexpression of miR-214 has shown to cause cardiac hypertrophy and dysfunction [23]. Therefore, short term induction of miR214 has protective effects but may have detrimental effects in the long run [24]. Recent studies demonstrated that synergistic actions of miRNA pairs on target genes have more efficient biological effect [25-29]. In 2016, Huang et al. demonstrated that a synergy effect of miR-21 and miR-146a led to better cardiomyocyte survival against ischemia or hypoxia-induced apoptosis both in vitro and in vivo as compared to their individual effect. It was found that the miR-21:miR-146a pair improved the inhibition of cardiomyocyte apoptosis by the miR-21— PTEN/AKT-p-p38-caspase-3 and miR-146a-TRAF6-p-p38caspase-3 signal pathways [30]. While improved cardiomyocyte survival reduces cell death during an injury, the potential of the cardiomyocytes to proliferate is pivotal in restoring the normal cardiac function. Study done by Eulalio et al. revealed that the synergistic effects of miR-590-3p and miR-199a-3p promoted the proliferation of already differentiated cardiomyocytes which led to improved cardiac function and regeneration after myocardial infarction in neonatal and adult rodents [31]. Similarly, another miRNA, miR-204 was found to have proproliferative effects on cardiomyocytes by downregulating Jarid2 [32]. The miRNA cluster, miR-17-92 which targets PTEN was demonstrated to promote cardiomyocyte proliferation during injury in embryonic, neonatal and adult hearts [33]. While miR-17-92 possesses the ability to promote cardiomyocyte


Table 2.

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List of transcription factors and miRNAs that promote cardiomyocyte reprogramming, survival, proliferation and cardiac repair. Transcription Factors/ miRNAs

References

Gata4, Mef2c, Tbx5 (GMT)

Ieda M, et al. [6]

Gata4, Hand2, Mef2c, Tbx5 (GHMT)

Song K, et al. [7]

MESP1, myocardin, GMT (GMTMM)

Wada R, et al. [11]

GATA4, HAND2, TBX5, myocardin, miR-1, miR-133

Nam YJ, et al. [12]

GMT, MESP1,ESRRG, myocardin, ZFPM2

Fu JD, et al. [10]

GHMT, miR-1, miR-133 (GHMT2m)

Zhao Y, et al. [13]

miR-499 and miR-133

Pisano F, et al. [29] Aurora AB, et al. [19]

miR-214

Lv G, et al. [20] Dong H, et al. [21]

miR-21 and miR-146a

Huang W, et al. [30]

miR-590-3p and miR-199a-3p

Eulalio A, et al. [31]

miR-204

Liang D, et al. [32]

miR-17-92

Chen J, et al. [33]

miR-24

Qian L, et al. [36]

proliferation, Danielson et al. demonstrated that overexpression of miR-17-92 results in hypertrophic and arrhythmogenic cardiomyopathy [34]. They conjectured that arrhythmias might have resulted from repression of connexin43 by miR-19a/b, a member of miR-17-19 cluster [34, 35]. It might be advantageous to conduct further studies on different members of the miR-17-92 cluster that can synergistically induce proliferation without causing arrhythmias. Overall, modulating the expression of miRNAs offers new therapeutic opportunities that can modify disease phenotypes by manipulating the pathophysiological process. Importantly, miRNAs can be used as a tool in gene therapy and also boost cell therapy in cardiac repair towards cardiomyocyte survival and proliferation. However, due to the extensive biological influence of miRNAs on multiple targets, it is imperative that a comprehensive study be done to fully understand their impacts and to prevent complications from potential off target side effects. Table (2) summarizes a list of transcription factors and miRNAs from recent studies that have demonstrated to promote cardiomyocyte reprogramming, survival, proliferation and cardiac repair. Other Common Molecular Interventions for Cardiac Repair Angiogenesis or neovascularization is an essential component in cardiac repair. They are needed to overcome ischemic insult and to transport nutrients, oxygen and other factors to cardiomyocytes. Hypoxia inducible factor-1 (HIF1) is the chief regulator of angiogenesis during hypoxic events [37]. HIF-1 activates angiogenic genes and their re-

ceptors such as VEGF, PIGF, PDGFB, ANGPT1, ANGPT2, MMP2, CATHD, KRT [38-40] which function to regulate proangiogenic chemokines and inducible nitric oxide synthase [41, 42]. HIF-1 contributes to the recruitment of endothelial progenitor cells and enhancement of endothelial cell proliferation [43]. Hence, HIF-1 modulation has tremendous therapeutic potential. Several studies have since demonstrated that modulation of HIF-1 promotes angiogenesis, reduces infarct size, and improves cardiac function after myocardial infarction in rodents [44, 45]. In addition, vascular endothelial growth factor (VEGF) is another crucial growth factor that mediates angiogenesis and can be targeted for cardiac repair. However, its role in clinical application is limited as unregulated expression of VEGF causes vascular permeability leading to plasma leakage [46, 47], angioma formation [48, 49] and acute cor pulmonale in myocardial infarction (MI) [50]. Nonetheless, the damaging effects of VEGF can be countered by angiopoietin-1 (ANG1) [46, 47] as demonstrated by Tao et al. where VEGF and ANGPT1 were co-expressed using adeno-associated viral vector (AAV) serotype 1. The presence of both VEGF and ANG1 induced angiogenesis and cardiomyocyte proliferation by the activation of Akt kinase and up-regulation of cyclin D2/cdk4 and cyclin A/cdk2 expression. There were also reduced cell apoptosis via the Akt/Bcl-2 pathway and improvement in cardiac function of the porcine MI heart [51]. Of late, there has been a growing interest in the roles of modified RNA (modRNA) as an effective and robust approach in cardiac repair. In particular, modRNA encoding human vascular endothelial growth factor-A (VEGF-A) are


of interest as they are able to expand, mobilize and differentiate epicardial progenitor cells into cardiovascular cell types thus inducing vascular regeneration leading to reinforced cardiac repair in a mouse MI model [52]. modRNA has been indicated to be superior to the classical cardiac gene therapy in terms of long term survival, efficiency as well as reducing side effects such as abnormal vascular permeability, immunogenicity and tumorigenesis due to their ability to spatially and temporally control VEGF-A expression [48, 52-57]. Several other studies have also shown the potency of fibroblast growth factor-1 (FGF-1) and neuregulin-1 (NRG-1) in cardiac repair although their efficacy remains a problem [58-63]. A new strategy uses biodegradable microparticles (MPs) that mediates sustained release of cytokines has evolved to overcome these limitations [64-66]. In 2013, Formiga et al. reported that FGF-1 and NRG-1 loaded poly(lactic-co-glycolic acid) (PLGA) MPs facilitate sustained treatment and significantly improved global cardiac function by promoting angiogenesis, cardiac proliferation and stem cell recruitment in MI rat model [67]. A similar result is observed in pigs using catheter based intramyocardial injection of FGF-1 or NRG-1 loaded MPs [68]. The later study indicates that percutaneous intramyocardial injection of MPs loaded with cytokines is clinically feasible in cardiac regenerative medicine for MI. Another approach has been to target inflammation. Inflammation is important for the removal of dead cells but if unregulated can cause worsening of myocardial damage [69, 70]. Hence, the regulation of inflammatory response by disrupting inflammatory pathways and the subsequent prevention of pathological cardiac remodeling may prove to be cardioprotective. Recent findings have shown that high mobility group box-1 protein (HMGB-1) formerly thought to be key mediator of systemic inflammation, has potential therapeutic roles in cardiac regeneration. HGMB-1 is involve in the recruitment, activation, proliferation and differentiation of stem cells in particular resident cardiac c-kit+ cells into cardiomyocytes, thus attenuating cardiac remodeling and improving cardiac function in mouse model [71-76]. In addition, interactions between HMGB-1 and its receptor for advanced glycation end products (RAGE) stimulates both mesoangioblasts and endothelial progenitor cell (EPCs) homing, suggesting their dual function in tissue regeneration and neovascularization at ischemic regions [77, 78]. Rossini et al. further demonstrated that HMGB-1 has paracrine actions by inducing release of growth factors, cytokines and chemokines including VEGF, IFN-, Mip-1, Il9, Il-10, Il-4, Il-1ra, GM-CSF, Il-1, and TNF- from human cardiac fibroblasts. These stimulate human and murine CSCs migration and proliferation [79]. Moreover, studies have reported that cardiac specific overexpression of HMGB-1 in transgenic mice showed smaller MI size and enhanced angiogenesis. HMGB-1 overexpression also works to counteract cardiac remodeling and dysfunction thus restoring cardiac function and further increasing their survival rate after MI as compared to wild type mice. On the contrary, treatment with anti-HMGB-1 antibodies worsened ischemic reperfusion injury in rat heart [80, 81]. Although HMGB-1 may appear to be a promising tool for cardiac repair, there seems to be conflicting opinions especially due to its inflammatory ef-


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fects in cardiac diseases [82, 83]. This discrepancy may be largely due to differences in dosage, time and routes of administration used between different studies [75, 84-86]. Therefore, in order to optimize immune targets and minimize myocardial damage, a better understanding of the exact mechanism of HMGB-1 immunomodulation is needed before their translation to the clinical arena. In 2015, Chen et al. reported that synergistic effects from the co-delivery of fibroblast growth factor-2 (FGF-2) and interleukin-10 (IL-10) by heparin-based coacervate through intramyocardial injection further enhanced ischemic heart repair with better long term left ventricle (LV) contractile function and LV myocardial elasticity. There were also reduced LV fibrosis, preserved infarct wall thickness, enhanced revascularization and notably, prevention of chronic inflammation particularly at the infarct area in MI mouse model [87]. Indeed, spatial and temporal release of FGF2/IL-10 using biocompatible polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) heparin-based coacervate has achieved high efficiency with sustained linear released that can maintain bioactivity and long term bioavailability allowing for long term synergistic cardiac regenerative effects [87, 88]. This indicates that targeting both angiogenic and immunoregulatory proteins are equally important therapeutically to achieve best results for cardiac repair. A list of important growth factors/cytokines documented to be critical for cardiac repair and remodeling has been provided in Table (3). Other factors which have been reviewed elsewhere include placental growth factor, stromal cell derived factor-1, platelet derived growth factor, insulin-like growth factor-1, thymosin beta-4, yes-associated protein-1, calcium cycling proteins and monocyte chemotactic protein3 [89-106]. Cellular Based Intervention The prospect of using cell based therapies has been widely researched in recent years in an effort to replace the option of heart transplantation from a donor. Despite the vast achievements, progress has been slow as many fundamental issues remain to be resolved. Nevertheless, the potential benefit of this approach has brought many refinements and improvements for clinical translation. Besides, the discovery of cardiac progenitor cells (CPCs) residing within the adult heart has shed some light on the regenerative properties of the heart [112-114]. Resident CPCs could be the ideal source of cardiac regeneration as compared to other types of stem cells as they are committed to cardiovascular lineages besides being autologous and tissue specific [115, 116]. There are different human CPCs populations characterized by their surface markers such as c-Kit+ CPCs [117], cardiospheres or cardiosphere derived CPCs (CDC) [118, 119], epicardium derived cells (EPDC) [120] and Islet-1 (Isl-1+) expressing CPCs [121]. Nonetheless, mesenchymal stem cells (MSCs) have also been regarded as another important potential therapeutic agent for cardiovascular regenerative therapy [122]. Table (4) shows a list of transplanted cell types that demonstrated improvement in cardiac repair in various animal models. c-Kit+ CPCs have been described in multiple studies to be capable of self-renewal and differentiation into cardiomyo-


Table 3.

List of important growth factors/cytokines previously reported to be important for cardiac repair and remodeling.

Growth Factors/ Cytokines

References Shyu KG, et al. [44]

Hypoxia Inducible Factor-1 Kido M, et al. [45] Formiga FR, et al. [66] VEGF Tang JM, et al. [107] VEGF and ANG-1

Tao Z, et al. [51] Zangi L, et al. [52]

Modified RNA (VEGF) Yla-Herttuala S, Aavik E [53] Fibroblast Growth Factor-1

Palmen M, et al. [58] Jabbour A, et al. [62]

Neuregulin-1 Gao R, et al. [63] Fibroblast Growth Factor-1 and Neuregulin-1

Formiga FR, et al. [67] Garbayo E, et al. [68] Limana F, et al. [75] Limana F, et al. [76]

High Mobility Group Box-1 Kitahara T, et al. [80] Oozawa S, et al. [81] Fibroblast Growth Factor-2

Chu H, et al. [88]

Interleukin-2

Zeng Z, et al. [108]

Interleukin-27

Tanaka T, et al. [109] Burchfield JS, et al. [110]

Interleukin-10 Krishnamurthy P, et al. [111] Fibroblast Growth Factor-2 and Interleukin-10

Chen WC, et al. [87]

Placental Growth Factor

Zhang J, et al. [102] Tilokee EL, et al. [103]

Stromal Cell derived Factor-1 Saxena A, et al. [104] Insulin-like Growth Factor-1

D'Amario D, et al. [91]

Insulin-like Growth Factor-1 and Hepatocyte Growth Factor

Ellison GM, et al. [92]

Thymosin beta-4

Bock-Marquette I, et al. [93]

Yes-associated Protein-1

Smart N, et al. [94] Del Re DP, et al. [99]

Monocyte Chemotactic Protein-3

Xin M, et al. [100] Schenk S, et al. [98]

cytes, smooth muscle cells and endothelial cells [117, 123, 124]. Beltrami et al. demonstrated that post-infarcted hearts of rats injected with Lin-c-kit+ cardiac cells from rats were capable of regenerating more than 50% of new cardiomyocytes and vascular cells leading to functionally improved overall cardiac performance. Notably, treated hearts showed

Tan et al.

improved ejection faction and end diastolic pressure, reappearance of synchronous motility in ventricular wall, increased wall thickness, reduced infarct size and cavitary dilatation [123]. Similarly, human c-kit+ cardiac cells injected in MI heart of immunosuppressed rodents are able to generate a chimeric heart composed of myocytes, capillaries and coronary arterioles. The presence of connexin-43 in these hearts also indicates that structural coupling between human and rodent myocytes occur. Connexin junctions are essential as they act to restore contractile and ventricular functions, increasing ejection fraction and reducing chamber dilatation [117]. Bolli et al. further demonstrated that autologous c-kit+ CPCs infusion after MI promotes cardiac regeneration, improved regional and global left ventricular function in pigs with chronic MI [125]. Intracoronary administration of exogenous autologous c-kit+ CPCs is thought to be the most applicable approach clinically for patients with chronic ischemic cardiomyopathy due to their ability to activate endogenous c-kit+ CPCs proliferation and differentiation into cardiomyocytes. This likely occurs through paracrine effects which alleviate left ventricular dysfunction and remodeling even after replacement of infarcted tissue by scar formation [125, 126]. It is also possible that the regenerative properties of c-kit+ CPCs is due to the role of CPC antigen, c-kit in stem cell factor (SCF) /c-kit signaling pathways and activation of their downstream PI3K-AKT and MEK-ERK pathways. All these may be responsible for the salutary effects of increased survival, proliferation and migration of c-kit+ CPCs. Hence, targeting these pathways may be critical in overcoming poor engraftment and survival issues in cardiac cell therapy [127]. Based on the many encouraging improvements observed from administration of Lin-c-kit+ CPCs into animals with post infarcted left ventricular (LV) dysfunction [117, 123126, 128-130], a randomized phase 1 trial, Stem Cell Infusion in Patients with Ischemic cardiomyopathy (SCIPIO) was carried out in 2011 [131]. Here, patients with LV ejection fraction (LVEF) less than or equal to 40% and evidence of myocardial scar were administered with autologous CPCs through intracoronary infusion. Patients showed improved LV systolic function and reduced infarct size, indicating that this approach is feasible, safe and that further larger phase II studies are warranted. However, recent studies have reported that endogenous ckit+ cells contribute minimally to new cardiomyocytes within the heart but abundantly generated cardiac endothelial cells [132-134]. Sultana et al. demonstrated that murine cardiac ckit+ cells are not stem cells but are in fact endothelial cells as co-localization of progenitor markers NKX2.5 or cTnT and c-kit rarely occurs. Instead, c-kit+ cells co-localized with the expression of the endothelial cell marker, PECAM. In addition, in vivo MI mouse models revealed that c-kit+ endothelial cells rarely undergo de-differentiation into CPCs to promote cardiac repair [133]. Genetic lineage tracing performed by Liu et al. concluded that most of the labeled cardiomyocytes were derived from pre-existing Kit-expressing cardiomyocytes immediately after tamoxifen induction as opposed to de novo lineage conversion of Kit+ CPCs reported by Ellison et al. [134, 135]. They also showed that MI does not


Table 4.


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List of transplanted cell types that demonstrated improvement in cardiac repair in various animal models. Transplanted cell types

Animal model

References

Mouse/ Rat

Fischer KM, et al. [129] Beltrami AP, et al. [123] Tang XL, et al. [126]

+

c-Kit

Rota M, et al. [130] Bearzi C, et al. [117] Ellison GM, et al. [135] Pig

Bolli R, et al. [125]

Human

Bolli R, et al. [131]

Mouse/ Rat

Messina E, et al. [118] Smith RR, et al. [119] Chimenti I, et al. [137] Sun Y, et al. [149] Li TS, et al. [162] Cheng K, et al. [181] Smith RR, et al. [182] Malliaras K, et al. [143]

Cardiosphere derived Cardiac Progenitor Cells /

Tseliou E, et al. [144] Reich H, et al. [147]

Cardiospheres

Cheng K, et al. [151] Vandergriff A.C, et al. [183] Cheng K, et al. [184] Pig

Johnston P.V, et al. [138] Yee K, et al. [145] Kanazawa H, et al. [146] Takehara N, et al. [185]

Human

Makkar RR, et al. [139] Malliaras K, et al. [140]

Islet-1+

Soh BS, et al. [175] Mouse Bartulos O, et al. [186] Oyama T, et al. [187]

Cardiac Side Population Cells

Rat Mouse

Toma C, et al. [177]

Mesenchymal Stem Cells

Human

Hare JM, et al. [179] Houtgraaf JH, et al. [180]

promote an increase in c-Kit+ cardiomyocytes after injury but instead increase the number of PECAM+ endothelial cells, CD11b+ macrophages and FSP1+fibroblasts in the infarcted regions [134]. Therefore, due to the conflicting results regarding the c-Kit+ CPCs in cardiac repair, the publication of SCIPIO trial results have been subjected to intense scrutiny. Taken together, the significant improvement of heart patients in clinical trials from c-kit+ CPCs therapy sug-

gest that the precise mechanisms of how c-kit+ cells work are still poorly understood which warrant further investigations. It is possible that endothelial mesenchymal transition of the endothelial cells into cardiomyocytes may explain the discrepancies seen in different studies mentioned earlier. Cardiosphere-derived CPCs (CDCs) are a mixture of stromal, mesenchymal and progenitor cells at different stages


of commitment. They are clonogenic and multipotent, and have the ability to differentiate spontaneously into cardiomyocytes and vascular cells [115, 136]. Chimenti et al. showed that injection of human CDCs in the infarcted hearts of severe combined immunodeficiency (SCID) mice was able to mediate paracrine effects by secreting pro-angiogenic growth factors such as VEGF, HGF, IGF-1. This not only promoted angiogenesis, and reduced post-ischemic apoptosis but also recruited endogenous c-kit+ and NKX2.5+ stem cells leading to the overall cardioprotective effects [137]. In this respect, the dual ability of CDCs to differentiate directly and also biologically secrete paracrine factors makes them an attractive candidate for cardiac repair. In addition, infusion of autologous CDCs four weeks after infarction in pigs showed reduced infarct size, slower adverse left ventricular remodeling and improved haemodynamic function as compared to placebo [138]. This shows that intracoronary delivery of CDCs previously isolated following MI is effective and useful for cardiac repair. A prospective, randomized human Phase 1 trial CADUCEUS (CArdiosphere-Derived autologous stem CElls to reverse ventricular dysfunction), which was carried out on patients with LVEF of 25–45% post-MI showed positive regenerative outcome although no differences in the change of cardiac output or stroke volume from baseline to 1 year was detected in CDC-treated patients as compared with control patients. Besides, there were also no changes seen in the end-diastolic and end-systolic volumes after 1 year in treated patients. Nevertheless, CDC-treated patients showed increased viable myocardial tissue, regional contractility, regional systolic wall thickening and reduced scar mass by 6 months with further improvement and recovery of systolic function at the treated infarcted segments over the period of one year after intracoronary infusion of autologous CDCs. This study indicated that intracoronary infusion of autologous CDC may be safe, and is currently being expanded into a phase II study [139, 140]. On the other hand, the Autologous Human Cardiacderived Stem Cell To Treat Ischemic cArdiomyocpathy (ALCADIA) trial, an open label, non-randomized, small, phase 1 safety/feasibility study that evaluates a hybrid cell therapy application of the administration of autologous cardiac stem cells via intramyocardial injections together with implantation of biodegradable gelatin hydrogel sheet containing the controlled release formulation of basic Fibroblast Growth Factor (bFGF) that covers the injection sites areas in patients with ischemic cardiomyopathy and heart failure has revealed favorable trends at 6 months after treatment which include the increase of LVEF and reduction of the infarct size although there was one case of serious adverse event related to worsening heart failure among the patients who were followed for 12 months. However, a larger prospective, randomized, placebo control clinical trials are required to further investigate the effectiveness of this treatment [141] Although using autologous CDCs as a potential approach for cardiac repair avoids immune rejection, it may not be practical clinically due to timing, logistic and economic constraints. In particular, the harvesting and processing of patient specific tissue while maintaining the quality of cell potency is difficult due to patient age and presence of comor-

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bidities [142]. Pre-clinical studies on animal and ongoing human trials have shown that allogeneic CDCs or cardiospheres are safe and may be as effective as autologous CDCs [143-147]. Besides, the transient presence of transplanted allogeneic CDCs or cardiospheres is enough to amplify endogenous cardiac repair and regeneration instead of directly generating lost tissues [143]. Therefore, allogeneic human CDCs or cardiospheres are potential off-the-shelf product for cardiac repair. Malliaras et al. reported that allogeneic rat CDCs elicit negligible lymphocyte proliferation that was comparable to syngeneic rat CDCs. Xenogeneic human CDCs on the other hand induced strong proliferative response in mixed lymphocyte reactions (MLR) in vitro. Similarly, levels of proinflammatory and anti-inflammatory cytokines were comparable between allogeneic and syngeneic rat CDC but were markedly increased in human CDCs. When transplanted into rats by intramyocardial injection after MI, cell survival was less than 1% after 3 weeks post-MI in both allogeneic and syngeneic group. The syngeneic group was found to have a higher residual number of surviving cells, indicating that allogeneic CDCs are cleared more rapidly. Allogeneic CDCs were shown to be hypoimmunogenic and were found to elicit cellular but not humoral immune memory response; inducing transient and mild local immune reaction without histologically evident rejection or systemic immunogenicity in vivo. Both allogeneic and syngeneic CDCs also exert comparable beneficial global cardiac effects. They were both found to promote endogenous cardiac regeneration and secrete paracrine factors such VEGF, IGF-1 and HGF, thus allowing cardiomyogenesis and angiogenesis. This indicates that allogeneic transplantation of CDCs without immunosuppression is safe. In addition, the fact that improvement of cardiac structure and function persists even after 6 months despite the low rate of transplanted cell survival implies that cardiac regeneration is due to an indirect mechanism whereby these transplanted cells may have already exerted their paracrine effects before their cell death [137, 143]. Intracoronary infusion of allogeneic CDCs post-reperfusion in pigs with acute MI and repeated intramyocardial injection of allogeneic CDCs in post-MI immunocompetent rats were shown to be cardioprotective and safe, consistent with known immunemodulatory and anti-inflammatory properties of CDCs [146, 147]. Moreover, the Phase 1 clinical trial, ALLSTAR (ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration) result has shown that intracoronary infusion of allogeneic CDCs in patients with LV dysfunction and scar size of more than 15% is safe and feasible [148]. In the one year trial, only 4 out of 14 adult subjects developed very low (MFI