Recently, emerging cell therapy strategies have sought to reverse post-infarction left ventricular dysfunction by direct transplantation of stem or progenitor cells. Enthusiasm for this therapeutic approach has been bolstered by a number of promising early phase human clinical trials [1
], but the precise mechanism(s) underlying the apparent beneficial effects have yet to be fully elucidated.
Progenitor cells have been variously hypothesized to regenerate myocardium by transdifferentiation and/or by coaxing non-cardiac or resident progenitor cells to a cardiac muscle fate, thus augmenting recovery of myocardial mass and retarding pathologic LV remodelling [5
]. Progenitor cells may also harbor beneficial paracrine or neurohormonal activity which thus far has been poorly characterized. In earlier studies, circulating progenitor cells cultured under conditions similar to the present investigation were shown to secrete various cytokines with potential cardiotrophic and neoangiogenic effects, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating growth factor (G-CSF), granulocyte-macrophage colony-stimulating growth factor (GM-CSF), stromal derived factor 1, and insulin like growth factor 1(IGF-1) [11
]. Soluble factors released by endothelial progenitor cells are known to augment migration of native endothelial cells and cardiac resident progenitor cells in vitro
]. Moreover, treatment with conditioned media derived from mesenchymal cells overexpressing the survival gene Akt1
demonstrated reduction in apoptosis of adult ventricular cardiomyocytes exposed to hypoxia and subsequent infarct size in an rodent acute myocardial infarction model [10
The major findings of the current study were: (1) progenitor cell therapy altered post-infarction remodeling by increasing infarct territory mass, associated with improved regional systolic function (2) therapy with conditioned media alone (derived from progenitor cells) altered infarctrelated remodeling in vivo in a similar fashion to cell therapy (3) significant increases in cardiomyocyte size in vivo were observed in the treated territory in both cell and conditioned media treated animals (4) a cardiotrophic effect of progenitor cell-derived conditioned media was confirmed in vitro, an effect that could in part be attributed to TGFβ1 secreted from both human and porcine CPC studied.
Our studies suggest that induction of cardiac hypertrophy in the infarct border zone may be a heretofore unrecognized mechanism contributing to progenitor cell therapy-mediated cardiac repair. Post-infarction cardiac hypertrophy is initially adaptive and usually develops in myocardial regions remote from the acute infarction. In contrast, we have observed cardiac hypertrophy occurring not only on serial measures of global LV mass but specifically including the infarct border zone. Importantly, these effects occurred as a result of a non-sustained stimulus in the early aftermath of acute infarction. Whether this intriguing pattern of postinfarction hypertrophy that includes the infarct territory is associated with more favorable long term repair is still unclear. It is intriguing to speculate whether early paracrine factor release in the infarct zone kick starts a program of cardiomyocyte hypertrophy.
Over the past decade there has been a growing appreciation of the role of vascular endothelium in regulating cell and organ size [30
]. Given the capacity for injected progenitor cells to induce significant neovascularisation [27
], it is interesting to consider the possibility that interplay between secreted proangiogenic growth factors (such as VEGF) and cardiotrophic factors (such as TGF 1 and IGF1) may also orchestrate progenitor cell-induced cardiac hypertrophy. It is conceivable that such an interplay may contribute to durable cardiac repair as disruption of coordinated cardiac hypertrophy and angiogenesis has been shown to mediate an eventual transition from compensated hypertrophy to heart failure [33
TGFβ1 has recently been shown to induce hypertrophic responses in cultured neonatal rat cardiomyocytes via PKC-dependent ATF-2 activation [20
]. Upregulation of TGFβ1 occurs in myocardium that has undergone hypertrophy as a result of pressure overload [34
] and following exposure to a hypertrophic agonist such as angiotensin II [21
]. Furthermore, in a rodent model of myocardial infarction upregulation of TGFβ1, its receptors TβR1 and TβR2, and the downstream signal transducer TGF-β-activated kinase has been detected in remote myocardial territories [38
]. Activation of this pathway paralleled the transcriptional upregulation of cardiac markers for ventricular hypertrophy, linking TGFβ1 signaling to the development of compensatory post-infarction myocardial hypertrophy [38
]. A multifaceted role for progenitor cell-derived TGFβ1 in cardiac repair might also include cardioprotection during ischemia-reperfusion [39
] and prosurvival signaling to endothelial cells during angiogenesis [40
Finally, a deleterious role for myocardial TGFβ1 signaling during post-infarction ventricular remodeling should also be considered. Using a mouse model of coronary ligation, Okada et al have demonstrated that inhibition of circulating TGFβ1 through adenoviral-mediated overexpression of the soluble TGFβ1 type 2 receptor attenuates post-MI fibrosis and infarct wall thinning, improving post-infarct contractile function and mortality [46
]. Timing of therapy to modulate TGFβ1 signaling in this setting may be of critical importance, as previous studies have demonstrated that antagonism of TGFβ1 at the time of infarction may attenuate late remodeling [48
], contrasting the findings of Okada et al where treatment was initiated at 3 days post-infarct [46
]. Further studies will be required to fully elucidate the complex role of this cytokine in post-MI regeneration and repair.
The use of progenitor cell nomenclature is challenging in this porcine model given that most of the accepted human antibodies used to discriminate human EPC populations such as CD133 and CD34 do no work in the pig. However, we have used a cell processing and culture protocol which was identical to that used in the human TOPCARE AMI trial which the current study attempted to model. Moreover the porcine MNC-derived cells in culture shared many antigens found in human EPCs such as Flk1, eNOS, CD105 as well as positivity for AcLDL and Isolectin B4. However, it must also be acknowledged that the progenitor cells used in this study were a heterogenous preparation and therefore the specific lineage of the progenitor cells contributing IGF1 and TGFβ secretion is still unclear.
Progenitor cell therapy in this animal model recapitulates the findings of previous human studies demonstrating preservation of infarct thickness, reduction in infarct size and improved regional systolic function following cell therapy [3
]. There are also a number of differences between this model and human cell therapy that are worthy of mention. First, progenitor cell function may be altered or impaired in patients receiving cell therapy, particularly the elderly and those with multiple risk factors for vascular disease [32
], whereas the animals studied here were all healthy prior to induction of myocardial infarction. Second, adjunctive post-infarction pharmacotherapy may interfere with the signalling pathways of the candidate paracrine factors which we have identified, modulating this component of the reparative response to cell therapy in humans. Specifically, angiotensin II blockade [42
], statins [43
] and β-blockers [45
] may affect growth factor expression and/or signaling in the heart. Interplay of this kind between cell and pharmacologic therapy for acute myocardial infarction has not yet been examined in human or experimental studies, but certainly merits greater attention as this treatment approach evolves. Finally the focus of this study was the cardiomyocyte size and LV mass changes after cell therapy (with particular emphasis on changes in the infarct and border zone) and not traditionally studied parameters such as angiogenesis and scar formation.
In conclusion, this study shows circulating progenitor cells exert a potent paracrine effect impacting cardiomyocyte size with direct consequences for infarct-related remodeling post myocardial infarction. These data strongly support a robust contribution of progenitor cells to regional post infarct repair with mechanistic and therapeutic implications for future human studies in the cell therapy field.