Eicosanoid-Induced Vascular Growth During Injury

TMS或CYP1B1shRNA可抑制Ang II和AA诱导的大鼠VSMC中NADPH氧化酶的激活，但不能阻止CYP1B1+/+小鼠VSMC中的NADPH氧化酶的激活。目的2b)研究细胞色素P1B1在血管紧张素转换酶II诱导的动脉粥样硬化、动脉瘤和新生内膜生长加速中的作用。3.探讨CYP1B1依赖Ang II和AA诱导高胆固醇血症小鼠VSMC产生NADPH氧化酶和ROS的机制。为了达到这些目标，我们计划使用许多最先进的体外和体内细胞和分子生物学技术和转基因小鼠。

DESCRIPTION (provided by applicant): The current proposal is an extension of our studies conducted over the previous grant period and is aimed to investigate the contribution of arachidonic acid (AA) metabolites generated via CYP1B1 and the underlying mechanism involved in Ang II-induced neointimal growth caused by vascular injury and atherosclerosis. It is based on our novel preliminary data that: a) the selective CYP1B1 inhibitor 2,4,32,52-tetramethoxystilbene (TMS) or adenovirus CYP1B1 shRNA (CYP1B1 shRNA) prevents Ang II-stimulated neointimal growth caused by balloon injury of rat carotid artery; b) Ang II increases neointimal growth in wire-injured carotid artery of wild type (Cyp1b1+/+),but not CYP1B1 deficient (Cyp1b1-/-), mice; c) treatment with TMS inhibits generation of fatty streak areas and their acceleration by Ang II in Apo E knockout (apoE-/-) mice fed a high fat diet (HFD); and d) Ang II and AA-induced NADPH oxidase activation in rat VSMCs is prevented by TMS or CYP1B1 shRNA, and in VSMC from Cyp1b1-/-, but not Cyp1b1+/+, mice. These findings have led us to the following central hypothesis: CYP1B1, through AA metabolites/lipid peroxides, results in activation of NADPH oxidase and production of ROS, which, by activating one or more signaling molecules, contribute to the pathogenesis of restenosis caused by Ang II during vascular injury and to atherosclerosis produced by hypercholesterolemia and its acceleration by Ang II. To test this hypothesis, we will address the following specific aims: Aim 1. Determine the contribution of CYP1B1 to neointimal growth stimulated by Ang II in balloon-injured rat and wire- injured mouse carotid artery; Aim 2a) Investigate the contribution of CYP1B1 to atherosclerosis and neointimal growth after wire injury of mouse carotid artery; Aim 2b) Examine the contribution of CYP1B1 to Ang II-induced acceleration of atherosclerosis, aneurysm, and neointimal growth; Aim. 3. Investigate the mechanism of CYP1B1-dependent Ang II- and AA-induced NADPH oxidase and ROS production in VSMCs from hypercholesterolemia in mice. To address these aims, we plan to use numerous state-of-the-art in vitro and in vivo cellular and molecular biology techniques and transgenic mice. These include using 1) adenovirus CYP1B1 shRNA; 2) apoE-/-/Cyp1b1+/+ and double knockout-apoE-/-/Cyp1b1-/- and knockin-apoE+/+/CYp1b1+/+ mice that we have generated in our laboratory; 3) isolated VSMCs from these transgenic animals; 4) morphological, histological, immunohistochemical and fluorescence, and biochemical techniques, and 5) HPLC-LC-ESI-MS for the identification of AA metabolites. The proposed studies should advance our current knowledge of the cellular and molecular mechanisms involved in the pathogenesis of restenosis and atherosclerosis and allow us to demonstrate CYP1B1 as a potential novel target for the development of more effective therapeutic drugs such as TMS for treating Ang II-induced restenosis caused by vascular injury and atherosclerosis by hypercholesterolemia.