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Abstract: Decreased endothelial nitric oxide (NO) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1) are early features of atherosclerosis. We investigated the effects of suppressing endogenous NO production by the NO synthase inhibitor l-mono-methyl-arginine (L-NMMA), given alone or in combination with interleukin(IL)-1α, on VCAM-1 expression by human umbilical vein endothelial cells (HUVEC). VCAM-1 expression (by enzyme immunoassay), barely detectable at baseline, was significantly increased by L-NMMA (by no more than 20% over control compared with IL-1α induction). This was paralleled by an increase in U937 monocytoid cell adhesion. When HUVEC incubated with L-NMMA were stimulated with low concentrations of IL-1α (0.05–0.5ng/mL), these determined a higher VCAM-1 expression than in the presence of L-NMMA or IL-1α alone. Northern analysis indicated that VCAM-1 mRNA was induced by L-NMMA alone, and that the effects of L-NMMA and IL-1α were, again, at least additive. Nuclear factor-κB (NF-κB), GATA, activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), transcription factors all involved in VCAM-1 gene expression, were all activated at electrophoretic mobility shift assay and at chromatin immunoprecipitation assay by L-NMMA, but additive effects with the combined administration of L-NMMA and IL-1α only occurred for NF-κB. These results support the view that endogenous NO mantains a normal endothelial non-reactivity towards circulating monocytes, and that suppression of this endogenous brake for endothelial activation results in the activation of multiple transcription factors even in the absence of other endothelial activators, with a prominent role of NF-κB in the presence or absence of other inflammatory mediators. [Copyright &y& Elsevier]

Abstract: Objective: Previous investigations have consistently shown that the piperazine derivative trimetazidine (TMZ, 1-[2,3,4-trimethoxybenzil] piperazine, dihydrocloride) has cardioprotective effects in the experimental ischemia–reperfusion model. We tested the hypothesis that cardioprotective effect of TMZ is partly mediated by preservation of the endothelial barrier of the coronary microcirculation. Methods: Isolated Wistar rat (250–300g) hearts were subjected to a 15min period of global ischemia and 180min reperfusion in the presence or absence of 1μM TMZ. Hemodynamic parameters, heart weight, creatinekinase (CK) release and microvascular permeability (FITC–albumin extravasation) were evaluated. In addition, eNOS gene expression was estimated by rt-PCR, and eNOS protein levels were assessed by Western analysis. In order to confirm the involvement of NO in mediating the cardioprotective effects of TMZ, 30μM Nω-nitro-l-arginine methylester (L-NAME), a specific inhibitor of nitric oxide synthase, was used. Results: After ischemia and reperfusion, TMZ produced a significant improvement of mechanical function associated with a reduction of CK release and FITC–albumin diffusion (P <0.001); the agent also resulted in improvement in coronary flow (at 45min+27% vs control). The eNOS mRNA and protein levels were significantly higher in TMZ-treated hearts compared to controls. The addition of L-NAME significantly reduced the beneficial effects of TMZ on contractile function, CK release and FITC–albumin diffusion. Conclusions: in the isolated rat heart, TMZ exerts a relevant, NO-dependent, cardioprotection against ischemia–reperfusion injury and preserves the endothelial barrier of the coronary circulation. This could contribute to explain the cardioprotective action of TMZ following ischemia and reperfusion. [Copyright &y& Elsevier]