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To determine the role of CYP1B1 in the gender difference in response to Ang II-induced hypertension, female Cyp1b1+/+ and Cyp1b1−/− mice were infused with Ang II (700 ng/kg/min) or vehicle with/without ovariectomy. In addition, mice were treated with the CYP1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene (TMS, 300 μg/kg, every 3rd day, i.p.), and 17-β-estradiol metabolites, 2-hydroxyestradiol (2-OHE), 4-OHE, or 2-methoxyestradiol (2-MeE2) (1.5 mg/kg/day, i.p., for 2 weeks), and systolic blood pressure (SBP) measured. Ang II increased SBP more in Cyp1b1−/− than in Cyp1b1+/+ mice (119±3 to 171±11 mmHg vs. 120±4 to 149±4 mmHg, P<0.05). Ang II caused cardiovascular remodeling and endothelial dysfunction, and increased vascular reactivity and oxidative stress in Cyp1b1−/− but not Cyp1b1+/+mice. The Ang II-induced increase in SBP was enhanced by ovariectomy and TMS in Cyp1b1+/+ but not Cyp1b1−/− mice. 2-OHE did not alter Ang II-induced increase in SBP in Cyp1b1+/+ mice but minimized it in Cyp1b1−/− mice, whereas 4-OHE enhanced Ang II-induced increase in SBP in Cyp1b1+/+ mice but did not alter the increased SBP in Cyp1b1−/− mice. 2-OHE-derived catechol-O-methyltransferase metabolite, 2-MeE2, inhibited Ang II-induced increase in SBP in Cyp1b1−/− mice. Ang II increased plasma levels of 2MeE2 in Cyp1b1+/+ but not Cyp1b1−/− mice, and increased activity of cardiac extracellular signal-regulated kinase 1/2, p38 mitogen-activated kinase, c-Src, and Akt in Cyp1b1−/− but not Cyp1b1+/+ mice. These data suggest that CYP1B1 protects against Ang II-induced hypertension and associated cardiovascular changes in female mice, most likely mediated by 2-MeE2-inhibiting oxidative stress and the activity of these signaling molecules.

Previously, we showed that Cyp1b1 gene disruption minimizes angiotensin II-induced hypertension and associated pathophysiological changes in male mice. This study was conducted to test the hypothesis that cytochrome P450 1B1-generated metabolites of testosterone, 6β-hydroxytestoterone and 16α-hydroxytestosterone, contribute to angiotensin II-induced hypertension and its pathogenesis. Angiotensin II infusion for 2 weeks increased cardiac cytochrome P450 1B1 activity and plasma levels of 6β-hydroxytestosterone, but not 16α-hydroxytestosterone, in Cyp1b1+/+ mice without altering Cyp1b1 gene expression; these effects of angiotensin II were not observed in Cyp1b1−/− mice. Angiotensin II-induced increase in systolic blood pressure and associated cardiac hypertrophy, and fibrosis, measured by intracardiac accumulation of α-smooth muscle actin, collagen and transforming growth factor-β, and increased nicotinamide adenine dinucleotide phosphate oxidase activity and production of reactive oxygen species; these changes were minimized in Cyp1b1−/− or castrated Cyp1b1+/+ mice, and restored by treatment with 6β-hydroxytestoterone. In Cyp1b1+/+ mice, 6β-hydroxytestosterone did not alter the angiotensin II-induced increase in systolic blood pressure; the basal systolic blood pressure was also not affected by this agent in either genotype. Angiotensin II or castration did not alter cardiac, angiotensin II type 1 receptor, angiotensin converting enzyme, Mas receptor, or androgen receptor mRNA levels in Cyp1b1+/+ or in Cyp1b1−/− mice. These data suggest that the testosterone metabolite, 6β-hydroxytestosterone, contributes to angiotensin II-induced hypertension and associated cardiac pathogenesis in male mice, most likely by acting as a permissive factor. Moreover, cytochrome P450 1B1 could serve as a novel target for developing agents for treating renin-angiotensin and testosterone-dependent hypertension and associated pathogenesis in males.

cytochrome P45 1B1 protects against angiotensin II-induced hypertension and associated cardiovascular changes in female mice, most likely via production of 2-methoxyestradiol. This study was conducted to determine if 2-methoxyestradiol ameliorates angiotensin II-induced hypertension, renal dysfunction, and end-organ damage in intact Cyp1b1−/−, ovariectomized female, and in Cyp1b1+/+ male mice. Ang II or vehicle was infused for 2 weeks and administered concurrently with 2-methoxyestradiol. Mice were placed in metabolic cages on day 12 of Ang II infusion for urine collection for 24 h. 2-Methoxyestradiol reduced angiotensin II-induced increases in systolic blood pressure, water consumption, urine output, and proteinuria in intact female Cyp1b1−/− and ovariectomized mice. 2-Methoxyestradiol also reduced Ang II-induced increase in blood pressure, water intake, urine output, and proteinuria in Cyp1b1+/+ male mice. Treatment with 2-methoxyestradiol attenuated Ang II-induced end-organ damage in intact Cyp1b1−/− and ovariectomized Cyp1b1+/+ and Cyp1b1−/− female mice, and Cyp1b1+/+ male mice. 2-Methoxyestradiol mitigated Ang II-induced increase in urinary excretion of angiotensinogen in intact Cyp1b1−/− and ovariectomized Cyp1b1+/+ and Cyp1b1−/− female mice but not in Cyp1b1+/+ male mice. The G-protein-coupled estrogen receptor 1 antagonist G-15 failed to alter Ang II-induced increases in blood pressure and renal function in Cyp1b1+/+ female mice. These data suggest that 2-methoxyestradiol reduces angiotensin II-induced hypertension and associated end-organ damage in intact Cyp1b1−/−, ovariectomized Cyp1b1+/+ and Cyp1b1−/− female mice, and Cyp1b1+/+ male mice independent of G protein-coupled estrogen receptor 1. Therefore, 2-methoxyestradiol could serve as a therapeutic agent for treating hypertension and associated pathogenesis in postmenopausal females, and in males.

Angiotensin II activates cPLA2α and releases arachidonic acid from tissue phospholipids which mediate or modulate one or more cardiovascular effects of angiotensin II and has been implicated in hypertension. Since arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2α might play a central role in the development of angiotensin II-induced hypertension. To test this hypothesis, we investigated the effect of angiotensin II infusion for 13 days by micro-osmotic pumps on systolic blood pressure and associated pathogenesis in wild type (cPLA2α+/+) and cPLA2α−/− mice. Angiotensin II-induced increase in systolic blood pressure in cPLA2α+/+ mice was abolished in cPLA2α−/− mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2α+/+ mice. Angiotensin II in cPLA2α+/+ mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction and increased vascular reactivity in cPLA2α+/+ mice; these changes were diminished in cPLA2α−/− mice. Angiotensin II also increased cardiac infiltration of F4/80+ macrophages and CD3+ T lymphocytes, cardiovascular oxidative stress, expression of endoplasmic reticulum stress markers p58IPK and CHOP in cPLA2α+/+ but not cPLA2α−/− mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2α+/+ but not cPLA2α−/− mice. These data suggest that angiotensin II-induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2α activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant pro-hypertensive effects and activation of one or more signaling molecules including ERK1/2 and cSrc.

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Cytochrome P450 (CYP) 1B1 is implicated in vascular smooth muscle cell migration, proliferation, and hypertension. We assessed the contribution of CYP1B1 to angiotensin (Ang) IIeinduced abdominal aortic aneurysm (AAA). Male Apoe À/À /Cyp1b1 þ/þ and Apoe À/À /Cyp1b1 À/À mice were infused with Ang II or its vehicle for 4 weeks; another group of Apoe À/À /Cyp1b1 þ/þ mice was coadministered the CYP1B1 inhibitor 2,3 0 ,4,5 0 -tetramethoxystilbene (TMS) every third day for 4 weeks. On day 28 of Ang II infusion, AAAs were analyzed by ultrasound and ex vivo by Vernier calipers, mice were euthanized, and tissues were harvested. Ang II produced AAAs in Apoe À/À /Cyp1b1 þ/þ mice; mice treated with TMS or Apoe À/À /Cyp1b1 À/À mice had reduced AAAs. Ang II enhanced infiltration of macrophages, T cells, and platelets and increased plateletderived growth factor D, Pdgfrb, Itga2, and matrix metalloproteinases 2 and 9 expression in aortic lesions; these changes were inhibited in mice treated with TMS and in Apoe À/À /Cyp1b1 À/À mice. Oxidative stress resulted in cyclooxygenase-2 expression in aortic lesions. These effects were minimized in Apoe À/À / Cyp1b1 þ/þ mice treated with TMS and in Apoe À/À /Cyp1b1 À/À mice and by concurrent treatment with the superoxide scavenger 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl. CYP1B1 contributed to the development of Ang IIeinduced AAA and associated pathogenic events in mice, likely by enhancing oxidative stress and associated signaling events. Thus, CYP1B1 may serve as a target for therapeutic agents for AAA in males. (Am J Pathol 2016 http://dx

cPLA2α contributes to renal dysfunction, inflammation, and end-organ damage, most likely via the action of pro-hypertensive eicosanoids and increased oxidative stress associated with Ang II-induced hypertension. Thus, cPLA2α could serve as a potential therapeutic target for treating renal dysfunction and end-organ damage in hypertension.