This proposal is aimed to test the hypothesis that: Cytochrome P450 (CYP) 1B1-generated metabolites of estradiol (E2) (2-methoxyestradiol, 2-ME) by inhibiting and testosterone (T) (6?-hydroxytestosterone, 6?-OHT) by enhancing angiotensin (Ang) II effect on cytosolic phospholipase A2? (cPLA2?) and generation of cyclooxygenase (COX)- arachidonic acid (AA)-derived prostaglandin (PG) E2 exerting prohypertensive effect via EP1 and EP3 receptors, protects the female but not males against hypertension and its pathogenesis. This hypothesis is based on our novel preliminary data that Ang II-induced hypertension in a) ovariectomized (OVX) or CYP1B1 gene disrupted (Cyp1b1-/-) female mice; and b) castrated (Cas) or Cyp1b1-/- male mice treated with 6?-OHT, is minimized by cPLA2? gene disruption (cPLA2?-/-), by AA metabolism inhibitor 5,8,11,14- eicosatetraynoic acid (ETYA), or COX-derived PGE2-EP1 and EP3 receptor antagonists. More importantly, our new observation shows that CYP1B1-generated E2 and T metabolites in the brain mediate the effect of Ang II on blood pressure (BP) by modulating the activity of cPLA2?AA system in the opposite direction in female (inhibitory) and male (stimulatory) mice. We will extend these observations and test our hypothesis by addressing the following specific aims.
AIM 1. To determine the interaction of CYP1B1 and cPLA2?/AA system in Ang II-induced hypertension and its pathogenesis in female mice. Sub-Aim 1. To investigate the contribution of central cPLA2?/AA system and CYP1B1 and their interaction in Ang II-induced hypertension and its pathogenesis in female mice.
Aim 2. To examine the interaction of CYP1B1 with cPLA2?/AA system in Ang II- induced hypertension and its pathogenesis in male mice. Sub-Aim 2. To determine the contribution of central CYP1B1 and its interaction with cPLA2?/AA system in Ang II-induced hypertension and its pathogenesis in male mice. To achieve these objectives, we will use the state-of-the-art techniques which include: 1) Radio- telemetry for measuring BP and for power spectral analysis, and Echocardiography for assessing cardiac function; 3) Cyp1b1+/+ and Cyp1b1-/- and cPLA2?+/+and cPLA2?-/- mice; 2) Adenovirus (Ad) CYP1B1 shRNA and Ad CYP1B1 DNA, and Adenovirus (Ad) cPLA2? shRNA and Ad cPLA2? DNA, and siRNA for EP1 and EP3, and steroid genomic and nongenomic receptors, and their respective controls; 4) UPLC/qTOFMS for the analysis of sex steroids and eicosanoids; 5) histological, immunohistochemical, and fluorescence microscopy and biochemical techniques; 6) Flow cytometry to determine immune cell population in the blood and tissues. The proposed studies should provide novel insights into the molecular mechanisms underlying sex differences as determined by the interaction of CYP1B1-generated metabolites of sex steroids and cPLA2?/AA system in Ang II-induced hypertension and its pathogenesis. Furthermore, these studies would allow us to demonstrate cPLA2? as a potential target for developing novel selective inhibitors of this enzyme for treating hypertension and associated pathogenesis in both sexes, and the detrimental effect of CYP1B1 inhibitors in females.
The proposed studies will provide novel insights into the molecular mechanisms underlying sex differences as determined by the interaction of cytochrome P450 (CYP) CYP1B1-generated metabolites of estrogen and testosterone and cytosolic phospholipase A2? (cPLA2?)/arachidonic acid system in the development and pathogenesis of high blood pressure. Moreover, these studies would demonstrate cPLA2? as a potential target for developing novel selective inhibitors of this enzyme for treating hypertension and its pathogenesis in both sexes, and the detrimental effect of CYP1B1 inhibitors in females.
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