Dean J. Kleinhenz

Rosiglitazone Attenuates Chronic Hypoxia–Induced Pulmonary Hypertension in a Mouse Model

Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARgamma with rosiglitazone would attenuate chronic hypoxia-induced pulmonary hypertension, in part, through suppressing NADPH oxidase-derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, Fi(O2) 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARgamma ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor beta (PDGFRbeta) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRbeta activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARgamma expression. Collectively, these findings indicate that PPARgamma ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARgamma activation in pulmonary hypertension.

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.

Disruption of endothelial peroxisome proliferator-activated receptor-γ reduces vascular nitric oxide production

Vascular endothelial cells express the ligand-activated transcription factor, peroxisome proliferator-activated receptor-gamma (PPARgamma), which participates in the regulation of metabolism, cell proliferation, and inflammation. PPARgamma ligands attenuate, whereas the loss of function mutations in PPARgamma stimulate, endothelial dysfunction, suggesting that PPARgamma may regulate vascular endothelial nitric oxide production. To explore the role of endothelial PPARgamma in the regulation of vascular nitric oxide production in vivo, mice expressing Cre recombinase driven by an endothelial-specific promoter were crossed with mice carrying a floxed PPARgamma gene to produce endothelial PPARgamma null mice (ePPARgamma(-/-)). When compared with littermate controls, ePPARgamma(-/-) animals were hypertensive at baseline and demonstrated comparable increases in systolic blood pressure in response to angiotensin II infusion. When compared with those of control animals, aortic ring relaxation responses to acetylcholine were impaired, whereas relaxation responses to sodium nitroprusside were unaffected in ePPARgamma(-/-) mice. Similarly, intact aortic segments from ePPARgamma(-/-) mice released less nitric oxide than those from controls, whereas endothelial nitric oxide synthase expression was similar in control and ePPARgamma(-/-) aortas. Reduced nitric oxide production in ePPARgamma(-/-) aortas was associated with an increase in the parameters of oxidative stress in the blood and the activation of nuclear factor-kappaB in aortic homogenates. These findings demonstrate that endothelial PPARgamma regulates vascular nitric oxide production and that the disruption of endothelial PPARgamma contributes to endothelial dysfunction in vivo.

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.

Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.

Attenuation of signaling and nitric oxide production following prolonged leptin exposure in human aortic endothelial cells.

Acute leptin exposure stimulates endothelial nitric oxide (NO) production in vitro. In contrast, chronic elevations in circulating leptin levels in patients with obesity are associated with endothelial dysfunction and impaired endothelial NO production. Therefore, the goal of the current study was to examine the direct effects of acute and more sustained leptin stimulation on endothelial nitric oxide synthase (eNOS) and NO production in human aortic endothelial cells (HAECs). HAECs were treated with vehicle or with leptin (5 or 60 ng/mL) acutely (30-60 minutes) or for 72 hours. HAEC NO release into culture media was measured with a chemiluminescence technique, and superoxide (O2 −.) production was measured with electron spin resonance (ESR) spectroscopy. HAEC eNOS activity was measured as the conversion of 3 H-arginine to 3 H-citrulline, and protein levels of eNOS, phospho-eNOS (serine 1177), Erk, phospho-Erk, suppressor of cytokine signaling (SOCS3), xanthine oxidase (XO), and the reduced nicotinamide adenine dinucleotide (NADPH) oxidase components p22phox, p67phox, Nox-4, and gp91phox were examined by Western blotting or immunoprecipitation. Acute leptin exposure increased eNOS serine 1177 phosphorylation and caused Erk activation. In contrast, prolonged leptin stimulation was not cytotoxic and failed to alter eNOS expression, phosphorylation, or HAEC NO release. Furthermore, prolonged leptin stimulation did not alter O2 −. production or NADPH oxidase or XO expression but increased SOCS3 expression. In contrast to acute stimulation, prolonged (72 hours) stimulation does not alter endothelial cell NO or O2 −. production. We postulate that chronic leptin stimulation, through increased SOCS3 expression, may attenuate the effects of leptin on vascular endothelial function.

The measurement of nitric oxide production by cultured endothelial cells

Nitric oxide (NO) produced by vascular endothelial cells (ECs) plays a critical role in normal vascular physiology. Important insights into mechanisms regulating the production of endothelial NO have been derived from in vitro studies employing cultured ECs. Although many techniques for the detection of NO have been described, many of these methods lack adequate sensitivity to detect the small amount of NO produced by cultured ECs. In this chapter, we describe three protocols that employ chemiluminescence, electron spin resonance, or electrochemical techniques to permit the reliable detection of EC NO production.

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.

ROZIGLITAZONE ATTENUATES CHRONIC HYPOXIA-INDUCED PULMONARY HYPERTENTION.: 170

increased by 30% (baseline; 439 6 59 3 1,000 mm vs saline infusion; 573 6 41 3 1,000 mm, n 5 6, p , .05), showing time-dependent progression of atherosclerosis. In marked contrast, there was no increase in lesion size in IGF-1-infused apoe null mice (baseline; 439 6 59 3 1,000 mm vs IGF infusion; 419 6 37 3 1,000 mm, n 5 6, p 5 .38), indicating IGF-1 prevented progression of preexisting atherosclerotic lesions. To determine mechanisms, we overexpressed IGF-1R using recombinant adenovirus in cultured macrophages (MW) exposed to 100 mg/mL native LDL (nLDL) or oxidized LDL (oxLDL) for 24 hours. IGF-1R up-regulation markedly inhibited MW lipid accumulation and foam cell formation (85 6 11% inhibition vs MW infected with GFP adenovirus, n 5 4, p , .05). Furthermore, flow cytometry analysis indicated that IGF-1 infusion increased levels of circulating Sca-1/Flk-1 positive endothelial progenitor cells (EPCs), which are thought to contribute to endothelial repair (3.46 6 2.1% in IGF-1-infused mice vs 0.96 6 0.28% in saline-infused mice, n 5 6). In conclusion, our data demonstrate that IGF-1 markedly suppresses atherosclerotic plaque progression in apoe null mice, potentially via prevention of foam cell formation and lipid accumulation in lesions and via promotion of endothelial repair by increasing circulating EPCs. Our findings establish a new paradigm for IGF-1 effects on the cardiovascular system.

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.