C. Caramelo

Mechanisms of the endothelial toxicity of cyclosporin A. Role of nitric oxide, cGMP, and Ca2+.

Cyclosporin A (CyA) is an efficient immunosuppressive agent, which, however, causes functional and structural alterations in endothelial cells. The aim of the present study was to examine the mechanisms of CyA-induced endothelial disfunction. CyA administration (Wistar rats, 25 mg/kg per day for 15 days) induced a significant inhibition of endothelium-dependent relaxation to acetylcholine on isolated femoral arteries. No changes with CyA were detected in the relaxation response to the endothelium-independent agent (sodium nitroprusside) or the endothelium-dependent receptor-independent agent (Ca2+ ionophore). The addition of L-arginine (10(-5) mol/L) shifted to the left the acetylcholine-mediated vasorelaxing response in CyA-treated segments, an effect that was accompanied by a marked increase of cGMP. 45Ca2+ uptake was higher in CyA-treated segments with respect to control segments but became normalized after incubation with L-arginine or sodium nitroprusside. De-endothelialization or incubation with the L-arginine competitive analogue N omega-nitro-L-arginine (NwNLA) increased 45Ca2+ uptake in control segments but not in CyA-treated segments. In conclusion, in isolated rat arteries, chronic CyA therapy affects endothelial function by uncoupling the acetylcholine-mediated relaxation and interfering with an endothelium-mediated pathway that regulates 45Ca2+ uptake by a mechanism reversed by an L-arginine-dependent cGMP generation.

Disruption of Cadherin-Related Junctions Triggers Autocrine Expression of Vascular Endothelial Growth Factor in Bovine Aortic Endothelial Cells: Effects on Cell Proliferation and Death Resistance

The mechanisms involved in the blockade of proliferation in confluent endothelial cells are insufficiently understood. In this regard, the continuity of intercellular junctions appears to be critical to the regulation of endothelial monolayer cell growth. The present study examined the hypothesis that the disruption of the intercellular adherens junctions will trigger both endothelial cell proliferation and autocrine production of growth factors. With this purpose, we assessed the changes in growth, death resistance, and expression of vascular endothelial growth factor (VEGF) under conditions of disruption of the intercellular junctions between endothelial cells. Disruption of cell junctions was produced by means of a specific anti-vascular endothelial cadherin monoclonal antibody, EGTA, or cytochalasin D. Our results disclosed that these maneuvers induce an increase in VEGF mRNA production, with transcription of the 121-, 165-, and 189-amino acid isoforms of VEGF. Further evidence of the relationship between endothelial cells monolayer continuity and VEGF protein expression was obtained by the demonstration of an increase in VEGF protein, as determined by Western blot, induced by the aforementioned maneuvers, as well as by immunocytochemical detection of increased VEGF staining in the areas surrounding a mechanical endothelial injury and in endothelial cells at subconfluence. In functional terms, the autocrine expression of VEGF was associated with growth-promoting and cytoprotective effects, as assessed by [(3)H]thymidine uptake, (51)Cr release, and flow cytometry. In conclusion, our results reveal that disruption of homophilic interendothelial junctions induces VEGF expression. Under these conditions, autocrine VEGF appears to have a relevant role in death inhibition and proliferation of endothelial cells.

Inhibition by L-arginine of the endothelin-mediated increase in cytosolic calcium in human neutrophils

The effect of endothelin (ET) on the cytosolic-free calcium [(Ca2+]i) changes in polymorphonuclear leukocytes (PMN) from normal humans and Wistar rats was investigated. ET induced a dose-related [Ca2+]i peak. This [Ca2+]i transient was blunted by TMB-8 (10(-5)M) and by Ca(2+)-free EGTA medium, therefore suggesting a role of both intracellular Ca2+ release and Ca2+ influx in the generation of the [Ca2+]i peak. Preincubation of PMN with the nitric oxide (NO)-donor L-arginine (L-Arg) markedly blocked the ET-induced [Ca2+]i transient in an enantiomerically-specific manner. A similar blunting effect of L-Arg on the fMLP (10(-7)M)-induced [Ca2+]i transient was detected. The L-Arg antagonist, NG-monomethyl-L-arginine (L-NMMA), reverted the L-Arg blocking effect on both ET- and fMLP-induced [Ca2+]i transients. These data suggest that ET has a potential role in activating Ca2+ mobilization in PMN, an effect that can be inhibited by L-Arg.