Rosuvastatin elicits KDR-dependent vasculogenic response of human placental stem cells through PI3K/AKT pathway.

The growth and plasticity of engrafted human mesenchymal stem cells is regulated by external stimuli. Rosuvastatin (RSV) promotes myocardial neovascularization and limits myocardial remodeling in patients with chronic heart failure (CHF). While these non-lipid benefits may in part depend on the activation of stem cells, experimental evidence that RSV directly elicits vasculogenic differentiation of human mesenchymal stem cells is still lacking. We assessed whether RSV may drive a gene program of vascular commitment and the secretion of trophic mediators with antiapoptotic, angiogenic and antifibrotic activities in human mesenchymal stem cells from full-term placentas (FMhMSCs). With real-time RT-PCR, immunofluorescence, chemiluminescence, Western blot analysis, and in vitro vasculogenesis assays, we show that RSV enhanced expression of vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), encoding a major VEGF receptor, hepatocyte growth factor (HGF), and platelet-derived growth factor-BB (PDGF-BB) in a time- and dose-dependent manner. GATA-4 and Nkx-2.5 transcription was not affected. RSV enhanced capillary-like formation in vitro, but capillary-embedded FMhMSCs lacked endothelial marker expression, suggesting a role of pericyte-like elements in tube formation. In HUVEC/FMhMSC cocultures, RSV increases PDGFRß expression in FMhMSCs, and enhanced capillary density and organizational efficiency, promoting a long-lasting survival of tubular networks. RSV also activated PI3K-Akt pathway; the vasculogenic effects of the statin were abrogated following PI3K inhibition by LY294002. In conclusion, RSV-induced increase in capillary formation was dependent on VEGF and KDR. RSV promotes the activation of paracrine signals for vascular commitment of FMhMSCs through PI3K-Akt pathway. This observation may pave the way to the use of RSV as a pharmacological enhancer of stem cell potential for cardiovascular cell therapy.

Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.

The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species.

Hyaluronan mixed esters of butyric and retinoic acid affording myocardial survival and repair without stem cell transplantation.

Possible cardiac repair by adult stem cell transplantation is currently hampered by poor cell viability and delivery efficiency, uncertain differentiating fate in vivo, the needs of ex vivo cell expansion, and consequent delay in transplantation after the onset of heart attack. By the aid of magnetic resonance imaging, positron emission tomography, and immunohistochemistry, we show that injection of a hyaluronan mixed ester of butyric and retinoic acid (HBR) into infarcted rat hearts afforded substantial cardiovascular repair and recovery of myocardial performance. HBR restored cardiac [(18)F]fluorodeoxyglucose uptake and increased capillary density and led to the recruitment of endogenous Stro-1-positive stem cells. A terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay demonstrated that HBR-treated hearts exhibited a decrease in the number of apoptotic cardiomyocytes. In isolated rat cardiomyocytes and Stro-1 stem cells, HBR enhanced the transcription of vascular endothelial growth factor, hepatocyte growth factor, kdr, akt, and pim-1. HBR also increased the secretion of vascular endothelial growth factor and hepatocyte growth factor, suggesting that the mixed ester may have recruited both myocardial and Stro-1 cells also. An increase in capillarogenesis was induced in vitro with medium obtained from HBR-exposed cells. In the infarcted myocardium, HBR injection increased histone H4 acetylation significantly. Acetyl-H4 immunoreactivity increased in rat cardiomyocytes and Stro-1 cells exposed to HBR, compared with untreated cells. In conclusion, efficient cardiac regenerative therapy can be afforded by HBR without the need of stem cell transplantation or vector-mediated gene delivery.

Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts.

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.

Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia.

Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth, and cell death can be tightly connected. In this paper, we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xL, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cardiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion, this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia.

H9c2 cardiac myoblasts undergo apoptosis in a model of ischemia consisting of serum deprivation and hypoxia: inhibition by PMA.

Cardiac myocytes undergo apoptosis under condition of ischemia. Little is known, however, about the molecular pathways that mediate this response. We show that serum deprivation and hypoxia, components of ischemia in vivo, resulted in apoptosis of rat ventricular myoblast cells H9c2. Hypoxia alone did not induce significant apoptosis for at least 48 h, but largely increased the proapoptotic action of serum deprivation. H9c2 cells apoptosis is evidenced by an increase in terminal (TdT)-mediated dUTP nick end-labeling-positive nuclei and by activation of caspases 3, 6, 7 and 9, and loss of mitochondrial functions. In this model of simulated ischemia, represented by serum deprivation plus hypoxia, cardiomyoblasts apoptosis was associated with a p53-independent Bax accumulation and with a down-regulation of Bcl-xL, whereas the levels of cIAP-1, cIAP-2 and X-IAP proteins did not change. Phorbol-12-myristate-13-acetate significantly reduced the induction of apoptosis, inhibiting caspase 3 cleavage, Bax accumulation, Bcl-xL down-regulation as well as restoring cell viability.