5-Azacytidine-treated bone marrow stromal cells transplanted into the myocardial infarct region formed islands of cardiac-like tissue, induced angiogenesis, prevented thinning and dilatation of the infarct region, and improved regional and global contractile function.

Background-Endothelin-1 (ET-1) plays an important role in the maintenance of vascular tone and pathological states such as ischemia/reperfusion (I/R) injury, coronary vasospasm, and cardiac allograft vasculopathy. We assessed the effects of elevated ET-1 levels as seen after I/R to determine if ET-1 modulates nitric oxide (NO) production via the translocation of specific protein kinase C (PKC) isoforms. Methods and Results-Human saphenous vein endothelial cells (HSVECs) (nϭ8) were incubated with ET-1 or phosphate-buffered saline (PBS) for 24 hours. NO production was determined in the supernatant by measuring nitrate/nitrite levels. Protein expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), caveolin-1 and PKC were determined. Lastly, PKC translocation and activity were assessed after exposure to the drug of interest. HSVECs exposed to ET-1 displayed decreased NO production. PKC inhibition reduced NO production, whereas PKC activation increased production. NO production was maintained when HSVECs exposed to ET-1 were treated with the PKC agonist, PMA. eNOS protein expression was reduced after ET-1 treatment. PKC inhibition also downregulated eNOS protein expression, whereas PMA upregulated expression. ET-1 exposure led to a significant increase in PKC␦ and PKC translocation compared with control, whereas translocation of PKC was inhibited. ET-1 exposure significantly reduced overall PKC activity compared with control. Conclusions-Our study demonstrates that high levels of ET-1 impair endothelial NO production via an isoform-specific PKC-mediated inhibition of eNOS expression. ET-1 antagonism with bosentan stimulates translocation of PKC and leads to increased PKC activity and NO production. ET-1 antagonism may provide a novel therapeutic strategy to improve vascular homeostasis.

Cells in which glucose uptake is rate limiting respond to hypoxic insults with an increase in glucose transport activity. To understand the underlying cellular mechanisms involved in this adaptive response, the effects of an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol (DNP), and of an inhibitor of the electron transport chain, rotenone, were compared with the effect of hypoxia in L6 muscle cells and 3T3-L1 adipocytes. All three conditions (DNP, rotenone, and 3% oxygen) elevated hexose uptake by approximately twofold in 4 h relative to control cells. All three insults decreased cellular ATP levels rapidly. A subsequent recovery was observed within 1-2 h in the presence of DNP or 3% oxygen, probably as a result of anaerobic production of ATP through increased glucose uptake and glycolysis. DNP and rotenone elevated the content of GLUT-1 protein in isolated plasma membranes and decreased it in intracellular light microsomes, suggestive of translocation of this transporter isoform. No change in GLUT-4 protein distribution was detected. In contrast, 3% oxygen caused a marked specific increase in GLUT-1 protein in both plasma membranes and microsomes. Consistently, cycloheximide had no effect on the hexose transport responses to DNP or rotenone, but prevented the response to hypoxia. However, GLUT-1 mRNA and the total cell content of GLUT-1 protein were elevated by all three treatments. It is proposed that within the time frame studied, reductions in the energy charge may activate the glucose transport system in L6 myotubes and 3T3-L1 adipocytes by GLUT-1 protein biosynthesis and translocation. When both responses exist, the biosynthetic pathway is dispensable, and posttranslational mechanisms, including transporter translocation suffice to sustain the adaptive elevation in glucose transport activity for several hours.

Ischaemic preconditioning can be induced in human cardiomyocytes independent of other cell types. The effect can be established in human cell cultures.

Immunosuppressants are necessary to prevent graft rejection after solid organ transplantation. However, they are also known to have significant side effects, including endothelial toxicity. Endothelial progenitor cells originate in the bone marrow and are recognized by their angiogenic and endothelial reparative properties. The effects of the immunosuppressants cyclosporine A (CyA), tacrolimus and rapamycin were analyzed on endothelial progenitor-like cells. Rapamycin induced rapid cell death, even at concentrations much lower than those used clinically, in peripheral blood mononuclear cells (PBMC) cultured to favor outgrowth of endothelial progenitors. Cyclosporine A and tacrolimus had no significant effects at clinical concentrations. The effect of rapamycin was specific to endothelial progenitor cells, in particular to the early stages of differentiation, as a lesser effect was observed in late outgrowth endothelial progenitors, mature aortic endothelial cells, and macrophages derived from the same PBMCs. The mechanism of cell death appeared to be apoptosis; however, its induction was probably multifactorial and did not depend on caspase or cathepsin activation. In conclusion, rapamycin induces endothelial progenitor cell death, possibly because it blocks survival signals given by growth factors critically required by these cells.

Our in vitro and in vivo studies taken together confirm that miR-17 directly regulates TIMP-1 and -2. Less dilated aortic BAV tissue may be in the initial stages of dilation under the control of miR-17-related miRNAs. New therapies that inhibit these miRNAs may prevent aortic dilation.

Background-Cyclosporine (CyA) is associated with many side effects, including endothelial dysfunction and transplant vasculopathy (TxV). We previously demonstrated that CyA results in impairment of nitric oxide bioavailability and enhanced sensitivity to endothelin-1 (ET-1). In this study, we evaluated rapamycin (SRL) for its effects on the endothelium. Methods and Results-Lewis rats (n ϭ8) were injected with SRL (1.5 mg/kg), CyA (5 mg/Kg), or saline (Con) intraperitoneally daily for 2-weeks. Thoracic aortic segments were assessed for endothelial-dependent (Edep) and independent (Eind) relaxation after exposure to acetylcholine and sodium nitroprusside by deriving the percent maximum relaxation (Emax). ET-1 plasma levels were also measured.

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