Salvatore Cuzzocrea

Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite.

DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) contribute to peroxynitrite-induced cellular injury. We investigated the role of PARS activation in the pathogenesis of endothelial dysfunction. In human umbilical vein endothelial cells (HUVEC), DNA strand breakage (alkaline unwinding assay), PARS activation (incorporation or radiolabeled NAD+ into proteins), mitochondrial respiration [conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan] and apoptotic index (cytoplasmatic release of histones) were measured. Endotoxin shock was induced in rats by bacterial lipopolysaccharide. Vascular reactivity of thoracic aortic rings were measured in organ chambers. In HUVEC, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, induced DNA strand breakage and caused an activation of PARS. Pharmacological inhibition of PARS reduced the acute and delayed suppression of mitochondrial respiration when cells were exposed to intermediate, but not high doses of peroxynitrite. Similarly, protection against the intermediate, but not high doses of peroxynitrite was seen in fibroblasts from the PARS-/- mice, when compared to wild-type controls. These data suggest that PARS plays a role in peroxynitrite-induced cytotoxicity, but at very high levels of oxidant exposure, PARS-independent cytotoxic mechanisms become predominant. Peroxynitrite-induced apoptosis was not affected by PARS inhibition. Vascular rings exposed to peroxynitrite and rings taken from rats subjected to endotoxic shock exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. The development of this endothelial dysfunction was ameliorated by the PARS inhibitor 3-aminobenzamide. Activation of PARS by peroxynitrite, therefore, may be involved in the development of endothelial dysfunction in endotoxemia.

Peroxynitrite-induced thymocyte apoptosis: The role of caspases and poly (ADP-ribose) synthetase (PARS) activation

The mechanisms by which immature thymocyte apoptosis is induced during negative selection are poorly defined. Reports demonstrated that cross‐linking of T‐cell receptor leads to stromal cell activation, expression of inducible nitric oxide synthase (iNOS) and, subsequently, to thymocyte apoptosis. Therefore we examined, whether NO directly or indirectly, through peroxynitrite formation, causes thymocyte apoptosis. Immuno‐histochemical detection of nitrotyrosine revealed in vivo peroxynitrite formation in the thymi of naive mice. Nitrotyrosine, the footprint of peroxynitrite, was predominantly found in the corticomedullary junction and the medulla of naive mice. In the thymi of mice deficient in the inducible isoform of nitric oxide synthase, considerably less nitrotyrosine was found. Exposure of thymocytes in vitro to low concentrations (10u2003μm) of peroxynitrite led to apoptosis, whereas higher concentrations (50u2003μm) resulted in intense cell death with the characteristics of necrosis. We also investigated the effect of poly (ADP‐ribose) synthetase (PARS) inhibition on thymocyte apoptosis. Using the PARS inhibitor 3‐aminobenzamide (3‐AB), or thymocytes from PARS‐deficient animals, we established that PARS determines the fate of thymocyte death. Suppression of cellular ATP levels, and the cellular necrosis in response to peroxynitrite were prevented by PARS inhibition. Therefore, in the absence of PARS, cells are diverted towards the pathway of apoptotic cell death. Similar results were obtained with H2O2 treatment, while apoptosis induced by non‐oxidative stimuli such as dexamethasone or anti‐FAS antibody was unaffected by PARS inhibition. In conclusion, we propose that peroxynitrite‐induced apoptosis may play a role in the process of thymocyte negative selection. Furthermore, we propose that the physiological role of PARS cleavage by apopain during apoptosis may serve as an energy‐conserving step, enabling the cell to complete the process of apoptosis.