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Cardiac repair depends on angiogenesis and cell proliferation. Previously we identified Canopy 2 (CNPY2) as a secreted angiogenic growth factor which promotes neovascularization. We investigated the role of CNPY2 in cardiac repair following myocardial infarction (MI) and the possible mediators involved using Cnpy2 knockout (KO) mice and human cardiac tissue. Cardiac tissue from patients with end-stage heart failure had significantly lower endogenous CNPY2 expression compared to samples from control patients. CNPY2 expression in mouse hearts significantly decreased following MI. Significantly less leukocyte and endothelial cell proliferation was found in Cnpy2 KO than wild-type (WT) mice post MI which contributed to impaired angiogenesis, tissue repair, and decreased cardiac function (fractional shortening: WT: 21.1 ± 2.1% vs. KO: 16.4 ± 1.6%, p <.01 at day 28 post MI). RT-qPCR revealed significantly increased p16INK4a expression in Cnpy2 KO mouse hearts (WT: 1.0 ± 0.04 vs. KO: 2.33 ± 0.11 [relative expression of p16 INK4a], p <.01) which was confirmed by immunostaining (WT: 8.47 ± 1.22 vs. KO: 12.9 ± 1.22 [% total cells], p <.05) for the p16INK4a protein. Expression of cell cycle-related proteins, cyclin D1, cyclin-dependent kinases 4 and 6, and phosphorylated retinoblastoma protein (pRb) was significantly decreased in Cnpy2 KO mouse hearts. The up-regulation of the p16INK4a/cyclin D1/Rb pathway by knockout of Cnpy2 was accompanied by attenuation of PDK1/Akt phosphorylation. MI exacerbated the detrimental effects of p16INK4a on tissue repair in Cnpy2 KO mice. Overexpression of CNPY2 in the cardiac tissue of transgenic mice reversed the inhibition of cell proliferation through suppression of the p16INK4a pathway. Cardiac injury and progressive heart failure were associated with decreased CNPY2 levels in both humans and mice. Knockout of Cnpy2 resulted in up-regulation of p16INK4a which impaired cardiac function and tissue repair. These data suggest that CNPY2 is an important regulator of p16INK4a and promotes cell proliferation and tissue repair through inhibition of the p16INK4a pathway. CNPY2 treatment may offer a new approach to restore cardiac function after an MI. • Lower CNPY2 expression in cardiac tissue from patients with end-stage heart failure. • CNPY2 expression in mouse hearts decreased significantly following an MI. • Cnpy2 KO mice showed impaired functional recovery post MI. • Cnpy2 KO upregulated p16INK4a to hinder leukocyte and endothelial cell proliferation and heart repair. • Overexpressing CNPY2 reversed inhibited cell proliferation by suppressing p16INK4a. [ABSTRACT FROM AUTHOR]

Abstract: Despite advances in the treatment of acute tissue ischemia significant challenges remain in effective cytoprotection from ischemic cell death. It has been documented that injected stem cells, such as mesenchymal stem cells (MSCs), can confer protection to ischemic tissue through the release of paracrine factors. The study of these factors is essential for understanding tissue repair and the development of new therapeutic approaches for regenerative medicine. We have recently shown that a novel factor secreted by MSCs, which we called HASF (Hypoxia and Akt induced Stem cell Factor), promotes cardiomyocyte proliferation. In this study we show that HASF has a cytoprotective effect on ischemia induced cardiomyocyte death. We assessed whether HASF could potentially be used as a therapeutic agent to prevent the damage associated with myocardial infarction. In vitro treatment of cardiomyocytes with HASF protein resulted in decreased apoptosis; TUNEL positive nuclei were fewer in number, and caspase activation and mitochondrial pore opening were inhibited. Purified HASF protein was injected into the heart immediately following myocardial infarction. Heart function was found to be comparable to sham operated animals one month following injury and fibrosis was significantly reduced. In vivo and in vitro HASF activated protein kinase C ε (PKCε). Inhibition of PKCε blocked the HASF effect on apoptosis. Furthermore, the beneficial effects of HASF were lost in mice lacking PKCε. Collectively these results identify HASF as a protein of significant therapeutic potential, acting in part through PKCε. [Copyright &y& Elsevier]

Abstract: Following a myocardial infarction (MI), fibroblasts differentiate to myofibroblasts, which possess some of the characteristics of smooth muscle cells (SMCs) and contribute to wound healing. Previous studies suggested that the miR-143/-145 cluster plays a critical role in SMC differentiation. Therefore, we determined whether miR-145 promoted differentiation of cardiac fibroblasts to myofibroblasts. Following coronary occlusion in mice, myocardial miR-145 expression was downregulated at 3days but was restored at 7days. In vitro studies showed that hypoxia also downregulated miR-145 in cardiac fibroblasts. The number of α-smooth muscle actin (α-SMA) positive cells in fibroblast cultures was employed to determine their transdifferentiation to cardiac myofibroblasts and was increased by 73.5% after transient transfection with miR-145. Ultrastructural analysis of α-SMA stress fibers revealed that ~95% of the α-SMA+ cells treated with miR-145 organized their actin-filament bundles with a specific orientation compared to only 15% in the scrambled control group. This orientation of the SMA bundles and their integration with the filamentous actin fibers of the cytoskeleton permit infarct wound contraction. Structural and functional studies showed that miR-145 induced a myofibroblast phenotype, and miR-145 also potentiated the production of mature collagen by myofibroblasts. Repression of KLF5, a target of miR-145, was validated by a chimeric luciferase construct tagged with the full-length 3′-UTR of KLF5. A dramatic decrease in KLF5 and a corresponding increase in myocardin expression were observed after transfecting cultured fibroblasts with miR-145. Similar results were found in vivo: the transient decrease in miR-145 expression 3days post-MI was associated with an increase in KLF5 and a decrease in myocardin. In addition, in vivo delivery of a miR-145 antagomir 1day prior to and 2 and 6days after MI decreased myofibroblast formation and increased scar size. The antagomir also reversed the suppressed expression of KLF5 protein in the scar region at day 7 after MI. In summary, we describe a novel association between miR-145 and fibroblast differentiation toward myofibroblasts. These observations provide a new approach to promote endogenous scar healing and contracture by stimulating the transdifferentiation of cardiac fibroblasts to myofibroblasts. [Copyright &y& Elsevier]

Bicuspid aortic valve (BAV) disease is a congenital abnormality that is associated with ascending aortic aneurysm yet many of the molecular mechanisms remain unknown. To identify novel molecular mechanisms of aneurysm formation we completed microarray analysis of the proximal (severely dilated) and distal (less dilated) regions of the ascending aorta from five patients with BAV. We identified 180 differentially expressed genes, 40 of which were validated by RT-qPCR. Most genes had roles in inflammation and endothelial cell function including cytokines and growth factors, cell surface receptors and the Activator Protein 1 (AP-1) transcription factor family (FOS, FOSB and JUN) which was chosen for further study. AP-1 was differentially expressed within paired BAV aneurysmal samples (n = 8) but not Marfan patients (n = 5). FOS protein was significantly enriched in BAV aortas compared to normal aortas but unexpectedly, ERK1/2 activity, an upstream regulator of FOS was reduced. ERK1/2 activity was restored when BAV smooth muscle cells were cultured in vitro. An mRNA-miRNA network within paired patient samples identified AP-1 as a central hub of miRNA regulation. FOS knockdown in BAV SMCs increased expression of miR-27a, a stretch responsive miRNA. AP-1 and miR-27a were also dysregulated in a mouse model of aortic constriction. In summary, this study identified a central role for AP-1 signaling in BAV aortic dilatation by using paired mRNA-miRNA patient sample. Upstream analysis of AP-1 regulation showed that the ERK1/2 signaling pathway is dysregulated and thus represents a novel chain of mediators of aortic dilatation in BAV which should be considered in future studies. [ABSTRACT FROM AUTHOR]

Background Mast cells (MCs) dynamically participate in wound healing after myocardial infarction (MI) by releasing cytokines. Indeed, MC-deficient mice undergo rapid left ventricular dilation post-MI. Mesenchymal stem cells (MSCs) are recruited to the injured region following an MI and have potential for cardiac repair. In the current study, we evaluate the effect of MCs on MSC proliferation and myogenic differentiation. Methods and results MCs were cultured from mouse bone marrow and MC granulate (MCG) was extracted from MCs via freeze-thaw cycles followed by filtration. α-SMA (smooth muscle actin) expression was examined as an indicator of myogenic differentiation. MSC/MC co-culture resulted in decreased MSC differentiation indicated by α-SMA suppression in MSCs. MCG also suppressed α-SMA expression and increased MSC migration and proliferation in a dose-dependent manner. Removal of MCG rescued α-SMA expression and MSC differentiation. Platelet derived growth factor (PDGF) receptor blockade using AG1296 also rescued MSC differentiation even after MCG treatment. Real-time PCR and Western blot showed that MCG exerted its effects on MSCs via downregulation of miR-145 and miR-143, downregulation of myocardin, upregulation of Klf4, and increased Erk and Elk1 phosphorylation. Conclusions MCs promote MSC proliferation and migration by suppressing their myogenic differentiation. MCs accomplish this via activation of the PDGF pathway, downregulation of miR-145/143, and modulation of the myocardin-Klf4 axis. These data suggest a potential role for MSC/MC interaction in the infarcted heart where MCs may inhibit MSCs from differentiation and promote their proliferation whereby increased cardiac MSC accumulation promotes eventual cardiac regeneration after MCs cease activity. [ABSTRACT FROM AUTHOR]

Abstract: We have demonstrated that mesenchymal stem cells overexpressing the survival gene Akt can confer paracrine protection to ischemic myocytes both in vivo and in vitro through the release of secreted frizzled related protein 2 (Sfrp2). However, the mechanisms mediating these effects of Sfrp2 have not been fully elucidated. In this study, we studied rat cardiomyoblasts subjected to hypoxia reoxygenation (HR) injury to test the hypothesis that Sfrp2 exerts anti-apoptotic effect by antagonizing pro-apoptotic properties of specific Wnt ligands. We examined the effect of Wnt3a and Sfrp2 on HR-induced apoptosis. Wnt3a significantly increased cellular caspase activities and TUNEL staining in response to HR. Sfrp2 attenuated significantly Wnt3a-induced caspase activities in a concentration dependent fashion. Using a solid phase binding assay, our data demonstrates that Sfrp2 physically binds to Wnt3a. In addition, we observed that Sfrp2 dramatically inhibits the beta-catenin/TCF transcriptional activities induced by Wnt3a. Impressively, Dickkopf-1, a protein that binds to the Wnt coreceptor LRP, significantly inhibited the Wnt3a-activated caspase and transcriptional activities. Similarly, siRNA against beta-catenin markedly inhibited the Wnt3a-activated caspase activities. Consistent with this, significantly fewer TUNEL positive cells were observed in siRNA transfected cells than in control cells. Together, our data provide strong evidence to support the notion that Wnt3a is a canonical Wnt with pro-apoptotic action whose cellular activity is prevented by Sfrp2 through, at least in part, the direct binding of these molecules. These results can explain the in vivo protective effect of Sfrp2 and highlight its therapeutic potential for the ischemic heart. [Copyright &y& Elsevier]