Angiotensins, Prostaglandins-Adrenergic Interactions

描述（由申请人提供）：该提案是先前工作的扩展，旨在检验IV组胞质磷脂酶A2（cPLA 2？）在高血压和高血压相关靶器官损伤的发病机制中起关键作用的假设。这一新的假设是基于有趣的初步数据，即小鼠中cPLA 2 <$$>基因破坏（cPLA 2 <$-/-）可改善Ang II和DOCA盐诱导的高血压。此外，cPLA 2？基因破坏可防止Ang II诱导的靶器官损伤（cPLA 2-/-小鼠心血管功能障碍、心脏和肾脏纤维化减少就是证据）。cPLA2基因破坏还改善了血管紧张素II诱导的巨噬细胞和T淋巴细胞的肾浸润，表明高血压诱导的免疫系统激活的改变。更重要的是，cPLA 2？基因破坏可防止与Ang II诱导的高血压相关的中枢神经系统穹隆下器官（SFO）中活性氧簇（ROS）的产生，这种作用可通过cPLA 2 ²-/-小鼠SFO中腺病毒（Ad）cPLA 2 ² DNA的表达来逆转。为了研究cPLA 2？参与高血压和终末器官损伤发病机制的创新概念，将解决以下具体目标。目标1.确定cPLA 2 <$在Ang II和DOCA盐诱导的高血压中的作用。目标2.确定cPLA 2对Ang II和DOCA盐诱导的靶器官损伤的作用。目标3.确定cPLA 2 ²对Ang II和DOCA盐诱导的免疫系统激活的贡献。目标4。确定cPLA 2对Ang II和DOCA盐诱导的中枢神经系统变化的作用。为了实现这些目标，将使用最先进的体内和体外细胞和分子生物学以及组织学和免疫组织化学技术。其中包括：a）1）cPLA2？+/+和cPLA 2？- /-小鼠; 2）腺病毒（Ad）cPLA 2 <$shRNA和Ad cPLA 2 <$DNA; 3）遥测血压和超声心动图评估心脏功能; 4）组织学，免疫组织化学，荧光显微镜和生化技术; 5）流式细胞术，以确定血液和组织中的免疫细胞群; 6）转基因小鼠脾脏的T细胞，以确定cPLA 2 <$DNA的机制。- 诱导的NADPH氧化酶活性和ROS产生;和7）HPLC-LC-ESI-MS以测量类花生酸和其他可能的AA代谢物。这些研究将为cPLA 2 <$与NADPH氧化酶激活和免疫系统在高血压发展及其发病机制中的作用机制提供新的见解。此外，这些研究应该允许证明cPLA 2 <$是开发新型的，选择性的，水溶性的这种酶的抑制剂的潜在目标，用于治疗高血压和相关的靶器官损伤，并刺激进一步的基础和临床研究，以确定cPLA 2 <$基因在高血压和心血管疾病和肾脏疾病中可能的多态性。

DESCRIPTION (provided by applicant): This proposal is an extension of the previous work and is aimed to test the hypothesis that Group IV cytosolic phospholipase A2 (cPLA2¿)plays a critical role in the pathogenesis of hypertension and hypertension- associated target-organ damage. This novel hypothesis is based on intriguing preliminary data that cPLA2¿ gene disruption in mice (cPLA2¿-/-) ameliorates both Ang II- and DOCA-salt-induced hypertension. Moreover, cPLA2¿ gene disruption prevents Ang II-induced target-organ damage (as evidence by reductions in cardiovascular dysfunction, cardiac and renal fibrosis in cPLA2¿-/- mice). cPLA2¿ gene disruption also ameliorates Ang II-induced renal infiltration of macrophages and T lymphocytes, suggesting alterations in hypertension-induced immune system activation. More imporantly, cPLA2¿ gene disruption prevents production of reactive oxygen species (ROS) in subfornical organ (SFO) in the central nervous system associated with Ang II-induced hypertension, effects that are reversed by expression of adenovirus (Ad) cPLA2¿ DNA in the SFO of cPLA2¿-/- mice. To investigate the innovative concept that cPLA2¿ is involved in the pathogenesis of hypertension and end organ damage, the following specific aims will be addressed. Aim 1. To determine the contribution of cPLA2¿ to Ang II- and DOCA-salt-induced hypertension. Aim 2. To determine the contribution of cPLA2¿ to Ang II- and DOCA-salt-induced target-organ damage. Aim 3. To determine the contribution of cPLA2¿ to Ang II- and DOCA-salt-induced immune system activation. Aim 4. To determine the role of cPLA2¿ to Ang II and DOCA-salt-induced changes in the central nervous system. To accomplish the objective of these aims, the state-of-the-art in vivo and in vitro cellular and molecular biology and histologicl and immunohistochemical techniques will be used. These include: a) 1) cPLA2¿+/+ and cPLA2¿- /- mice; 2) Adenovirus (Ad) cPLA2¿ shRNA and Ad cPLA2¿ DNA; 3) telemetry for measuring BP and echocardiography for assessing cardiac function; 4) histological, immunohistochemical, and fluorescence microscopy and biochemical techniques; 5) flow cytometry to determine immune cell population in the blood and tissues; 6) T cells from the spleen of transgenic mice to determine the mechanism of cPLA2¿-induced NADPH oxidase activity and ROS production; and 7) HPLC-LC-ESI-MS to measure eicosanoids and other possible AA metabolites. The proposed studies will provide novel insights into the mechanism linking cPLA2¿ to activation of NADPH oxidase and the immune system in the development of hypertension and its pathogenesis. Moreover, these studies should allow to demonstrate that cPLA2¿ is a potential target for developing novel, selective, water-soluble inhibitors of this enzyme for treating hypertension and associated target-organ damage and for stimulating further basic and clinical research to determine possible polymorphisms in the cPLA2¿ gene in hypertension and cardiovascular and renal diseases.