Alterations in zinc homeostasis underlie endothelial cell death induced by oxidative stress from acute exposure to hydrogen peroxide.

Oxidative stress has been associated with multiple pathologies and disease states, including those involving the cardiovascular system. Previously, we showed that pulmonary artery endothelial cells (PAECs) undergo apoptosis after acute exposure to H(2)O(2). However, the underlying mechanisms regulating this process remain unclear. Because of the prevalence of H(2)O(2) in normal physiological processes and apparent loss of regulation in disease states, the purpose of this study was to develop a more complete understanding of H(2)O(2)-mediated adverse effects on endothelial cell survival. Acute exposure of PAECs to H(2)O(2) caused a dose-dependent increase in cellular release of lactate dehydrogenase and a significant increase in production of superoxide ions, which appear to be generated within the mitochondria, as well as a significant loss of mitochondrial membrane potential and activity. Subsequent to the loss of mitochondrial membrane potential, PAECs exhibited significant caspase activation and apoptotic nuclei. We also observed a significant increase in intracellular free Zn(2+) after bolus exposure to H(2)O(2). To determine whether this increase in Zn(2+) was involved in the apoptotic pathway induced by acute H(2)O(2) exposure, we developed an adenoviral construct for overexpression of the Zn(2+)-binding protein metallothionein-1. Our data indicate that chelating Zn(2+), either pharmacologically with N,N,N',N-tetrakis(2-pyridylmethyl)ethylene diamine or by overexpression of the Zn(2+)-binding protein metallothionein-1, in PAECs conferred significant protection from induction of apoptosis and cell death associated with the effects of acute H(2)O(2) exposure. Our results show that the acute toxicity profile of H(2)O(2) can be attributed, at least in part, to liberation of Zn(2+) within PAECs. We speculate that regulation of Zn(2+) levels may represent a potential therapeutic target for cardiovascular disease associated with acute oxidative stress.

Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression.

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn(2+)) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn(2+) with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl(2)) to PAEC. We observed that Zn(2+)-exposed PAECs exhibited a dose-dependent increase in superoxide (O(2)(-).) generation that was localized to the mitochondria. Furthermore, we found Zn(2+)-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn(2+)-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn(2+)-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn(2+)-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn(2+) homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn(2+) levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.