Rationale Paracrine secretions appear to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia as well as in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains pro-angiogenic, membrane-bound nano-vesicles called exosomes (CD34Exo). Objective Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. Methods and Results When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function and their amputation. CD34Exo were found to be enriched with pro-angiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb, but did not affect the endogenous synthesis of miR-126-3p suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1; simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF, ANG1, ANG2, MMP9, TSP1 etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. Conclusions Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled angiomiRs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC-mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.angiogenesis | stem cell | vasculogenesis

Rationale Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. Objective To offset age and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). Methods and Results When injected into the border zone of mice following acute myocardial infarction (AMI), Shh-modified CD34+ cells (CD34Shh) protected against ventricular dilation and cardiac functional declines associated with AMI. Treatment with CD34Shh also reduced infarct size and increased border zone capillary density compared to unmodified CD34 cells or cells transfected with the empty vector. CD34Shh primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34Shh revealed that; 1) exosomes transfer Shh protein to other cell types and, 2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. Conclusions Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34Shh cells.

The morphogen Sonic Hedgehog (Shh) promotes neovascularization in adults by inducing proangiogenic cytokine expression in fibroblasts; however, the direct effects of Shh on endothelial cell (EC) function during angiogenesis are unknown. Our findings indicate that Shh promotes capillary morphogenesis (tube length on Matrigel ™ increased to 271±50% of the length in untreated cells, p=0.00003), induces EC migration (modified Boyden chamber assay, 191±35% of migration in untreated cells, p=0.00009), and increases EC expression of matrix metalloproteinase 9 (MMP-9) and osteopontin (OPN) mRNA (real-time RT-PCR), which are essential for Shhinduced angiogenesis both in vitro and in vivo. Shh activity in ECs is mediated by Rho, rather than through the "classic" Shh signaling pathway, which involves the Gli transcription factors. The Rho dependence of Shh-induced EC angiogenic activity was documented both in vitro, with dominantnegative RhoA and Rho kinase (ROCK) constructs, and in vivo, with the ROCK inhibitor Y27632 in the mouse corneal angiogenesis model. Finally, experiments performed in MMP-9-and OPNknockout mice confirmed the roles of the ROCK downstream targets MMP-9 and OPN in Shhinduced angiogenesis. Collectively, our results identify a "non-classical" pathway by which Shh directly modulates EC phenotype and angiogenic activity.

Humans with a rare gene mutation in SERPINE1 live longer and show evidence of protection from aging-related morbidity.

Background Bicuspid aortic valve (BAV) is a heritable condition that has been linked by an unknown mechanism to a predisposition for ascending aortic aneurysm. Matrix metalloproteinases have been implicated in this predisposition. Metallothionein is a poorly characterized, metal-binding protein that regulates matrix metalloproteinases and is an antioxidant known to be upregulated under oxidative stress. Methods and Results To determine putative factors involved in the pathogenesis of aortic aneurysm in BAV patients, our first goal was to identify genes that are dysregulated in ascending aortic aneurysms of BAV patients compared with tricuspid aortic valve patients and nondiseased (control) donors. By microarray analysis (22 000 probe sets), 110 dysregulated genes were identified in BAV compared with tricuspid aortic valve patients and control donors; 8 were genes of the metallothionein family. Metallothionein gene expression and protein expression were significantly lower in aortic tissue and cultured aortic smooth muscle cells from BAV patients compared with control subjects. Matrix metalloproteinase-9 expression was increased in BAV aortic samples relative to controls. BAV aorta was more susceptible to oxidative stress, and induction of metallothionein under oxidative stress was reduced in BAV patients compared with control subjects. Conclusions These results demonstrate dysregulated metallothionein expression in ascending aortic smooth muscle cells of BAV patients that may contribute to an inadequate response to oxidative stress and provoke aneurysm formation. We hypothesize that metallothionein plays a pivotal role in the response of ascending aortic smooth muscle cells to oxidative stress cues normally involved in the maintenance of the extracellular matrix, including the regulation of matrix metalloproteinase expression.

Background CXC-chemokine receptor 4 (CXCR4) regulates the retention of stem/progenitor cells in the bone marrow (BM), and the CXCR4 antagonist AMD3100 improves recovery from coronary-ligation injury by mobilizing stem/progenitor cells from the BM to the peripheral blood. Thus, we investigated whether AMD3100 also improves recovery from ischemia-reperfusion (IR) injury, which more closely mimics myocardial infarction in patients, because blood flow is only temporarily obstructed. Methods and Results Mice were treated with single subcutaneous injections of AMD3100 (5 mg/kg) or saline after IR injury. Three days later, histological measurements of the infarct-area/area-at-risk ratio were smaller in AMD3100-treated mice than in mice administered saline, and echocardiographic measurements of left-ventricular function were greater in the AMD3100-treated mice at week 4. CXCR4+ cells were mobilized for just 1 day in both groups, but the mobilization of sca1+/flk1+ cells endured for 7days in AMD3100-treated mice compared to just 1 day in the saline-treated mice. AMD3100 upregulated BM levels of endothelial nitric oxide synthase (eNOS) and two targets of eNOS signaling, matrix-metalloproteinase 9 and soluble Kit ligand. Furthermore, the loss of BM eNOS expression abolished the benefit of AMD3100 on sca1+/flk1+ cell mobilization without altering the mobilization of CXCR4+ cells, and the cardioprotective effects of AMD3100 were retained in eNOS-knockout mice that had been transplanted with BM from wild-type mice, but not in wild-type mice with eNOS-knockout BM. Conclusions AMD3100 prolongs BM progenitor mobilization and improves recovery from IR injury, and these benefits appear to occur through a previously unidentified link between AMD3100 and BM eNOS expression.

The average age of the US population continues to increase. Age is the most important determinant of disease and disability in humans, but the fundamental mechanisms of aging remain largely unknown. Many age-related diseases are associated with an impaired fibrinolytic system. Elevated plasminogen activator inhibitor-1 (PAI-1) levels are reported in age-associated clinical conditions including cardiovascular diseases, type 2 diabetes, obesity and inflammation. PAI-1 levels are also elevated in animal models of aging. While the association of PAI-1 with physiological aging is well documented, it is only recently that its critical role in the regulation of aging and senescence has become evident. PAI-1 is synthesized and secreted in senescent cells and contributes directly to the development of senescence by acting downstream of p53 and upstream of insulin-like growth factor binding protein-3. Pharmacologic inhibition or genetic deficiency of PAI-1 was shown to be protective against senescence and the aging-like phenotypes in kl/kl and N(ω)-nitro-l-arginine methyl ester-treated wild-type mice. Further investigation into PAI-1's role in senescence and aging will likely contribute to the prevention and treatment of aging-related pathologies.