Administration of endothelial progenitor cells (EPC) is a promising therapy for post-infarction cardiac repair. However, the mechanisms that underlie apparent benefi cial effects on myocardial remodeling are unclear. In a porcine model of acute myocardial infarction, we investigated the therapeutic effects of a mixed population of culture modifi ed peripheral blood mononuclear cells (termed hereafter porcine EPC). Porcine EPC were isolated using methods identical to those previously adopted for harvest of EPC in human cell therapy studies. In addition the therapeutic effects of paracrine factors secreted by these cells was evaluated in vitro and in vivo. Intracoronary injection of autologous porcine EPC was associated with increased infarct territory mass and improved regional ventricular systolic function at 2 months compared to control. Treatment with conditioned media derived from autologous EPC was associated with similar improved effects on infarct territory mass and function. Histologic analysis of the infarct territory revealed signifi cantly increased cardiomyocyte size in EPC and conditioned media treated groups, when compared to controls. A paracrine EPC effect was also verifi ed in a pure myocardial preparation in which cardiomyocytes devoid of fi broblast, neuronal and vascular elements directly responded by increasing cell mass when exposed to the same conditioned media. Analysis of conditioned media revealed elevated levels of TGFβ1 (human 267.3±11.8 pg/ml, porcine 57.1±6.1 pg/ml), a recognized mediator of hypertro-phic signaling in the heart. Neutralizing antibodies to TGFβ1 attenuated the pro-hypertrophic effect of conditioned media, and use of recombinant TGFβ1 added to fresh media replicated the pro-hypertrophic effects of conditioned media in vitro. These data demonstrate the potential of paracrine factors secreted from endothelial progenitor cells to induce cardiomyocyte hypertrophy contributing to increased infarct territory LV mass, with favorable medium term effects on regional function following myocardial infarction. STEM CELLS AND DEVELOPMENT 17:941-952 (2008)

Rationale: Smooth muscle precursor cells have previously been reported to reside in bone marrow and in the circulation, but little is currently known regarding the proximate stimuli for smooth muscle cell differentiation of these putative progenitors. Objective: Because local thrombin generation occurs as an initial response to vascular injury, we hypothesized that thrombin may influence the differentiation of circulating smooth muscle progenitor cells. Methods and Results: Peripheral blood mononuclear cells were cultured on type I collagen using a protocol optimized to stimulate smooth muscle cell outgrowth. Thrombin-stimulated upregulation of the transcription factor myocardin and smooth muscle myosin heavy chain, and both were inhibited by hirudin or the RhoA inhibitor Y27632. After 10 days of culture, smooth muscle outgrowth colonies formed, which stained positive for ␣-smooth muscle actin, smooth muscle myosin heavy chain, and calponin, in addition to having a contractile response to 100 nmol/L angiotensin II. Coincubation of peripheral blood mononuclear cells with thrombin, 10 mol/L protease-activated receptor-1, but not protease-activated receptor-4 activating peptide significantly increased the number of smooth muscle outgrowth colonies formed. Thrombin-induced enhancement of smooth muscle outgrowth colony formation was inhibited by hirudin, Y27632, and an antibody against protease-activated receptor-1. Conclusions: These data illustrate a novel thrombininduced pathway for smooth muscle differentiation from putative smooth muscle progenitors in peripheral blood. W e have previously described circulating smooth muscle outgrowth cells (SOCs). 1,2 Moreover, we have shown in human subjects who have undergone bone marrow transplantation, smooth muscle cells (SMCs) of donor origin are markedly enriched in coronary atherosclerotic plaque compared to the nondiseased vessel wall. 3 A number of potential agents may mediate SMC differentiation from blood borne progenitors including transforming growth factor-␤; plateletderived growth factor-BB 4 ; and sphingosine-1-phosphate, which induces RhoA-dependent myocardin expression and SMC differentiation in mesenchymal stem cells. 5 Thrombin generation occurs at sites of injury and could conceivably contribute to smooth muscle differentiation of circulating progenitor cells based on several lines of evidence. Under various in vitro conditions in mature SMCs, thrombin stimulates upregulation of the SMC marker genes smooth muscle myosin heavy chain (SM-MHC) 6 and calponin. 7 Thrombin also enhances the angiogenic potential of endothelial progenitor cells 8 and may influence vascular progenitor phenotype and function. 9 In the context of circulating progenitors, no study to date has fully investigated the downstream effects of thrombin on circulating mononuclear cells. Here, we show that thrombin induces robust sequential upregulation of the master smooth muscle cell transcription factor myocardin, as well as smooth muscle specific protein expression in peripheral blood mono...

Insulin-like growth factor-1 within the EPC CM mediates potent acute myocardial repair and chronic remodelling effects post-MI. These findings may provide a rationale for comparative trials of specific growth factors vs. current progenitor cell strategies.

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Background-Insulin-like growth factor-1 (IGF-1) is recognized as an important regulator of cardiac structure and cardiomyocyte homeostasis. The prosurvival and antiapoptotic effects of IGF-1 have been investigated in vitro and in rodent models of myocardial infarction (MI). However, the clinical application of IGF-1 has been hampered by dose-dependent side effects both acutely and during chronic administration. We hypothesized that single, low-dose IGF-1 (LD-IGF-1) administered locally and early in the reperfusion phase after acute MI in a large animal model would avoid significant side effects but would have prosurvival effects that would manifest in long-term structural and functional improvement after MI treatment. Methods and Results-Forty-four female Landrace pigs underwent intracoronary administration of LD-IGF-1 or saline 2 hours into the reperfusion phase of acute left anterior descending artery occlusion MI. In the area of infarction, IGF-1 receptor and signaling responses were activated at 30 minutes and cardiomyocyte cell death attenuated at 24 hours after LD-IGF-1 but not saline treatment. Hemodynamic and structural studies using pressure-volume loop, CT, and triphenyltetrazolium chloride analysis 2 months post-MI confirmed a marked reduction in infarct size, attenuation of wall thinning, and augmentation of wall motion in the LD-IGF-1-treated but not in the saline-treated animals. These regional structural benefits were associated with global reductions in left ventricular volumes and significant improvement in left ventricular systolic and diastolic function. Conclusions-One-time LD-IGF-1 effects potent acute myocardial salvage in a preclinical model of left anterior descending artery occlusive MI, extending to long-term benefits in MI size, wall structure, and function and underscoring its potential as an adjunctive therapeutic agent. (Circ Cardiovasc Interv. 2011;4:327-335.)