Thursday, November 21, 2024
11/21/2024
This proposal is aimed to test our novel hypothesis that the cytochrome P450 (CYP)1B1-generated metabolites of 17b-estradiol (E2) (2-methoxyestradiol, 2-ME) and testosterone (T) (6b-hydroxytestosterone, 6b-OHT) inhibit and enhance, respectively, Ang II-induced production of prohypertensive arachidonic acid (AA)-12/15 lipoxygenase (ALOX15)/12(S)-hydroxyeicosatetraenoic acid (HETE) and Cyp4a/20-HETE metabolites in the paraventricular nucleus (PVN) and that this mechanism accounts for the sex differences in Ang II-induced hypertension, i.e., a lesser increase in blood pressure (BP) in females than males. This hypothesis is based on our compelling preliminary data that transduction of PVN with adenovirus (Ad)- ALOX15, and Ad-Cyp4a10/Cyp4a12a-shRNA, respectively, a) ameliorate Ang II-induced increase in BP in both wild-type (WT) female and male mice; b) attenuate the effect of Ang II to enhance BP, in CYP1B1 gene disrupted (Cyp1b1–/–) female mice that lack 2-ME; and c) minimize the 6b-OHT effect to restore Ang II-induced increase in BP that is diminished in Cyp1b1–/– male mice lacking endogenous 6b-OHT. We will expand these observations to test our hypothesis by addressing the following specific aims. Aim 1. To determine contribution of the central AA/LOX15 system to Ang II-induced hypertension and its pathogenesis in female and male mice. Sub-Aim 1. To examine interaction of the central AA/LOX15 system and CYP1B1-generated E2 and T metabolites in Ang II-induced hypertension and its pathogenesis in female and male mice. Aim 2. To examine contribution of the central AA/Cyp4a10 and Cyp4a12a systems to Ang II-induced hypertension and its pathogenesis in female and male mice. Sub-Aim 2. To determine interaction of the central AA/Cyp4a10 and Cyp4a12a systems and CYP1B1-generated E2 and T metabolites in Ang II-induced hypertension and its pathogenesis in females and male mice. To achieve these aims, we will use the state-of-the-art techniques, which include: a) Radiotelemetry for measuring BP; b) wild-type and Alox15–/–, Cyp1b1–/–, and cPLA2a–/– female and male mice; c) Ad-ALOX15-shRNA, Ad-Cyp4a10, and Ad-Cyp4a12a-shRNA and respective Ad- scrambled shRNA controls, and their Ad-DNA and Ad-GFP-DNA control, and 12(S)-HETE Gpr31 and Ltb4r2 and 20-HETE Gpr75 receptor siRNAs and their non-target siRNA; d) Histological, immunohistochemical, and fluorescence microscopy and biochemical techniques; e) Flow cytometry to determine immune cell infiltration in aorta, kidney and brain; f) UPLC/qTOF MS for the analysis of sex steroids and eicosanoids. The proposed studies will provide insights into the molecular mechanism underlying sex differences determined by the interaction of CYP1B1-generated metabolites of sex steroids and AA/ALOX15- and Cyp4a systems in the PVN in Ang II-induced hypertension and associated pathogenesis. Moreover, these studies should identify central ALOX15 and Cyp4a as a potential site of action of the selective inhibitors of these enzyme systems and 12(S)- HETE and 20-HETE receptor antagonists for treating hypertension and its pathogenesis in both sexes.
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