John A. Polikandriotis

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.

231 CHRONIC ETHANOL INGESTION CAUSES ALVEOLAR MACROPHAGE DYSFUNCTION: THE ROLE OF PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA.

Rationale Chronic ethanol (ETOH) ingestion increases the incidence of the acute respiratory distress syndrome (ARDS), a severe form of lung injury with a high mortality rate. Recent studies have clarified numerous alcohol-induced derangements in lung cell function. The current study focuses on the impact of chronic ETOH ingestion on alveolar macrophage function and explores the peroxisome proliferator-activated receptor gamma (PPARg) as a potential therapeutic target 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 maltose-dextrin for ETOH (control) for 6 weeks. After sacrifice, alveolar macrophages were isolated and their ability to phagocytose and internalize fluorescent microorganisms was evaluated using confocal microscopy with and without ex vivo treatment with the PPARg agonist rosiglitazone. In addition, expression of PPARg was examined in isolated macrophages and lung homogenates. Results Chronic ETOH ingestion in the rat decreased the capacity of alveolar macrophages to bind and internalize infectious particles. In contrast, ex vivo treatment with rosiglitazone restored the phagocytic capacity of the macrophages. Chronic EtOH ingestion also significantly reduced PPARg expression in lung homogenates. Conclusions These results indicate that chronic EtOH ingestion disrupts macrophage function and reduces PPARg expression in the lung. PPARg agonists such as rosiglitazone may improve the capacity of the alcoholic lung to clear infectious particles and decrease the risk or severity of respiratory infections.

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.

161 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGAND 15d-PGJ2 REPRESSES PROINFLAMMATORY RESPONSES IN VASCULAR ENDOTHELIAL CELLS: THE ROLE OF NITRIC OXIDE.

Purpose Peroxisome proliferator-activated receptor gamma (PPARg) activation prevents atherosclerotic vascular disease and reduces vascular dysfunction and inflammation in diabetic and nondiabetic subjects. The precise molecular mechanisms for this vascular protection remain to be defined. We have previously shown that PPARg ligands enhance endothelial nitric oxide (NO) bioavailability, in part, by reducing superoxide anion (O2 2.) generation and suppressing expression of selected subunits of NADPH oxidase and by enhancing eNOS activity at the level of post-translational regulation. We hypothesized that PPARg-induced increases in endothelial NO production contribute to previously described vascular anti-inflammatory effects following PPARg activation. Methods Human umbilical vein endothelial cells (HUVECs) were treated with 10 μM 15d-PGJ2 with or without human recombinant TNFa (100 U for 2 hours) followed by analysis of cytokine production, adhesion molecule expression, and monocyte adhesion. In separate studies, HUVECs were treated with the NOS inhibitor L-NAME prior to treatment with PPARg ligands. Results 15d-PGJ2 attenuated TNFa-mediated induction of IL-6, IL-8, MCP-1, and IP-10, endothelial-monocyte adhesion, and ICAM, VCAM, and E-selectin expression. The NOS inhibitor L-NAME reduced 15d-PGJ2-mediated NO production and attenuated 15d-PGJ2 repression of TNFa-mediated ICAM expression. Conclusions These data indicate that treatment with 15d-PGJ2 suppressed TNFa-stimulated expression of inflammatory genes in vascular endothelial cells. L-NAME-mediated attenuation of the ability of 15d-PGJ2 to suppress ICAM expression suggests that PPARg-stimulated NO production may contribute to the anti-inflammatory effects of PPARg ligands in vascular endothelial cells.

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.