Dean A. Wiseman

Asymmetric dimethylarginine inhibits HSP90 activity in Pulmonary Arterial Endothelial Cells: Role of Mitochondrial Dysfunction

Increased asymmetric dimethylarginine (ADMA) levels have been implicated in the pathogenesis of a number of conditions affecting the cardiovascular system. However, the mechanism(s) by which ADMA exerts its effect has not been adequately elucidated. Thus the purpose of this study was to determine the effect of increased ADMA on nitric oxide (NO) signaling and to begin to elucidate the mechanism by which ADMA acts. Our initial data demonstrated that ADMA increased NO synthase (NOS) uncoupling in both recombinant human endothelial NO synthase (eNOS) and pulmonary arterial endothelial cells (PAEC). Furthermore, we found that this endothelial NOS (eNOS) uncoupling increased 3-nitrotyrosine levels preferentially in the mitochondria of PAEC due to a redistribution of eNOS from the plasma membrane to the mitochondria. This increase in nitration in the mitochondria was found to induce mitochondrial dysfunction as determined by increased mitochondrial-derived reactive oxygen species and decreased generation of ATP. Finally, we found that the decrease in ATP resulted in a reduction in the chaperone activity of HSP90 resulting in a decrease in its interaction with eNOS. In conclusion increased levels of ADMA causes mitochondrial dysfunction and a loss of heat shock protein-90 chaperone activity secondary to an uncoupling of eNOS. Mitochondrial dysfunction may be an understudied component of the endothelial dysfunction associated with various cardiovascular disease states.

Progressive dysfunction of nitric oxide synthase in a lamb model of chronically increased pulmonary blood flow : a role for oxidative stress

Cardiac defects associated with increased pulmonary blood flow result in pulmonary vascular dysfunction that may relate to a decrease in bioavailable nitric oxide (NO). An 8-mm graft (shunt) was placed between the aorta and pulmonary artery in 30 late gestation fetal lambs; 27 fetal lambs underwent a sham procedure. Hemodynamic responses to ACh (1 microg/kg) and inhaled NO (40 ppm) were assessed at 2, 4, and 8 wk of age. Lung tissue nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), neuronal NOS (nNOS), inducible NOS (iNOS), and heat shock protein 90 (HSP90), lung tissue and plasma nitrate and nitrite (NO(x)), and lung tissue superoxide anion and nitrated eNOS levels were determined. In shunted lambs, ACh decreased pulmonary artery pressure at 2 wk (P < 0.05) but not at 4 and 8 wk. Inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). In control lambs, ACh and inhaled NO decreased pulmonary artery pressure at each age (P < 0.05). Total NOS activity did not change from 2 to 8 wk in control lambs but increased in shunted lambs (ANOVA, P < 0.05). Conversely, NO(x) levels relative to NOS activity were lower in shunted lambs than controls at 4 and 8 wk (P < 0.05). eNOS protein levels were greater in shunted lambs than controls at 4 wk of age (P < 0.05). Superoxide levels increased from 2 to 8 wk in control and shunted lambs (ANOVA, P < 0.05) and were greater in shunted lambs than controls at all ages (P < 0.05). Nitrated eNOS levels were greater in shunted lambs than controls at each age (P < 0.05). We conclude that increased pulmonary blood flow results in progressive impairment of basal and agonist-induced NOS function, in part secondary to oxidative stress that decreases bioavailable NO.

A novel role for caveolin-1 in regulating endothelial nitric oxide synthase activation in response to H2O2 and shear stress

Previous studies have shown that acute increases in oxidative stress induced by the addition of hydrogen peroxide (H(2)O(2)) can increase endothelial nitric oxide synthase (eNOS) catalytic activity via an increase in the phosphorylation of eNOS at serine 1177. However, it is unclear how increased H(2)O(2) affects nitric oxide (NO) signaling when endothelial cells are exposed to biomechanical forces. Thus, the purpose of this study was to evaluate the acute effects of H(2)O(2) on NO signaling in the presence or absence of laminar shear stress. We found that acute sustained increases in cellular H(2)O(2) levels in bovine aortic endothelial cells did not alter basal NO generation but the NO produced in response to shear stress was significantly increased. This amplification in NO signaling was found to correlate with an H(2)O(2)-induced increase in eNOS localized to the plasma membrane and an increase in total caveolin-1 protein levels. We further demonstrated that overexpressing caveolin-1 increased eNOS localized to the plasma membrane again without altering total eNOS protein levels. We also found that caveolin-1 overexpression increased NO generation in response to shear stress but only in the presence of H(2)O(2). Conversely, depleting caveolin-1 with an siRNA decreased eNOS localized to the plasma membrane and abolished the enhanced NO generation. Finally, we found that expressing a caveolin-1 binding-site deletion mutant of eNOS in COS-7 cells decreased its plasma membrane localization and resulted in attenuated NO production in response to calcium activation. In conclusion, we have identified a new role for caveolin-1 in enhancing eNOS trafficking to the plasma membrane that seems to be involved in priming eNOS for flow-mediated activation under conditions of oxidative stress. To our knowledge, this is the first report that H(2)O(2) modulates eNOS activity by altering its subcellular location and that caveolin-1 can play a stimulatory role in NO signaling.

Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17β-Estradiol

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERalpha) within the plasma membrane of endothelial cells (EC). ERalpha is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERalpha to the plasma membrane. However, the role played by caveolin 1 in regulating 17beta-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17beta-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERalpha localization to the plasma membrane in EC. Further, before estrogen stimulation ERalpha associates with caveolin 1, whereas stimulation promotes a pp60(Src)-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERalpha-PI3 kinase interactions and disrupting caveolin 1-ERalpha interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERalpha/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62-73 of caveolin 1 as the ERalpha-binding site. Delivery of a synthetic peptide based on this sequence decreased ERalpha plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17beta-estradiol-induced NO production by promoting ERalpha to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.

Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of Sp1 activity.

We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H(2)O(2)), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H(2)O(2) treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840 bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650 bp) was also inhibited by H(2)O(2). A similar effect was observed when the 650 bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H(2)O(2). Exposure of PAECs caused a significant increase in labile zinc levels in response to H(2)O(2). As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H(2)O(2). As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H(2)O(2) decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H(2)O(2). Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H(2)O(2)-induced inhibition of eNOS promoter activity.

Elevated zinc induces endothelial apoptosis via disruption of glutathione metabolism: role of the ADP translocator

Zinc is the second-most abundant transition metal within cells and an essential micronutrient. Although adequate zinc is essential for cellular function, intracellular free zinc (Zn2+) is tightly controlled, as sustained increases in free Zn2+ levels can directly contribute to apoptotic endothelial cell death. Moreover, exposure of endothelial cells to acute nitrosative and/or oxidative stress induces a rapid rise of Zn2+ with mitochondrial dysfunction and the initiation of apoptosis. This apoptotic induction can be mimicked through addition of exogenous ZnCl2 and mitigated by zinc-chelation strategies, indicating Zn2+-dependent mechanisms in this process. However, the molecular mechanisms of Zn2+-mediated mitochondrial dysfunction are unknown. Here we report that free Zn2+ disrupts cellular redox status through inhibition of glutathione reductase, and induces apoptosis by redox-mediated inhibition of the mitochondrial adenine nucleotide transporter (ANT). Inhibition of ANT causes increased mitochondrial oxidation, loss of ADP uptake, mitochondrial translocation of bax, and apoptosis. Interestingly, pre-incubation with glutathione ethyl ester protects endothelial cells from these observed effects. We conclude that key mechanisms of Zn2+-mediated apoptotic induction include disruption of cellular glutathione homeostasis leading to ANT inhibition and decreases in mitochondrial ATP synthesis. These pathways could represent novel therapeutic targets during acute oxidative or nitrosative stress in cells and tissues.

Mass Spectroscopy and Molecular Modeling Predict Endothelial Nitric Oxide Synthase Dimer Collapse by Hydrogen Peroxide Through Zinc Tetrathiolate Metal-Binding Site Disruption

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

Perinatal changes in superoxide generation in the ovine lung: Alterations associated with increased pulmonary blood flow.

Although alterations in ROS generating systems are well described in several vascular disorders, there is very limited information on the perinatal regulation of these systems in the lung both during normal development and in pulmonary hypertension. Thus, this study was undertaken to explore how the two predominant superoxide generating systems, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) and xanthine oxidase (XO), are developmentally regulated in control lambs and in our established lamb model of increased pulmonary blood flow (Shunt) over the first 2months of life. We found that the levels of p47(phox), p67(phox), and Rac1 subunits of NADPH oxidase complex were altered. During the first two months of life there was no change in p47(phox) protein levels in either normal or Shunt lambs. However, both p67(phox) and Rac1 protein levels decreased over time. In addition, p47(phox) protein levels were significantly increased in shunt lambs at 2- and 4-, but not 8-weeks of age compared to age-matched controls while levels of the p67(phox) subunit were decreased at 8-weeks of age in the Shunts but unchanged at other time periods. Furthermore, Rac1 protein expression was significantly increased in the Shunts only at 4weeks of age. These data correlated with a significant increase in NADPH oxidase dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. During normal development XO levels significantly increased over time in normal lambs but significantly decreased in the Shunts. In addition, XO protein levels were significantly increased in the Shunt at 2- and 4-weeks of age but significantly decreased at 8-weeks. Again this correlated with a significant increase in XO dependent superoxide generation at 2- and 4-, but not 8-weeks of age in the Shunts. Collectively, our findings suggest that NADPH oxidase and XO are major contributors to superoxide generation both during the normal development and during the development of pulmonary hypertension.

Alterations in lung arginine metabolism in lambs with pulmonary hypertension associated with increased pulmonary blood flow.

Previous studies demonstrate impaired nitric oxide (NO) signaling in children and animal models with congenital heart defects and increased pulmonary blood flow. However, the molecular mechanisms underlying these alterations remain incompletely understood. The purpose of this study was to determine if early changes in arginine metabolic pathways could play a role in the reduced NO signaling demonstrated in our lamb model of congenital heart disease with increased pulmonary blood flow (Shunt lambs). The activities of the arginine recycling enzymes, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) were both decreased in lung tissues of Shunt lambs while arginase activity was increased. Associated with these alterations, lung L-arginine levels were decreased. These changes correlated with an increase in NO synthase-derived reactive oxygen species (ROS) generation. This study provides further insights into the molecular mechanisms leading to decreased NO signaling in Shunt lambs and suggests that altered arginine metabolism may play a role in the development of the endothelial dysfunction associated with pulmonary hypertension secondary to increased pulmonary blood flow.

Microtubules growth rate alteration in human endothelial cells.

To understand how microtubules contribute to the dynamic reorganization of the endothelial cell (EC) cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks microtubule plus-ends. Using this model, we were able to measure microtubule growth rate at the centrosome region and near the cell periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of microtubules in EC are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the centrosome. Growth rate of microtubule plus-ends decreases from the cell center toward the periphery. Our data suggest the existing mechanism(s) of local regulation of microtubule plus-ends growth in EC. Microtubule growth rate in the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory effect of cell-cell contacts. Centrosomal microtubule growth rate distribution in single EC indicated the presence of two subpopulations of microtubules with “normal” (similar to those in monolayer EC) and “fast” (three times as much) growth rates. Our results indicate functional interactions between cell-cell contacts and microtubules.