International Journal of Cardiology 173 (2014) 12–19

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

Review

Application of stem cell/growth factor system, as a multimodal therapy approach in regenerative medicine to improve cell therapy yields Fatemeh Pourrajab a,b,⁎, Mojtaba Babaei Zarch a, Mohammad Baghi Yazdi a, Abolfazl Rahimi Zarchi c, Abbas Vakili Zarch a a b c

School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran Department of Clinical Biochemistry and Molecular Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran School of Nursing, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 2 October 2013 Received in revised form 26 December 2013 Accepted 8 February 2014 Available online 20 February 2014 Keywords: Regenerative medicine Growth factors Stem cell

a b s t r a c t Stem cells hold a great promise for regenerative medicine, especially for replacing cells in infarcted organ that hardly have any intrinsic renewal capacity, including heart and brain. Signaling pathways that regulate pluripotency or lineage-specific gene and protein expression have been the major focus of stem cell research. Between them, there are some well known signaling pathways such as GF/GFR systems, SDF-1α/CXC4 ligand receptor interaction and PI3K/Akt signaling, and cytokines may regulate cell fate decisions, and can be utilized to positively influence cell therapy outcomes or accentuate synergistic compliance. For example, contributing factors in the progression of heart failure are both the loss of cardiomyocytes after myocardial infarction, and the absence of an adequate endogenous repair signaling. Combining cell engraftment with therapeutic signaling factor delivery is more exciting in terms of host progenitor/donor stem cell survival and proliferation. Thus stem cell-based therapy, besides triggering signaling pathways through GF/GFR systems can become a realistic option in regenerative processes for replacing lost cells and reconstituting the damaged organ, as before. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction 1.1. Desirable application of chemotactic factors during the recovery processes Despite advances in medical and surgical procedures, cardiovascular diseases remain the leading cause of cardiovascular morbidity and mortality. The death of large numbers of cardiomyocytes results in development of heart failure; thus, restoring lost myocardium would be desirable for the treatment of cardiomyopathy. Human stem cell-derived cardiomyocytes (hSC-DCMs) hold a great promise for myocardial regeneration after infarction. However, existing strategies are restricted by low cell survival and engraftment, and significant obstacles still exist with consistent derivation of hSC-DCMs populations [1]. It was found that long-term culture increases mesenchymal stem cell (MSC)-cellular stress and causes expression of more cell cycle inhibitors, p16(INK), p21 and p19(ARF), whereas the presence of vascular endothelial growth factor (VEGF) reduces cellular stress besides improving MSC viability in infarcted hearts, by increasing pro-survival ⁎ Corresponding author at: Department of Clinical Biochemistry and Molecular Biology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Professor Hessabi BLV, Shohadaye Gomnam BLV, Yazd, Iran, P.O. 8915173149; Fax: +98 351 8202632. E-mail address: [email protected] (F. Pourrajab).

http://dx.doi.org/10.1016/j.ijcard.2014.02.006 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

factors such as phosphorylated-Akt and Bcl-xL. Co-injection of MSCs with VEGF to MI hearts increases cell engraftment and results in better improvement of cardiac function than alone injection of VEGF or MSCs [2–4]. It has been also reported that MSCs overexpressing insulin-like growth factor (IGF)-1 not only improved survival and engraftment in the infarcted heart, but also promoted stem cell incorporation through paracrine release of stromal cell-derived factor (SDF)-1α. The presence of IGF-I has a pivotal role in attracting stem cells to the injured heart and their differentiation via release of paracrine factors, besides activating molecular pathways of cell survival [4,5]. On the other hand, MSCs overexpressing IGF-1, exhibit massive stem cell mobilization via SDF-1α signaling and culminate in extensive angiomyogenesis in the infarcted heart. Clusters of stem cell-like cells have been identified in the human adult heart, which contribute to organ regeneration, as well. These cells express stem cell markers such as Sca-1 and c-Kit, and also harbor telomerase activity which is only present in replicating cells [3,4]. Tissue injuries are also associated with local increases in mediators, the chemotactic factors particularly relevant to bone marrow mesenchymal stem cell (BMMSC) mobilization and homing for tissue repair. BMMSCs are able to migrate in response to a large set of chemotactic factors, including both growth factors (GFs) and chemokines [6,7].

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Some of the GFs have been known to be produced by MSCs and known for their autocrine activity, promigratory, proliferating, or differentiating activity on MSCs. The most chemoattractive GFs have been reported to be platelet-derived growth factor (PDGF-AB), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1), which are already reported as potent chemoattractants for MSCs [7–9]. Data also indicate that the migration capacity of stem cells is under the control of tyrosine kinases and CXC chemokine receptors (a large family of G protein-linked receptors that are known as seven transmembrane). One of the parameters that have to be taken into account in cell therapy protocols, is that the chemokines are mostly effective on tumor necrosis factor-α (TNF-α)-primed stem cells and MSC subsequent homing to injured tissues depends on the systemic and local inflammatory state [6,10,11]. MSCs pre-incubation with inflammatory cytokines such as TNF-α, lead to increased MSC mobilization towards chemokines especially RANTES (regulated on activation, normal T cell expressed and secreted/CCL5, a chemotactic cytokine that plays an active role in recruiting leukocytes into inflammatory sites) [12]. Notably, MSC's pre-incubation with inflammatory cytokines does not change the migration capacity of cells in response to the major GFs; VEGF, PDGF, IGF-1, HGF and EGF. For example, VEGF as a potent angiogenic agent underlining a major chemotactic activity of MSCs and endothelial cells is not responding to TNF-α. Whereas the growth factor Ang-1 not only promotes angiogenesis from pre-existing blood vessels, but has also been shown to respond to proinflammatory conditions, Ang-1 is a crucial chemotactic factor for endothelial cells through its receptor Tie-2 [13–15]. BM-derived hematopoietic stem cells (HSCs) and MSCs express Tie-2 and Tie-1, respectively and respond to Ang-1 under proinflammatory conditions (i.e., in the presence of TNF-α) [7,14]. Generally, the chemotactic activity of chemokines on stem cells is appeared to be less efficient than that of GFs. Exceptionally, three chemokines RANTES/CCL5, SDF 1/CXCL12 and macrophage-derived chemokine (MDC)/CCL22 display clearly a significant activity. Although, in comparison to PDGF-AB and IGF-1 which have the most potent activity on stem cells, the three mentioned chemokines, display limited effects [16]. MDC, as an active chemokine on mature hematopoietic cells, and also as a potent chemoattractant for BMMSCs, even displays the strongest activity among the chemokines tested on steady state cells [7,16]. A dramatic increase has been observed in MSC sensitivity to RANTES, MDC, and SDF-1, after TNF-α stimulation. It was indicated that MSCs express the receptors for RANTES (CCR3, CCR4, and CCR5, but not CCR1), MDC (CCR4), and SDF-1 (CXCR4). Some cytokines like granulocyte-colony stimulating factor (G-CSF) and SCF cause an increase in the release of stem cells from the bone marrow into the peripheral blood circulation. The administration of G-CSF and/or SCF could contribute to myocardial regeneration, if circulating stem cells accumulate in the myocardium and differentiate into cardiomyocyte [7]. Notably, the migration capacity of MSCs clearly stands in contrast to that of HSCs whose migration is induced mainly by the single chemokine, SDF-1 [17], whereas hematopoietic GFs display little or no chemotactic activities [12]. Consider that MMPs and CD26/DPPIV proteases are able to favor cell locomotion and tissue reconstitution by breaking down the extracellular matrix and also by degrading locally a number of chemokines. Their local administration along the chemotactic factors can accentuate MSC homing and incorporation into the injured tissue through the extracellular matrix, as recently reported with IGF-1, HGF, or PDGF after myocardial damage [18]. Consider that systemic administration may also trigger endogenous MSC mobilization towards tissue injuries, while avoiding ex vivo stem cell amplification. In addition, the local or systemic administrating of inflammatory mediators might influence not only MSC mobilization [19], but also MSC proliferation and differentiation [20] and MSC engraftment [7,21].

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1.2. The IGF-1/IGF-1R system poses a greater degree of angiogenesis in the heart IGF-1 is an autocrine/paracrine growth factor that circulates at high levels in the plasma and is expressed in most cell types. IGF-1 has major effects on development, cell growth and differentiation [22,23]. Intramyocardial transplantation of MSCs overexpressing IGF-1/GF-1R during the acute phase of MI in some studies has ensured optimal participation of IGF-1/IGF1R system for enhanced cardiac repair [24]. Through IGF-1/IGF1R system, significant progress has been made to promote donor cell survival, engraftment and differentiation in the transplanted heart [25–27]. The main function of IGF-1 in the heart is known to stimulate cardiac growth and contractility [28]. Although there are reports showing left ventricular (LV) hypertrophy caused by IGF-1 administration [29], decreased level of IGF-1 has been indicated in different pathological conditions and implies its critical roles in tissue protection and repair [30]. IGF-1 has distinct beneficial effects on cardiomyocytes including their survival and proliferation. Besides activating molecular pathways of cell survival, IGF-1 accelerates stem cell migration and plays a pivotal role in attracting stem cells toward the heart. Part of its function is through potent activation of paracrine factors. It was reported that MSCs overexpressing IGF-1, showed improved survival and engraftment in the infarcted heart and promoted stem cell recruitment through the paracrine release of SDF-1α. The autocrine and particularly paracrine bioactivity of IGF-1 released from stem cells in terms of cytoprotection has been determined under sever conditions [5]. 1.3. IGF-I signaling pathways play a pivotal role in cell survival and homing MSCs secrete a plethora of angiogenic and mitogenic cytokines, as well as growth factors in normoxic conditions. The secretion of these factors is increased significantly in response to anoxia for reducing the infracted size. The findings imply that MSCs play a crucial role in improving regional blood flow in scar tissue. Indeed, this therapeutic effect of MSCs was overlooked by most studies which then are emphasizing multipotential characteristics of the cells [31]. It has also been shown that concomitant overexpression of Ang-1 and Akt in MSCs promotes their survival in the infarcted heart. It is important to note that the IGF-1/IGF-1R system has a wide distribution in the heart inside the myocytes, cardiac progenitor cells (CPCs), and cardiac fibroblasts, and its activation regulates many functions such as telomerase activity, hinders replicative senescence, and preserves functionally competent CPCs [32]. IGF-1 promotes multiple growth factor expression and releasing, including hepatocyte growth factor (HGF), basic fibroblast growth factor (b-FGF), and vascular endothelial growth factor (VEGF), besides SDF-1α, which stimulate bone marrow and endothelial progenitor cell mobilization towards the ischemic area [5]. Although multiple signaling pathways have been reported downstream of IGF-1/IGF-1R interaction, the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a major role in cytoprotection. IGF-1/IGF1R interaction activates PI3K to the cell membrane that in turn, activates the Akt kinase, thus activating its downstream substrates such as Bcl.xL and inhibiting glycogen synthase kinase (GSK)3β in stem cells (Fig. 1). In other words, overexpression of Akt increases Akt phosphorylation (pAkt) level which in turn brings on the release of bioactive molecules such as SDF-1α to act in a paracrine/autocrine fashion to exert cytoprotective and ionotropic effects [5,33]. Besides its role in cell survival, pAkt negatively regulates the kinase activity of GSK3β (a negative regulator of cell growth). These intracellular changes, for example inactivation of GSK-3β are associated with the expression of muscle specific proteins and potentiate myogenesis, in addition to their pivotal role in cell survival. SDF1-α/CXCR4 ligand/receptor system is a potent activator of stem cell mobilization and homing [34], as well as a modulator of several

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Fig. 1. IGF-1/IGF-1R signaling pathway and its role in promoting stem cell proliferation or inhibiting stem cell senescence. IGF-1 binds to IGF1R and activates PI3K to the cell membrane that, in turn, activates the Akt kinase. Activation of the Akt kinase results in activating its downstream substrates such as Bcl.xL and inhibiting glycogen synthase kinase (GSK)3β, which in turn causes increased cell proliferation and decreased differentiation. Also, activation of PI3K/Akt pathway brings on the level of Akt phosphorylation (pAkt), switches on SDF-1α/CXCR4 signaling pathway and results in accelerated mobilization of progenitor/stem cells and extensive neovascularization.

biological functions including increased cell growth, proliferation, antiapoptosis, and emigrational and transcriptional activation. SDF1-α is also a “retention factor” and ensures retention of the mobilized CXCR4-expressing cells long enough to ensure their participation in the repair process [24]. IGF-1 also promoted multiple growth factor expression, including SDF-1α and VEGF, which stimulate stem progenitor cell migration to the ischemic site. Prolonged expression of SDF-1α has been observed in response to overexpression of VEGF/IGF-I under ischemia condition, which supports the declining intrinsic SDF-1α levels for a long-drawn-out duration and provides a cue for stem cells to mobilize and home into the ischemic site [5,25,35]. Then, SDF-1α levels become significantly heightened after overexpressed-IGF-1/MSC transplantation in the ischemic organ, until a significant presence of mobilized progenitor cells is observed. These results highlight the biological implications of IGF-1 on stem cell mobilization through SDF1α/CXCR4 interaction, in agreement with a recent report that resident and mobilized progenitor/stem cells express SDF-1α receptor CXCR4, affluently. The accelerated mobilization of stem cells results in extensive neovascularization and myogenesis in the infarcted heart. Subsequently, induced expression of CXCR4 and its interaction with the elevated levels of SDF-1α contribute to the antiapoptotic effects in resident cells. The reports also highlight the biological implications of IGF-I on stem cell mobilization through the SDF-1α/CXCR4 system [5]. 1.4. VEGF, another therapeutic target where angiogenesis and cell survival strategy come into play Vascular endothelial growth factor (VEGF) is originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. VEGF, a homodimeric 34–42 kDa protein that is chemotactic for monocytes and immune cells into the site of inflammation and tumors [36,37], increases vascular permeability [38]. VEGF acts as a survival factor to protect stem cells from stresses such as hypoxia and chemotherapy. It plays various roles in normal human physiology,

angiogenesis and cell survival, in particular. The growth factor is up-regulated in response to hypoxia in a physiological condition including wound healing, ovulation and atherosclerosis and is suggested as an attractive target for cell therapy strategies [3,37]. The VEGF/VEGF receptor system is a crucial regulator of the growth and development of the blood vessels in the heart under hypoxic condition [3].The VEGF is one of the most important cytokines participating in the recovery of microvascular injury. It was demonstrated that VEGF directly stimulates to mobilize bone marrow progenitor cells, to induce stem cells and to activate endothelial progenitor cells. VEGF can per se be secreted by MSCs and boost the activity for cardiovascular reconstitution [39]. High levels of VEGF stimulate PDGFR expression, to trigger cell migration and proliferation [40]. Transactivation of VEGF is stimulated by a hypoxic condition which provokes revascularization [41,42]. VEGF plays an important role in embryonic development, as well as angiogenesis during wound healing and menstrual cycle in healthy adults. VEGF is also important in a number of both malignant and non-malignant pathologies. VEGFs are a highly basic heparin binding growth factor, which is structurally similar to VEGF-A and the placental growth factor (PLGF). It is highly abundant in tissues such as heart, skeletal muscle and pancreas, which may act in a paracrine fashion to regulate endothelial cell function [43]. The main functions of VEGF are to promote survival, induce proliferation and enhance migration, besides invasion of endothelial cells for contributing to angiogenesis. It regulates the above functions by interacting with its tyrosine kinase receptors and transmitting signals to various downstream proteins [37]. In particular, the elevation levels of VEGF after the onset of acute myocardial infarction (AMI) have been reported to be associated with cardiovascular protection and improvements of clinical outcomes [44]. VEGF has favorable effects on both vascular endothelial survival and endothelial healing in patients with AMI. VEGF would have cardiovascular protective roles in AMI patients through both promoting endothelial survival under ischemic conditions and inducing compensatory

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processes in the infarcted myocardium [45,46]. Accordingly, low plasma VEGF levels after the onset of AMI would be associated with a significantly increased risk for major adverse cardiovascular and cerebrovascular events [44]. The evidence that VEGF augments perfusion of ischemic myocardium together with reduction in the size of defects, has been reported by several previous clinical trials [47,48]. All of these trial findings clarify that VEGF successfully rescues foci of hibernating myocardium [49,50]. Besides, more recent clinical trials show that intramyocardial VEGF gene transfer is safe and can potentially augment myocardial perfusion and target vessel revascularization in patients with severe chronic ischemic heart disease [49–51]. Also, other clinical documents indicate the beneficial effects of autologous freshly isolated BMC transplantation that would lead to a significant reduction of infarct size and an increase of global ejection fraction (EF) as well as infarct wall movement velocity after a month of follow-ups [52,53]. Additionally transplantation of autologous freshly isolated BMCs has been found which leads to a significant increase of migratory response to SDF-1 and VEGF [54]. Under hypoxia for example, the factor secretion by stem cells can accelerate the development of microvessels and enhance regional blood flow in ischemic area [55]. Although hypoxia can induce VEGF and angiogenesis in ischemic myocardium, on its own it is insufficient to allow to recover cardiomyocytes in ischemic heart [31,56]. Stem cell transplantation significantly increased VEGF level in the scar, border or normal zone of ischemic heart, while the ratio of differentiation into cardiomyocyte is limited. Transplanted VEGF/MSCs exhibit dual functions in cardiac repair after MI by reducing the infarction size and significantly improving the cardiac function through the left ventricular ejection fraction (LVEF) [3]. Marked enhancement of LVEF is due in part to the stem cell integration into the healthy host tissue to form new myocardium [31]. Stem cell integration is beneficial in preventing scar expansion. Functional studies have shown that VEGF/MSC transplantation stimulates extensive angio/myogenesis in the injured hearts as has been indicated by the expression of cardiac troponin T, CD31, and von Willebrand factor, thereby improving the left ventricular performance. VEGF/MSC transplantation system has demonstrated significant increases in the blood vessel formation by the stimulation of the SDF-1α pathway. Activation of the SDF-1α pathway, in turn

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stimulates cardiac stem cells and then leads to their differentiation into endothelial cells in infarcted myocardium. On the other hand, blockage of SDF-1α or its receptor by RNAi or antagonists significantly diminishes the beneficial effects of the VEGF/MSC transplantation system [3,57].

1.5. The paracrine/autocrine activity of VEGF/VEGFR system VEGF is a homing and survival factor expressed by stem cells and MSCs. The MSC-conditioned medium markedly promoted cardiac stem cell (CSC) migration at least in part via the SDF-1α/CXCR4 pathway which is proposed to be involved by the VEGF/VEGFR-1 and VEGFR-3 systems (Fig. 2). In vivo experiments have emerged that VEGF/MSC-stimulated SDF-1 α expression in the infarcted hearts, results in massive mobilization and homing of bone marrow stem cells, as well as growth of CSCs. SDF-1α exerts biological activities through binding to its receptor CXCR4. In the atrioventricular groove, the VEGF-induced SDF-1a/CXCR4 activation also guides the exogenously introduced stem cells to migrate to the infarcted area, which leads to a reduction in the infarcted size [3,5]. Additionally through the SDF-1 α/CXCR4 pathway, the VEGFcontaining medium significantly increases the number of CSCs that express CXCR4, purposely to enhance CSC migration. Quantitative analysis has indicated high expression of SDF-1α in peri-infarction areas where VEGF and MSC have been transplanted [3]. It should be noted that the beneficial effects of MSCs in heart recovery after MI, have been associated with the stimulation of the SDF-1α/CXCR4 pathway in CSCs. So MSCs first express VEGF, to activate the SDF-1a/CXCR4 pathway. Activated SDF-1α then induces mobilization and migration of CSCs into the infarcted areas [3,10]. The VEGF/VEGFR-2 binding system activates the pathway of phosphatidyl inositol kinase (PI3-kinase) and the phosphorylation of Akt/protein kinase B (PKB). VEGF/VEGFR-2-induced cell migration is triggered through activation of the focal adhesion kinase (FAK) and also via the PI3-kinase/Akt pathway. FAK activation is mediated by the c-terminal region of VEGF-R2 [58]. Akt is a serine kinase involved in antiapoptotic signaling. However, VEGF/VEGFR-1 interaction does not activate these pathways and is supposed to be not involved in VEGF mediated cell survival [35].

Fig. 2. The VEGF-triggered stem cell migration is dependent on paracrine/autocrine activation of SDF-1/CXCR4 signaling. Besides having the potential to increase significantly cell survival and proliferation, VEGF/VEGFR system causes at least in part rapid activation of SDF-1α/CXCR4 pathway, in a manner mediating cardiac stem cell (CSC) migration. Moreover, the VEGF/VEGFR system promotes cardiac stem cell migration through either pathway: FAK/PI3-kinase signaling or p38/MAPK pathway.

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VEGF activation of the p38/MAPK stress pathway is also implicated in cell migration, while p38 inhibitors prevent cell migration [59]. 1.6. Which molecular factors are contributing to the VEGF angiogenesis? VEGF has been shown to elevate intracellular inositol 1,4,5trisphosphate and calcium levels, and to stimulate tyrosine phosphorylation and von Willebrand factor release (vWf, a glycoprotein protein which signals the extent of damage in the vascular endothelium), in human umbilical vein endothelial cells [60,61]. VEGF effects on permeability and vascular tone are coupled to nitric oxide (NO) production [62–64]. The angiogenic process is accompanied by vasodilation and involvement of many angiogenic-vasodilator factors such as vasodilator NO. Some studies have shown that chemical mediators, which activate the constitutive NO synthase, as well as NO donors, such as sodium nitroprusside, promote endothelial cell proliferation and migration in vivo and in vitro, while inhibitors of NO synthase suppress these responses [65,66]. The endothelium-derived NO relaxes the vascular smooth muscle cell by inducing cyclic guanosine monophosphate (GMP) in these cells (Fig. 3) [67]. In addition, human monocyte-

induced angiogenesis requires an L-arginine/NO synthase-dependent mechanism [68]. VEGF stimulates the NO synthase pathway, enzyme activity and the levels of cyclic GMP. Subsequently, NO synthase and soluble guanylatecyclase heighten, in turn the activity of VEGF. Sodium nitroprusside and prostaglandin E1 (PGE1) are positive stimulators of cyclic GMP and cyclic AMP, respectively (Fig. 3). Imposingly, the inhibition of NO synthase blocks the in vivo angiogenesis induced by VEGF and by a highly angiogenic carcinoma population. The NO synthase inhibitor L-NMMA blocks the effect of VEGF on cyclic GMP elevation, thus demonstrating that VEGF is linked to the NO synthase pathway. VEGF causes an increase of the calciumdependent NO synthase isoform activity about two fold and the calcium-independent isoform by 36% [69], consistent with the GF signaling through intracellular elevation of inositol 1,4,5-trisphosphate and calcium levels, which lead to the beginning of tyrosine phosphorylation of the pathway and NO release [60,61,69]. Recent reports have indicated that VEGF-stimulated angiogenesis proceeds by an integrin-mediated pathway distinct from that stimulated by bFGF. Conversely, bFGF does not affect either isoform of the

Host /donor stem cell

Fig. 3. Mechanism of VEGF-induced angiogenesis, by triggering tyrosine phosphorylation of the pathway endothelial cell and release of the angiogenic-vasodilator factor NO. VEGF stimulates the NO synthase pathway, which heightens the level of intracellular cyclic guanosine monophosphate (cGMP) to promote endothelial cell proliferation and migration in vivo. The endothelium-derived NO relaxes the vascular smooth muscle cells, as well as promoting progenitor stem cell to replicate by inducing cGMP in these cells, while inhibitors of NO synthase suppress these responses. VEGF causes an increase of the NO synthase isoform activity, through intracellular elevation of the inositol 1,4,5-trisphosphate and calcium levels.

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enzyme and has no effect on cyclic GMP accumulation but slightly increased cyclic AMP production. Inhibition of the soluble guanylatecyclase impairs the adhesion induced by either VEGF or bFGF and this effect is possibly involved in the reduction of DNA synthesis and migration. The data presented here clearly demonstrate that NO production significantly contributes to the growth-promoted effect of VEGF, but not for the growthpromoted effect of bFGF [69,70]. The findings also exhibit an important role for erythropoietin (EPO) in VEGF-induced neovascularization. In heart failure, the paracrine action of both factors, VEGF and EPO on cardiomyocytes, is crucial for cardiac improvement through myocardial angiogenesis. EPO heightens VEGF expression in various ischemic tissues, which is strongly correlated with new vessel formation [71–73]. VEGF expression is especially apparent in cardiomyocytes, under ischemic conditions in the heart. Subsequent EPO treatment of rats with post-MI HF, results in an increased protein expression of VEGF in the LV wall. The mice that lack an EPO-receptor (EPO-R) in the heart, display defective VEGF expression, and dramatically accelerated development of left ventricular (LV) dysfunction during pressure overload [71,74]. EPO treatment also increases the VEGF-immunoreactive area, suggesting that EPO additionally increases the number of cells that produced VEGF [74]. Notably, neutralization of VEGF does not revoke the effects of EPO on aortic sprouting, while completely abrogates the salutary effects of EPO on cardiac function and microvascularization [71,74]. Under ischemic conditions, EPO stimulates VEGF-production predominantly through the JAK2/STAT-3 signal transduction pathway. Considerably, EPO does not stimulate VEGF transcription in fibroblasts or endothelial cells and the VEGF-independent angiogenic effect of EPO in the aortic ring is modest [74]. 2. Discussion The short biological half-life and low-produced level of chemotactic factors including growth factors and chemokines warrant a steady source to ensure their non-fluctuating and consistent levels during regenerative processes in the biological system. Injected stem cells would continue to secrete required factors and proteins until several days of post-engraftment in the target tissue. Given that the cell therapy has limitations, we surveyed the literature for the relevant articles which have considered a combined transplantation system consisting of stem cells beside growth factors. Previous studies have shown that growth factor expressions are elevated as part of the intrinsic repair mechanism after heart infarction or organ injury. Transplantation of MSCs/GFs during the acute phase of MI in the studies has ensured optimal participation of GF/GFR signaling pathways for enhanced cardiac repair [75]. Additionally, donor cell attrition is a crucial factor, and successful engraftment is conversely dependent on cell maintenance and survival. Combining cell therapy with therapeutic chemotactic factors is supposed to overcome this problem especially during the acute phase of engraftment, and is more exciting in terms of donor cell survival and differentiation. Studies have also shown that concomitant overexpression of signaling molecules such as angiopoietin-1 and Akt in MSCs promoted stem cell survival in the infarcted heart [76]. It is important to note that GF/GFR systems have a wide distribution on various cells at different organs such as the heart on myocytes and cardiac progenitor cells (CPCs). The activation of such systems regulates many functions such as telomerase activity, hinders replicative senescence, and preserves functionally competent organ progenitor cells [32,75]. Preconditioning of stem cells with the proper chemotactic factors has enhanced their paracrine/autocrine activity and shows multiple fold increase in their response to GF/GFR systems, as well as GF expression [5]. However, stem cell (SC)/GF model of transplantation is expected to yield more persistent supply of the chemotactic factors in the infarcted organ at the site of cell engraftment, for a longer time duration as compared

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with the preconditioned cell engraftment [27,77,78]. The major growth factor systems mentioned in this review, the IGF-I/IGF-IR and VEGF/VEGFR systems exert their biological effects not only by activating Akt phosphorylation through the PI3K signaling pathway which serves as an essential mediator of the GF/GFR systems [5,79], but also by the SDF-1α/CXCR4 interaction which improved the survival of host progenitor/donor stem cells and has been attributed to the antiapoptotic effects of IGF-I/IGF-IR and VEGF/VEGFR systems. The presence of paracrine pathways in progenitor stem cells is an important aspect which contributes to the maintenance of normal stem cell property through the function of IGF-I/VEGF [10,72,73,80]. Then, the proper local concentration of bioactive molecules provides an alternative or supportive mechanism for host progenitor/donor stem cell replicating/homing during tissue regeneration. Compared to the other angiogenic growth factors such as bFGF, IGF-1 and VEGF are more potent inducers of proper angiogenic response. Moreover, pro-angiogenic activity of IGF-1 is associated with VEGF up-regulation; conversely active VEGF release is following IGF-1 overexpression [5,81]. Finally, the combined effect of these possible contributory factors is significantly dependent on the mobilized progenitor cells or in particular the donor integrated stem cells. It is, however, difficult to delineate the independent role of each one of the contributory factors toward the improvement in regeneration of the organ and proper function in general and that of the mobilized cells, in particular. In summary, the SC/GF model of transplantation serves as a reservoir of chemokines, which act in autocrine and paracrine fashion to activate survival signaling in host/donor progenitor stem cells. The combined admission of the engrafts also promotes multiple signaling pathways, including SDF-1α/CXCR4 ligand/receptor, IGF-I/IGF-IR and VEGF/VEGFR pathways, which stimulate BMMSC and endothelial progenitor stem cell mobilization to the ischemic niches, besides mediated cell survival and proliferation in the injected site. The accelerated mobilization of stem cells resulted in extensive neo-vascularization and regeneration of the tissues in the infarcted organ. Several reports highlight the beneficial implications of special growth factors on stem cell homing through chemokine signaling, in particular, through the SDF-1α/CXCR4 interaction pathway. 3. Conclusion The important findings of this review are as follows: (1) in the combined model of SC/GF transplantation, host progenitor/donor stem cells engraft and survive better under anoxia as compared with stem cell admissions; (2) release of chemokines, specifically SDF1-α from the SC/GF system contributes to massive host stem cell mobilization and homing; (3) extensive presence of newly formed blood vessels and tissue repair have been observed after SC/GF transplantation; and (4) in the infarcted heart, GF-localized overexpression significantly preserves LV wall thickness and contractile function. Acknowledgment We are indebted to the Shahid Rahnemun Research Center, Shahid Sadoughi University of Medical Sciences, for their kind support. References [1] Da Silva JS, Hare JM. Cell-based therapies for myocardial repair: emerging role for bone marrow-derived mesenchymal stem cells (MSCs) in the treatment of the chronically injured heart. Methods Mol Biol 2013;1037:145–63. [2] Pons J, Huang Y, Arakawa-Hoyt J, et al. VEGF improves survival of mesenchymal stem cells in infarcted hearts. Biochem Biophys Res Commun 2008;376(2):419–22. [3] Tang JM, Wang JN, Zhang L, et al. VEGF/SDF-1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart. Cardiovasc Res 2011;91(3):402–11. [4] Orlic D, Kajstura J, Chimenti S, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A 2001;98(8):10344–9.

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