PPAR gamma and Nox4 in pulmonary hypertension

描述(由申请人提供)：肺动脉高压(PH)是一种复杂的疾病，会导致显著的发病率和死亡率。越来越多的证据表明，代谢紊乱是PH的病理生理学基础。这项建议关注的是过氧化物酶体增殖物激活受体γ(PPARγ)，这是一种降低PH值的主要代谢调节因子。低氧和其他原因引起的PH降低了PPARγ的表达，从而增加了NADPH氧化酶4的表达和活性。NOX4产生活性氧簇(ROS)，参与肺血管细胞的增殖和PH的发病。另一方面，刺激PPARγ减少NOX4的表达和活性，减轻缺氧诱导的血管重构、右室肥厚和PH。PPARγ调节PH值的机制仍在研究中。初步数据表明，PPARγ的减少降低了PPARγ辅活化子1α(Pgc1α)和解偶联蛋白2(UCP2)的表达，这两种蛋白质分别调节线粒体(MT)的生物发生和活性氧(ROS)的产生。这些研究将验证这样一种假设：PPARγ耗竭可减少肺动脉平滑肌细胞(PASMC)Pgc1、α和UCP2，并刺激MT功能障碍和ROS的产生。研究人员进一步推测，MT ROS激活ERK1/2-NF-κB轴，增加NOX4的表达和过氧化氢的产生，从而促进PASMC增殖、肺血管重塑和PH。为了验证这一假设，特定目标1将探索PPARγ活性降低在MT功能障碍、NOX4诱导、 并采用体外和体内互补的PASMC增殖模型。基因或药物降低的PPARγ活性将在体外用于人PASMC，而可诱导的、以平滑肌为靶点的PPARγ基因敲除小鼠(γKO)将用于体内。具体目标2将研究PPARγ减少在低氧诱导的PGC1UCP2和α改变、MT功能障碍、NOX4诱导和PASMC增殖中的作用。在体外和体内，PPARγ功能模型的得失将暴露在典型的对照或低氧条件下，并将测定MT功能、ROS产生和PASMC增殖。将通过测量右室收缩压、右室肥厚和心功能(超声心动图)来评估缺氧性肺动脉高压。采用免疫组织化学方法检测PASMC增殖细胞核抗原，肺切片形态计量学分析α-平滑肌肌动蛋白染色。将测试全部和部分(10-硝基-油酸)PPARγ配体抑制低氧增殖和PH的能力。从特发性肺动脉高压患者分离的PASMC中，关键的观察结果将得到证实。这项拟议的研究由一个多产和合作的研究团队进行，将促进对PPARγ在健康和疾病期间调节PASMC表型的作用的理解，并确定靶向PPARγ可以阻断PH发病的新策略。

DESCRIPTION (provided by applicant): Pulmonary hypertension (PH) is a complex disorder that causes significant morbidity and mortality. Mounting evidence suggests that metabolic derangements account for the pathophysiology underlying PH. This proposal focuses peroxisome proliferator-activated receptor gamma (PPARγ), a major metabolic regulator that is decreased PH. Hypoxia and other causes of PH decrease PPARγ expression which increases NADPH oxidase 4 (Nox4) expression and activity. Nox4 generates reactive oxygen species (ROS) that contribute to pulmonary vascular cell proliferation and PH pathogenesis. On the other hand, stimulating PPARγ reduces the expression and activity of Nox4 and attenuates hypoxia-induced vascular remodeling, right ventricular hypertrophy, and PH. The mechanisms by which PPARγ modulates PH continue to be defined. Preliminary data suggest that reductions in PPARγ reduce the expression of PPARγ coactivator 1 alpha (PGC1α) and uncoupling protein 2 (UCP2), proteins that regulate mitochondrial (MT) biogenesis and reactive oxygen species (ROS) generation, respectively. The proposed studies will test the hypothesis that PPARγ depletion reduces pulmonary artery smooth muscle cell (PASMC) PGC1α and UCP2 and stimulates MT dysfunction and ROS production. The investigators further postulate that MT ROS activate the ERK 1/2-NF-κB axis to increase Nox4 expression and H2O2 generation which promote PASMC proliferation, pulmonary vascular remodeling, and PH. To test this hypothesis, Specific Aim 1 will explore the role of reduced PPARγ activity in MT dysfunction, Nox4 induction, and PASMC proliferation using complementary in vitro and in vivo models. Genetic or pharmacological reductions in PPARγ activity will be used in human PASMC in vitro, and inducible, smooth muscle-targeted PPARγ knockout mice (smPPARγKO) will be employed in vivo. Specific Aim 2 will examine the role of reductions in PPARγ in hypoxia-induced alterations in PGC1α and UCP2, MT dysfunction, Nox4 induction, and PASMC proliferation. In vitro and in vivo gain and loss of PPARγ function models will be exposed to well characterized control or hypoxic conditions, and MT function, ROS production, and PASMC proliferation will be determined. Hypoxia-induced PH will be assessed with measures of right ventricular systolic pressure, right ventricular hypertrophy, and ventricular function (echocardiography). PASMC proliferation will be examined with immunohistochemistry for proliferating cellular nuclear antigen and morphometric analysis of lung sections stained for α-smooth muscle actin. The ability of full and partial (10- nitro-oleic acid) PPARγ ligands to attenuate hypoxic proliferatin and PH will be tested. Critical observations will be confirmed in PASMC isolated from patients with idiopathic pulmonary arterial hypertension. The proposed studies, conducted by a productive and collaborative research team, will advance understanding of the role of PPARγ in regulating PASMC phenotype during health and disease and identify novel strategies by which targeting PPARγ can interrupt PH pathogenesis.