Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor g (PPARg) contribute to hypoxiainduced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxiaexposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O 2 ) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARg, and transforming growth factor (TGF)b1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O 2 ) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARg and TGF-b1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H 2 O 2 release, proliferation, and TGF-b1 expression and blunted reductions in PPARg in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-b1 and reductions in PPARg expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxiainduced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.Keywords: rosiglitazone; PPARg; TGF-b; pulmonary hypertension Pulmonary hypertension (PH) is a progressive disorder associated with significant morbidity and mortality. Although recent therapeutic advances have improved survival for patients with PH, the prognosis remains poor (1). The pathobiology of PH is complex, and factors that contribute to endothelial dysfunction have been implicated in pathogenesis (2, 3). Among these factors, NADP reduced (NADPH) oxidase enzymes that produce reactive oxygen species (ROS) contribute to the development of a variety of vascular diseases, such as atherosclerosis (4) and systemic (5) and pulmonary hypertension (6). NADPH oxidases catalyze the reduction of molecular oxygen to generate superoxide (O 2 .2 ), hydrogen peroxide (H 2 O 2 ), or secondary oxidants (7). Seven isoforms of the catalytic moiety of the nonphagocytic NADPH oxidase enzyme have been described (Nox1-5, Duox1-2). These subunits are homologous to the catalytic moiety of the prototype phagocytic NADPH oxidase Nox2 (or gp91 phox ) but differ from each other regarding cellular localization, tissue distribution, regulation, activation, and expression (7,8). For example, although both Nox1 and Nox4 are expressed in vascular smooth muscle cells (VSMCs), they are targeted to discreet intracellular locations, are differe...

Objective Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O2•−) and hydrogen peroxide (H2O2) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O2•− (mtO2•−) and H2O2 (mtH2O2) increases Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Approach and Results Exposure of human pulmonary artery endothelial cells to hypoxia for 72 hours increased mtO2•− and mtH2O2. To assess the contribution of mtO2•− and mtH2O2 to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (TghSOD2) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 weeks. Compared to hypoxic control mice, MCAT mice developed less hypoxia-induced increases in RVSP, α-SMA staining, extracellular H2O2 (Amplex Red), Nox2 and Nox4 (qRT-PCR and western blot), or cyclinD1 and PCNA (western blot). In contrast, TghSOD2 mice experienced exacerbated responses to hypoxia. Conclusions These studies demonstrate that hypoxia increases mtO2•− and mtH2O2. Targeting mtH2O2 attenuates PH pathogenesis, whereas, targeting mtO2•− exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H2O2 release. These studies suggest that targeted reductions in mtH2O2 generation may be particularly effective at preventing hypoxia-induced PH.

Kang BY, Kleinhenz JM, Murphy TC, Hart CM. The PPAR␥ ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol 301: L881-L891, 2011. First published September 16, 2011 doi:10.1152/ajplung.00195.2011.-Peroxisome proliferatoractivated receptor (PPAR) ␥ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPAR␥ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O 2) for 72 h and treated with or without the PPAR␥ ligand rosiglitazone (RSG, 10 M) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxiaactivated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1␣ (chetomin) or nuclear factor (NF)-B (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 wk with or without gavage with RSG (10 mg·kg Ϫ1 ·day Ϫ1 ) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1␣, NF-B activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPAR␥ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxiaresponsive transcription factors. Targeting PPAR␥ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis. pulmonary hypertension; hypoxia; peroxisome proliferator-activated receptor ␥; endothelin; endothelial cells PULMONARY HYPERTENSION (PH), defined as an elevation of the mean pulmonary artery pressure Ͼ25 mmHg at rest or 30 mmHg with exercise causes significant morbidity and mortality (13, 31). The pathogenesis of PH involves endothelial dysfunction with increased production of vasoconstrictors, e.g., endothelin-1 (ET-1), and reduced production of vasodilators, e.g., prostacyclin and nitric oxide (NO) (5,14). Despite the availability of existing PH therapies that attenuate these derangements, PH morbidity and mortality remains unacceptably high (20, 44), indicating an urgent need for novel therapeutic strategies. Recent studies indicate that the nuclear hormone receptor peroxisome proliferator-activated receptor ␥ (PPAR␥), may play an important role in the pathophysiology of PH. Treatment with thiazolidinedione (TZD) PPAR␥ ligands attenuates PH in several experimental models (6,19,27,36,39). PPAR␥ ligands not only attenuate the development ...

Background-The oxidized low-density lipoprotein receptor (LDLR) LOX-1 plays a crucial role in atherosclerosis. We sought to detect and assess atherosclerotic plaque in vivo by using single-photon emission computed tomography/ computed tomography and magnetic resonance imaging and a molecular probe targeted at LOX-1. Methods and Results-Apolipoprotein E Ϫ/Ϫ mice fed a Western diet and LDLR Ϫ/Ϫ and LDLR