Heidi L. Rupnow

Peroxisome Proliferator-Activated Receptor γ Ligands Stimulate Endothelial Nitric Oxide Production Through Distinct Peroxisome Proliferator-Activated Receptor γ–Dependent Mechanisms

Objective—We recently reported that the peroxisome proliferator-activated receptor γ (PPARγ) ligands 15-deoxy-&Dgr;12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPARγ-ligand–stimulated increases in endothelial cell NO production. Methods and Results—Treating human umbilical vein endothelial cells (HUVEC) with PPARγ ligands (10 &mgr;mol/L 15d-PGJ2, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPARγ ligands and with the PPARγ antagonist GW9662 (2 &mgr;mol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPARγ, which reduced HUVEC PPARγ expression. Treatment with either small interfering RNA to PPARγ or GW9662 inhibited 15d-PGJ2-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ2, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ2- and rosiglitazone-stimulated NOS activity and NO production. Conclusions—These findings further clarify mechanisms involved in PPARγ-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPARγ-dependent mechanisms.

The Role of NADPH Oxidase in Chronic Intermittent Hypoxia-Induced Pulmonary Hypertension in Mice

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.

Fatty Acids Differentially Modulate Insulin-Stimulated Endothelial Nitric Oxide Production by an Akt-Independent Pathway

Background Insulin increases endothelial nitric oxide (NO) production by activating endothelial nitric oxide synthase (eNOS) through protein kinase B (Akt)-mediated phosphorylation of serine residue 1179 (p-eNOS serine 1179). Because fatty acids modulate insulinstimulated Akt signaling cascades in smooth muscle cells, we hypothesized that fatty acids would differentially regulate endothelial Akt signaling, eNOS phosphorylation, and NO production. Methods Porcine pulmonary artery endothelial cells (PAECs) were treated for 3 hours with 100 μM oleic (18:1) or eicosapentaenoic (20:5) acids or with an equivalent volume of ethanol vehicle (0.1%). PAECs were then treated with graded concentrations (10−9-10−5 M) of insulin or incubated overnight (24 hours) in culture medium without fatty acids before insulin treatment. Activation and phosphorylation of Akt and eNOS were determined by immunoblotting. NO production was measured with a chemiluminescence NO analyzer or with a NO-selective carbon fiber microelectrode. Results Insulin-stimulated Akt phosphorylation, eNOS phosphorylation, and NO production. The phosphatidylinositol-3 kinase inhibitor wortmannin attenuated insulin-stimulated Akt activation and NO production. Treatment with the ω-3 fatty acid 20:5, but not 18:1, enhanced insulin-stimulated NO production but failed to alter insulin-stimulated Akt activation or eNOS serine 1179 phosphorylation. Conclusion Individual fatty acyl species have distinct effects on insulin-stimulated endothelial NO production. Although fatty acids alter Akt signaling in muscle cells, the current results indicate that fatty acids do not modulate endothelial NO production through alterations in insulin-stimulated, Akt-mediated eNOS phosphorylation.

2 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGAND, ROSIGLITAZONE, ATTENUATES VASCULAR OXIDATIVE STRESS IN A MOUSE MODEL OF TYPE 2 DIABETES.

Purpose We have previously shown that peroxisome proliferator-activated gamma (PPARg) ligands reduce superoxide anion (O2 2 ×) generation in vascular endothelial cells in vitro by suppressing expression of selected subunits of NADPH oxidase and by increasing the expression and activity of Cu/Zn superoxide dismutase (SOD). The current study was designed to determine if PPARg ligands modulate vascular endothelial O2 2 × generation in vivo through these same mechanisms. Methods Lean control (db +/db 2) and obese, leptin receptor-deficient (db 2 /db 2) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7 days. Aortas were prepared for analysis of O2 2 × production using ESR spectroscopy and for RNA analysis, and serum was collected for analysis of metabolic parameters. Results Compared to db +/db 2 mice, obese, db 2 /db 2 mice had higher serum glucose, insulin, leptin, triglyceride, and fatty acid levels and lower adiponectin levels. Rosiglitazone had no effect on these metabolic derangements. Aortic O2 2 × generation measured with ESR spectroscopy was significantly increased in db 2 /db 2 mice. Aortic tissue from these mice also demonstrated higher relative mRNA levels of the NADPH oxidase subunits, Nox-1 and Nox-4, as measured by real-time PCR analysis and lower mRNA levels of PPARg. Rosiglitazone treatment decreased O2 2 × generation and mRNA levels of Nox homologues in db 2 /db 2 mice. Conclusions These data indicate that short-term treatment with the PPARg agonist rosiglitazone suppressed vascular NADPH oxidase expression and O2 2 × production in an animal model of vascular oxidative stress. Because these findings occurred in the absence of significant metabolic effects, these results indicate that rosiglitazone and other PPARg ligands may exhibit direct vascular protective effects.

304 CHRONIC ETHANOL INGESTION INCREASES ENDOTHELIAL NITRIC OXIDE SYNTHASE EXPRESSION AND ACTIVITY IN THE LUNG.

Purpose Chronic ethanol (EtOH) ingestion increases the incidence of the acute respiratory distress syndrome (ARDS), a severe form of acute lung injury. Based on previous evidence that chronic EtOH stimulation increased endothelial nitric oxide synthase (eNOS) expression and activity in pulmonary endothelial cells in vitro, we hypothesize that chronic EtOH ingestion would increase lung eNOS expression and activity in a well-established rat model of chronic EtOH ingestion. Methods Male Sprague-Dawley rats were fed liquid diets containing EtOH (36% of calories) or maltose-dextrin as an isocaloric substitution for EtOH (control) for 6 weeks. Selected animals were also treated with the angiotensin-converting enzyme (ACE) inhibitor lisinopril (3 mg/L) for 6 weeks. Lung tissue was prepared for analysis of H2O2 production, NOS activity, cGMP production, eNOS expression, and eNOS interactions with its regulatory proteins. Results Compared with control rats, H2O2 production, eNOS expression, lungNOS activity, and cGMP levels were significantly increased in EtOH-treated rats, effects attenuated by lisinopril. Chronic EtOH ingestion also increased lung levels of heat shock protein 90 (hsp90) while having no effect on caveolin 1 expression, and in co-immunoprecipitation studies decreased caveolin 1-eNOS interactions while having no effect on hsp90-eNOS interaction. Conclusions These results indicate that chronic EtOH ingestion increases lung eNOS expression and activity. The increased eNOS activity appears related in part to EtOH-induced alterations in the interaction of eNOS with regulatory proteins. Along with our previous reports, these findings provide new insight into mechanisms by which EtOH ingestion alters cell function in vivo and implicates EtOH-mediated increases in reactive oxygen and nitrogen species as important mediators.

195 THE RENIN ANGIOTENSIN SYSTEM MEDIATES CHRONIC ETHANOL-INDUCED INCREASES IN SUPEROXIDE PRODUCTION AND NADPH OXIDASE EXPRESSION IN THE LUNG

Purpose Chronic ethanol (EtOH) ingestion increases the incidence of the acute respiratory distress syndrome and causes oxidative stress and cellular dysfunction in the lung. The mechanisms of EtOH-induced oxidative stress in the lung remain to be defined. We sought to determine if chronic EtOH ingestion alters the expression of lung NADPH oxidase, a major enzymatic source of superoxide generation, in an in vivo rat model of chronic EtOH ingestion. Methods Male Sprague-Dawley rats were fed liquid diets containing EtOH (36% of calories) or an isocaloric diet substituting maltin-dextrin for EtOH (control) with or without the angiotensin-converting enzyme (ACE) inhibitor lisinopril for 6 weeks. Prior to sacrifice, blood pressure was monitored for 15-20 minutes. After sacrifice, lung frozen sections were stained with dihydroethidium (DHE) to detect superoxide production. Expression of specific components of the renin-angiotensin system (RAS) and specific NADPH oxidase components were then examined in lung homogenates. Results Chronic EtOH ingestion in the rat had no significant effect on blood pressure but increased superoxide formation in lung parenchyma measured as DHE fluorescence, an effect inhibited by lisinopril. Chronic EtOH ingestion failed to increase lung ACE expression, but increased angiotensinogen, angiotensin II type 1 (AT1) and type 2 (AT2) receptor expression. Chronic EtOH ingestion also increased expression of the NADPH oxidase subunit, gp91phox, an effect inhibited by lisinopril. Conclusions These results indicate that chronic EtOH ingestion alters superoxide production and specific NADPH oxidase subunit expression in the lung by a RAS-dependent pathway. These findings provide new insights into mechanisms by which EtOH causes oxidative stress in the lung.

417 EFFECTS OF CHRONIC ETHANOL ON NITRIC OXIDE PRODUCTION IN PORCINE PULMONARY ARTERY ENDOTHELIAL CELLS

Purpose Chronic ethanol (EtOH) ingestion increases the incidence and severity of the acute respiratory distress syndrome (ARDS), a pulmonary disorder characterized by disruption of the alveolar-capillary barrier. Nitric oxide (NO) has been implicated in regulation of endothelial barrier function. Because previous reports have demonstrated that exposure to EtOH for periods up to 6 hours increases endothelial NO production, we sought to determine if more chronic EtOH exposure could directly modulate endothelial nitric oxide synthase (eNOS) activity and endothelial NO production, thereby contributing to altered lung responses during chronic EtOH ingestion. Methods To test this hypothesis, porcine pulmonary artery endothelial cells (PAEC) were treated with 0.1% (w/v) EtOH for 72 hours in sealed chambers to prevent evaporation. Results EtOH caused dose-dependent increases in NO production and increased eNOS expression, effects that were attenuated by wortmannin, a specific PI-3 kinase inhibitor. EtOH also increased heat shock protein 90 (hsp90)-eNOS interaction, and geldanamycin, an hsp90 inhibitor, attenuated ETOH-stimulated eNOS-hsp90 interaction as well as NO production. In contrast to acute EtOH stimulation, chronic EtOH treatment did not stimulate Akt activation or phosphorylation of eNOS ser1177. Conclusions These results indicate that chronic EtOH exposure increases endothelial NO production via PI-3 kinase-mediated increases in eNOS expression and enhanced binding of hsp90 to eNOS. This study highlights potential differences in the mechanisms regulating endothelial NO production during acute and chronic EtOH exposure.