Myocardial protection

An immediate paracrine effect of stem cells in an ischemic environment is the release of cytoprotective molecules that increase cardiomyocyte survival (Figure 3). Our group characterized the spectrum of BM stem cell paracrine actions and demonstrated that MSCs exert direct cytoprotective action on ischemic cardiomyocytes. In particular, we showed that cell culture medium conditioned by hypoxic MSCs can reduce apoptosis and necrosis of isolated rat cardiomyocytes exposed to low oxygen tension²⁵. The cytoprotective effect was greatly enhanced in MSCs overexpressing the gene Akt-1 (Akt-MSCs) in vitro. To further validate the protective properties of the MSCs, we studied the effect of the CM in vivo, using a rat experimental model of coronary occlusion. Concentrated CM was injected into the heart at the infarct border zone 30 min after left coronary occlusion. After 72 hours, the infarct size and the cardiomyocyte apoptotic index were reduced, albeit modestly, in animals treated with CM from MSCs (MSC-CM) compared with those treated with saline. In contrast, a dramatic reduction of infarct size and cardiac apoptosis was observed after administration of CM from Akt-MSCs (AKT-MSCs-CM). These data showed that MSCs exert cardiac protective effect through the release of paracrine factors and that the activation of the Akt pathway markedly enhances the production/release of these putative molecules. In a follow-up study we confirmed our previous results and documented how the limitation of the infarct size was matched by preservation of cardiac function²⁶. To verify whether Akt overexpression upregulated the expression of secreted factors, we tested by quantitative RT-PCR some candidate genes encoding for molecules known to be released by MSCs. Our data showed that VEGF, bFGF, HGF, insulin growth factor 1 (IGF-1), and thymosin β4 (TB4) were significantly up-regulated in the Akt-MSCs at baseline normoxia and increased further after exposure to hypoxia. After exposure to hypoxia, native MSCs also up-regulated VEGF, bFGF, HGF, and TB4 albeit less than the Akt-MSCs. Recently, our original findings in rodents have been successfully replicated by others also in a large animal model. Indeed, Akt-MSCs injected into pig infarcted hearts led to limitation of infarct size and preservation of heart function³⁴.

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Figure 3

Proposed factors and signalling pathways involved in stem cell-mediated myocardial protection^{9, 26, 90–95}

It has been shown that ASCs mediate cardioprotection by producing and releasing soluble mediators with known cytoprotective properties. Most of these factors act through the activation of the pro-survival PI-3 kinase/Akt pathway. Factors like FGF-2 and EPO can activate the protein kinase C pathway, which has been shown to mediate cardioprotection. Interleukin 11 (IL11) also activates survival signalling cascades in PI3K/Akt and ERK1/2-STAT3 dependent fashion. The mechanisms of action through which Sfrp2 and TB4 lead to cardioprotection are partially undetermined and need to be further clarified. Finally, other factors and pathways are certainly involved but are still unidentified. Abbreviations: IL11, interleukin 11; HGF, hepatocyte growth factor; IGF-1, insulin growth factor 1; VEGF, vascular endothelial growth factor; EPO, erythropoietin; FGF-2, fibroblast growth factor-2; TB4, thymosin beta 4; GF, growth factor; C, cytochrome c; GLUT, glucose transporter; VDAC, voltage-dependent anion channel.

Other groups have confirmed the paracrine cytoprotective effects exerted by BM-derived stem cells on ischemic cardiomyocytes^{32, 35, 36}. Uemura and colleagues recapitulated our in vitro data adopting a different approach³⁵. Freshly isolated adult mouse cardiomyocytes were exposed for 3 hours to hypoxia. Positive annexin V staining and DNA laddering were both indicative of apoptosis. However, if the cardiomyocytes were cocultured with BM-MSCs hypoxia induced apoptosis was significantly reduced. High levels of VEGF, bFGF, IGF-1, and SDF-1 were present in the MSC-CM, especially after exposure to hypoxia. To confirm the cytoprotective effect in vivo, the authors injected GFP-labelled MSCs into infarcted mouse hearts. Three groups were considered: sham, MSCs and hypoxia-preconditioned MSCs. The cells engrafted mostly in the border area. However, the frequency of GFP-positive cardiomyocytes was very low and did not differ between the 2 cell groups. TUNEL staining showed that the number of apoptotic cardiomyocytes was significantly reduced in MSC-treated animals, particularly when preconditioned cells were administered. Accordingly, infarcted area and ventricular remodeling were significantly prevented in animals treated with hypoxic MSCs compared with those receiving normoxic MSCs.

Takahashi and colleagues showed that also rat BM-MNCs produce and release various cytoprotective factors, including VEGF, PDGF, IL-1β, and IGF-1, some of which are significantly upregulated by hypoxia³². Administration of CM from BM-MNCs cultured under normoxia or hypoxia inhibited apoptosis in vitro and reduced infarct size in vivo. Also human BM-MNCs exert potent cytoprotective effects³⁷. Kubal and collaborators exposed muscle cells from the atrial appendage of patient undergoing elective cardiac surgery to hypoxia/reperfusion stress. When cardiac cells were coincubated with BM-MNCs, harvested from the same patients, reduction of cell necrosis and apoptosis was documented. Of note, coincubation with control ECs or keratinocytes did not result in cytoprotection, suggesting that the effect was specific for BM-MNCs. Finally, it has been shown that also hBMSCs induce cardiac protection³⁰.

Neovascularization

Another important biological process positively influenced by stem cells in a paracrine fashion is neovascularization. Despite evidences that BM-derived stem or progenitor cells incorporate into vascular structures, several studies suggest that only a small number of vessels contain donor cells. The molecular processes leading to angiogenesis and arteriogenesis involve mediators such as nitric oxide, VEGF, bFGF, HGF, angiopoietin and others. These molecules lead to EC and VSMC migration, proliferation, vessel enlargement and maturation, and synthesis of extracellular matrix. As described earlier, it has been shown that BM stem cells can express proangiogenic molecules²⁹. Accordingly, it has been hypothesized that the release of putative proangiogenic factors may play an important role in determining the increase in capillary density and collateral development observed in ischemic tissues of animals treated with stem cells. For example, it has been reported that the injection of BM-MNCs into a zone of swine heart made ischemic by coronary ligation resulted in a significant increase of regional blood flow and capillary density at 3 weeks³⁸. The authors showed that the BM-MNCs injected expressed bFGF, VEGF, and angiopoietin-1 and that the cardiac levels of these angiogenic ligands were significantly increased 3 weeks after stem cell injection compared with controls. Furthermore, the cardiac levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were markedly increased after BM-MNC administration. Since also IL-1β and TNF-α were shown to have angiogenic activity, the authors concluded that the released factors likely contributed to stimulate angiogenesis in BM-MNC treated animals. The proangiogenic paracrine action of BM-MNCs has been validated by the documentation of increased microvessel density in ischemic hearts injected solely with CM from MNCs³².

We and others have demonstrated that MSC represent a source of paracrine proangiogenic and proarteriogenic factors^{26, 29, 39}. Data from Epstein and collaborators have suggested that in vivo local delivery of MSCs augments collateral perfusion through paracrine mechanisms²⁷. These authors injected 1×10⁶ MSCs in the adductor muscle of mice 24 hours after femoral artery ligation. Compared with controls injected with medium or mature ECs, distal limb perfusion improved and conductance vessels increased in number and total cross-sectional area. Surprisingly, labelled MSCs were dispersed between muscle fibers but not seen incorporated into mature collaterals. On the other hand, protein levels of VEGF and bFGF were significantly increased in the muscle of MSC-treated animals compared with controls; furthermore, colocalization of VEGF and transplanted MSCs within adductor tissue was documented. The authors concluded that MSCs contributed to collateral remodeling through paracrine mechanisms. Gene expression profiling of MSCs grown under normal conditions or under hypoxia stimulation allowed to document that these cells express a wide range of arteriogenic cytokines at baseline and that several of them are upregulated by hypoxia²⁹. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC-CM. Furthermore, it was shown that MSC-CM promoted proliferation and migration of ECs and VSMCs in a dose-dependent manner in vitro and enhanced collateral flow recovery and remodeling in a model of hind limb ischemia in vivo. Other studies, testing MSC transplantation in experimental infarcted hearts, reported an increase in capillary density in treated animals compared with controls, despite the presence of few ECs of donor origin^40–42. In these cases, even though not directly proven, a proangiogenic paracrine action seems the most reasonable explanation to the effects observed.

Finally, it has been shown that EPCs can enhance angiogenesis by releasing cytokines and chemokines. In particular, VEGF and stromal derived factor 1 (SDF-1) released in the CM by EPCs seem to promote the migration of mature ECs in vitro through differentiation-indipendent mechanisms⁴³. In addition, several reports showed that transplantation of EPCs into ischemic hearts promotes angiogenesis and arteriogenesis in vivo^44–46. Taken together, all these reports show that, besides direct vasculogenesis, ASCs improve neovascularization of ischemic tissues through proangiogenic and proarteriogenic paracrine effects (Figure 4).

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Figure 4

Mechanisms involved in neovascularization

In contrast to the embryonic heart vasculature, the adult heart vessels are quiescent. Only when under stress or in the presence of pathologic conditions, like myocardial infarction, the coronary vascular bed expands. ASCs are involved in postnatal neovascularization which encompasses 3 different mechanisms: the first is referred to as postnatal vasculogenesis, that consists in the assembly of new blood vessels by fusion and differentiation of endothelial precursor cells originating from the bone marrow. The second mechanism is angiogenesis and consists in the sprouting of new vessels from pre-existing vessels. The third mechanism is collateral enlargement and muscolarization, namely arteriogenesis. The release of proangiogenic and proarteriogenic factors by transplanted stem cells positively influences neovascularization in a paracrine fashion.

Cardiac remodeling

Beside cytoprotection and neovascularization, paracrine factors released by transplanted stem cell may alter the extracellular matrix, resulting in more favorable post-infarction remodeling and strengthening of the infarct scar. It has been shown that direct hMSC injection into ischemic rat hearts decreases fibrosis, apoptosis and left ventricular dilatation while increases myocardial thickness; this results in the preservation of systolic and diastolic cardiac function without evidence of myocardial regeneration⁴⁷. In animal models, MSC transplantation decreases fibrosis in the heart³¹ and other organs such as lung⁴⁸, liver⁴⁹ and kidney⁵⁰. It has been show that MSCs express a number of molecules involved in biogenesis of extracellular matrix such as collagens, metalloproteinases (MMPs), serine proteases and serine protease inhibitors, suggesting that transplanted MSCs can inhibit the fibrosis through paracrine actions³⁹. Xu and colleagues evaluated the effects of grafting MSCs on extracellular matrix in infracted rat hearts⁵¹. They showed that MSC transplantation significantly attenuated the increased cardiac expression of collagen types I and III, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor growth factor beta (TGF-β) observed in infarcted control hearts. Nagaya’s group⁵² found that MSC-CM significantly attenuated cardiac fibroblast proliferation and inhibited type I and type III collagen expression in cardiac fibroblasts; in addition, they showed that collagen activity in the MSC-CM was as high as the activity in the medium conditioned by cardiac fibroblasts. The authors concluded that MSCs exerted paracrine anti-fibrotic effects at least in part through inhibition of cardiac fibroblast proliferation and types I and III collagen synthesis.

Important data supporting the anti-remodeling effect exerted via paracrine action by BM-derived cells come from studies testing stem cell therapy to treat nonischemic cardiomyopathy. Nagaya and collaborators injected BM-MSCs into the myocardium of rats in which dilated cardiomyopathy was produced by inducing experimental myocarditis³¹. Compared with vehicle injection, MSC administration significantly increased capillary density and, most importantly, decreased the collagen deposition in the myocardium, resulting in decreased left ventricular end-diastolic pressure and increased left ventricular maximum dP/dt. The importance of paracrine mechanisms in this particular experimental model was underscored by the limited number of MSC-derived cardiomyocytes, which was clearly insufficient to result in the recovery of cardiac function reported. Of note, relative quantification of cardiac proteins showed that MMP-2 and MMP-9 significantly increased in DCM whereas in MSC treated hearts MMP level were comparable to sham. Even though the underlying mechanism remains unclear, taken together these data suggest that BM-derived ASC transplantation may influence extracellular remodeling in heart failure.

Stem cells may also produce and release local signaling molecules that limit local inflammation when injected into injured tissues. This hypothesis seems to be supported by the fact that expression profiling of adult progenitor cells has revealed expression of genes typically associated with enhanced DNA repair, antioxidant enzymes, and genes linked with detoxifier systems⁵³. Ohnishi and coworkers⁵⁴ demonstrated that MSC transplantation into a rat model of acute myocarditis attenuated the increase in CD68⁺ inflammatory cells and monocyte chemoattractant protein-1 (MCP-1) expression in the myocardium and improved cardiac function. Isolated adult rat cardiomyocytes (ARVCs) cultured in standard conditions were injured by MCP-1, which may play an important role in myocarditis⁵⁵; in contrast, in the presence of MSC-CM the MCP-1-induced injury was significantly attenuated.

Cardiac contractility and metabolism

Some evidences suggest that the administration of ASCs positively influences also cardiac contractility. For example, with regard to the early functional improvement of acutely infarcted hearts after injection of Akt-MSC-CM, the effect may be the consequence of myocardial protection leading to infarct size limitation and/or the direct action of inotropic factors released by the cells. As for the former mechanism, it has been well documented that therapies able to limit myocardial infarct size can prevent ventricular dysfunction⁵⁶. In support of the latter, we observed, in the presence of the CM from hypoxic Akt-MSCs, a marked increase in spontaneous contractile activity in ARVCs exposed to prolonged hypoxia²⁶. Such contractile activity was very different from the typical spontaneous contractility observed when the ARVCs are maintained in standard growth conditions. In this case, the vigorous and synchronized contraction observed may suggest the presence of inotropic factors in the CM able to positively influence the contractile properties of ARVCs. Preliminary experiments on freshly isolated adult rat cardiomyocytes exposed to standard growth medium or to different concentrations of MSC-CM or Akt-MSC-CM seem to confirm that MSCs do release inotropic factors that positively modulate cell contractility. Indeed, using an edge-to-edge detection system we observe that cell shortening, maximal rate of relenghtening (+dL/dt), and maximal rate of shortening (−dL/dt) are significantly improved in the presence of CM from hypoxic MSCs, particularly Akt-MSCs, compared with standard conditions (unpublished data). Results from other groups would confirm the paracrine action of BM-derived stem cells on cardiomyocyte contractility. For example, Takahashi and collaborators showed that CM from BM-MNCs preserved the contractility capacity of ARVCs³². Fractional shortening and ± dL/dt of ARVCs cultivated for 72 hours in standard growth medium were significantly lower compared with those recorded at baseline. Conversely, cell contractility parameters did not decrease significantly when ARVCs were cultured in the presence of CM from BM-MNCs pre-exposed to normoxia or hypoxia. Of note, the supernatant from hypoxic BM-MNCs was more effective compared with CM from normoxic cells in preserving ARVC contractility. This observation further suggests that production and release of the factors mediating inotropic paracrine effects are increased under hypoxia. More detailed experiments are needed in order to elucidate the pathways and the mediators involved. At the moment, the nature and identity of putative molecules can only be hypothesized. For example, it is known that, in addition to its growth-promoting and anti-apoptotic actions, IGF-I can increase cardiomyocyte contractility in vitro⁵⁷ and improve myocardial function both in normal and infarcted adult rat hearts^{58, 59}. The possibility of nonpeptidic molecules mediating this effect cannot be excluded.

Interestingly, Dhein and collaborators have reported that administration of BM-derived stem cells in doxorubicine induced decompensated rabbit hearts significantly limited the down-regulation of β-adrenoceptor normally present in this heart failure model and significantly increased cardiac contractility measured in vivo⁶⁰. It is well known that β-adrenoceptors couple to adenylate cyclase and mediate positive inotropic effects of catecholamines. In this particular case, the positive and significant correlation between β-adrenoceptor density and contractility described would support the hypothesis on a relationship between these two parameters. Interestingly, in failing hearts receiving BM cell transplantation β-adrenoceptor density was nearly normalized in the septum while was still below the normal level in the left ventricle. Considering that the cells were injected in the free wall and that the number of engrafted cells was low, the authors proposed that the effects observed might be due to paracrine action. However, it cannot be excluded that the improved hemodynamic situation resulted in decreased catecholamine levels, and that this consequently led to attenuation of β-adrenoceptor down-regulation. Experiments on isolated cardiomyocytes cocultured with BM-derived stem cells or in the presence of CM may help to clarify this issue.

It has also been suggested that administration of BM-derived cells may positively affect cardiac metabolism in ischemic hearts. Feygin et al. examined whether mesenchymal stem cell transplantation-induced beneficial effects are secondary to paracrine-associated improvements in left ventricular (LV) contractile performance, wall stress, and myocardial bioenergetics in hearts with postinfarction LV remodeling in pigs. They demonstrated that hearts in the AMI group developed severe contractile dyskinesis in the infarct zone and border zone, while MSC transplantation significantly improved contractile performance from dyskinesis to active contraction. Border zone systolic wall stress was severely increased in infarcted hearts but significantly improved after MSC transplantation. The border zone demonstrated profound bioenergetic abnormalities in infarcted hearts, which were attenuated after MSC transplantation. Because of low cell engraftment, the authors postulated that MSCs did not provide a structural contribution to the damaged heart and concluded that the observed beneficial effects likely resulted from paracrine repair mechanisms⁶¹.

Enhancing beneficial paracrine effects through genetic modification

The observation that BM-derived stem cells, particularly those which are genetically modified, may release of therapeutic paracrine factors provide an important conceptual advancement in that one can enhance stem cell therapeutic actions through gene transfer. In this context, Akt-MSCs revealed themselves as a useful model to identify possible novel therapeutic molecules, suggesting that genetic modification of stem or progenitor cells may represent an important strategic advancement in regenerative medicine. By combining gene with cell therapy, one may be able to enhance stem cell function and viability. Indeed, genetic modification can improve survival, metabolic characteristics, proliferative capacity, or differentiation of the stem cells. Furthermore, the cells may become a vehicle for gene therapy whose secreted gene products can exert paracrine or endocrine actions that may result in further therapeutic benefits. Our group was the first to conceptualize this approach^{76, 77} and showed that genetically modified EPCs had greater reparative potential and that MSCs overexpressing the prosurvival gene Akt-1 had greater post-implantation survival and functional benefits than did wild-type cells. Thought as a strategy to increase cell viability, the overexpression of Akt turned out to have many more effects on MSCs such as the production of secreted factors capable of protecting ARVCs both in vitro and in vivo. The salutary effects of this approach have been further validated and extended by several groups. BCL-2 engineered MSCs were shown to increase the survival of the MSCs after AMI in rats, leading to higher expression level of VEGF, increased capillary density and smaller infarct size relative to controls⁷⁸. Tang and collaborators⁷⁹ found that a higher number of MSCs transduced with heme oxygenase (HO)-1 survived when they are injected into infarcted hearts, leading to more efficient healing compared with control MSCs. Based on the data reported, the beneficial effects obtained with the HO-1-MSCs appear also to be primarily due to paracrine protective action, even though this hypothesis was not directly addressed in this study. Indeed, the low differentiation rate of MSCs into cardiomyocytes suggested by the authors could not alone explain the structural and functional improvements reported. Thus, as in the case of the Akt-MSCs, the original intervention, intended to increase stem cell viability, might have induced paracrine effects on adjacent myocardium. Similarly, HGF⁸⁰, bFGF⁸¹, adrenomedullin⁸² or VEGF-engineered MSCs⁸³ were shown to survive better and to exert more powerful cytoprotection and/or pro-neovascularization effect compared with control MSCs.

This work was supported by grants from the National Heart, Lung, and Blood Institute (RO1 HL35610, HL81744, HL72010, and HL73219 to Victor J. Dzau), the Edna Mandel Foundation and the Foundation Leducq (to Victor J. Dzau). Massimiliano Gnecchi is supported by the Fondazione IRCCS Policlinico San Matteo Pavia, Italy and by grants from the Ministero Italiano dell’Università e della Ricerca (MIUR), from the Ministero Italiano della Sanità, from the Fondazione Cariplo, and from the Fondazione Banca del Monte di Lombardia.