Nayaab S. Khan

Estrogen Metabolism by Cytochrome P450 1B1 Modulates the Hypertensive Effect of Angiotensin II in Female Mice

To determine the role of cytochrome P450 (CYP) 1B1 in the sex difference in response to angiotensin II (Ang II)–induced hypertension, female Cyp1b1+/+ and Cyp1b1−/− mice were infused with Ang II (700 ng/kg per minute) or vehicle with or without ovariectomy. In addition, mice were treated with the CYP1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene (TMS; 300 &mgr;g/kg IP, every third day), and 17-&bgr; estradiol metabolites, 2-hydroxyestradiol (2-OHE), 4-OHE, or 2-methoxyestradiol (1.5 mg/kg per day IP, for 2 weeks) and systolic blood pressure (SBP) measured. Ang II increased SBP more in Cyp1b1−/− than in Cyp1b1+/+ mice (119±3–171±11 versus 120±4–149±4 mm Hg; P<0.05). Ang II caused cardiovascular remodeling and endothelial dysfunction and increased vascular reactivity and oxidative stress in Cyp1b1−/− but not in Cyp1b1+/+mice. The Ang II–induced increase in SBP was enhanced by ovariectomy and TMS in Cyp1b1+/+ but not in 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-methoxyestradiol, inhibited Ang II–induced increase in SBP in Cyp1b1−/− mice. Ang II increased plasma levels of 2-methoxyestradiol in Cyp1b1+/+ but not in 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 in 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-methoxyestradiol–inhibiting oxidative stress and the activity of these signaling molecules.

6β-Hydroxytestosterone, a Cytochrome P450 1B1 Metabolite of Testosterone, Contributes to Angiotensin II–Induced Hypertension and Its Pathogenesis in Male Mice

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, 6&bgr;-hydroxytestosterone and 16&agr;-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 6&bgr;-hydroxytestosterone, but not 16&agr;-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 &agr;-smooth muscle actin, collagen, and transforming growth factor-&bgr;, 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 6&bgr;-hydroxytestoterone. In Cyp1b1+/+ mice, 6&bgr;-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, 6&bgr;-hydroxytestosterone, contributes to angiotensin II–induced hypertension and associated cardiac pathogenesis in male mice, most probably 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.

Cytosolic Phospholipase A2α Is Critical for Angiotensin II–Induced Hypertension and Associated Cardiovascular Pathophysiology

Angiotensin II activates cytosolic phospholipase A2&agr; (cPLA2&agr;) and releases arachidonic acid from tissue phospholipids, which mediate or modulate ≥1 cardiovascular effects of angiotensin II and has been implicated in hypertension. Because arachidonic acid release is the rate limiting step in eicosanoid production, cPLA2&agr; 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 (cPLA2&agr;+/+) and cPLA2&agr;−/− mice. Angiotensin II–induced increase in systolic blood pressure in cPLA2&agr;+/+ mice was abolished in cPLA2&agr;−/− mice; increased systolic blood pressure was also abolished by the arachidonic acid metabolism inhibitor, 5,8,11,14-eicosatetraynoic acid in cPLA2&agr;+/+ mice. Angiotensin II in cPLA2&agr;+/+ mice increased cardiac cPLA2 activity and urinary eicosanoid excretion, decreased cardiac output, caused cardiovascular remodeling with endothelial dysfunction, and increased vascular reactivity in cPLA2&agr;+/+ mice; these changes were diminished in cPLA2&agr;−/− 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 cPLA2&agr;+/+ but not cPLA2&agr;−/− mice. Angiotensin II increased cardiac activity of ERK1/2 and cSrc in cPLA2&agr;+/+ but not cPLA2&agr;−/− mice. These data suggest that angiotensin II–induced hypertension and associated cardiovascular pathophysiological changes are mediated by cPLA2&agr; activation, most likely through the release of arachidonic acid and generation of eicosanoids with predominant prohypertensive effects and activation of ≥1 signaling molecules, including ERK1/2 and cSrc.

Cytochrome P450 1B1 Contributes to the Development of Atherosclerosis and Hypertension in Apolipoprotein E–Deficient Mice

Cytochrome P450 (CYP) 1B1 contributes to vascular smooth muscle cell growth and hypertension in male mice. This study was conducted to determine the contribution of CYP1B1 to the development of atherosclerosis and hypertension and associated pathogenesis in 8-week-old male apolipoprotein E–deficient (ApoE−/−/Cyp1b1+/+), and ApoE- and CYP1B1-deficient (ApoE−/−/Cyp1b1−/−) mice fed a normal or atherogenic diet for 12 weeks. A separate group of ApoE−/−/Cyp1b1+/+ mice on an atherogenic diet was injected every third day with the CYP1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene (300 &mgr;g/kg), or its vehicle, dimethyl sulfoxide (30 &mgr;L, IP); systolic blood pressure was measured by the tail cuff method. After 12 weeks, mice were euthanized, blood collected for lipid analysis, and aortas harvested for measuring lesions and remodeling, and for infiltration of inflammatory cells by histological and immunohistochemical analysis, respectively, and for reactive oxygen species production. Blood pressure, areas of lipids and collagen deposition, elastin breaks, infiltration of macrophages and T lymphocytes, reactive oxygen species generation in the aorta, and plasma lipid levels were increased in ApoE−/−/Cyp1b1+/+ mice on an atherogenic diet; these changes were minimized in mice given 2,3′,4,5′-tetramethoxystilbene, and in ApoE−/−/Cyp1b1−/− mice on an atherogenic diet; absorption/production of lipids remained unaltered in these mice. These data suggest that aortic lesions, hypertension, and associated pathogenesis in ApoE−/−/Cyp1b1+/+ mice on an atherogenic diet are most likely dependent on CYP1B1-generated oxidative stress and increased plasma lipid levels independent of blood pressure and absorption of lipids. CYP1B1 could serve as a novel target for developing drugs to treat atherosclerosis and hypertension caused by hypercholesterolemia.

Cytosolic Phospholipase A2α Is Essential for Renal Dysfunction and End-Organ Damage Associated With Angiotensin II-Induced Hypertension

BACKGROUND The kidney plays an important role in regulating blood pressure (BP). cPLA2α in the kidney is activated by various agents including angiotensin II (Ang II) and selectively releases arachidonic acid (AA) from tissue lipids, generating pro- and antihypertensive eicosanoids. Since activation of cPLA2α is the rate-limiting step in AA release, this study was conducted to determine its contribution to renal dysfunction and end-organ damage associated with Ang II-induced hypertension. METHODS cPLA2α(+/+) and cPLA2α(-/-) mice were infused with Ang II (700 ng/ kg/min) or its vehicle for 13 days. Mice were placed in metabolic cages to monitor their food and water intake, and urine was collected and its volume was measured. Doppler imaging was performed to assess renal hemodynamics. On the 13th day of Ang II infusion, mice were sacrificed and their tissues and blood collected for further analysis. RESULTS Ang II increased renal vascular resistance, water intake, and urine output and Na(+) excretion, decreased urine osmolality, and produced proteinuria in cPLA2α(+/+) mice. Ang II also caused accumulation of F4/80(+) macrophages and CD3(+) T cells and renal fibrosis, and increased oxidative stress in the kidneys of cPLA2α(+/+) mice. All these effects of Ang II were minimized in cPLA2α(-/-) mice. CONCLUSION 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.

Clotrimazole–cyclodextrin based approach for the management and treatment of Candidiasis – A formulation and chemistry-based evaluation

Clotrimazole (CT) is a poorly soluble antifungal drug that is most commonly employed as a topical treatment in the management of vaginal candidiasis. The present work focuses on a formulation approach to enhance the solubility of CT using cyclodextrin (CD) complexation. A CT–CD complex was prepared by a co-precipitation method. Various characterization techniques such as differential scanning calorimetry, infrared (IR) and X-ray spectroscopy, scanning electron microscopy and nuclear magnetic resonance (NMR) spectroscopy were performed to evaluate the complex formation and to understand the interactions between CT and CD. Computational molecular modeling was performed using the Schrödinger suite and Gaussian 09 program to understand structural conformations of the complex. The phase solubility curve followed an AL-type curve, indicating formation of a 1:1 complex. Molecular docking studies supported the data obtained through NMR and IR studies. Enthalpy changes confirmed that complexation was an exothermic and enthalpically favorable phenomenon. The CT–CD complexes were formulated in a gel and evaluated for release and antifungal activity. The in vitro release studies performed using gels demonstrated a sustained release of CT from the CT–CD complex with the complex exhibiting improved release relative to the un-complexed CT. Complexed CT–CD exhibited better fungistatic activity toward different Candida species than un-complexed CT.

2-Methoxyestradiol Reduces Angiotensin II–Induced Hypertension and Renal Dysfunction in Ovariectomized Female and Intact Male Mice

Cytochrome P450 1B1 protects against angiotensin II (Ang II)–induced hypertension and associated cardiovascular changes in female mice, most likely via production of 2-methoxyestradiol. This study was conducted to determine whether 2-methoxyestradiol ameliorates Ang II–induced hypertension, renal dysfunction, and end-organ damage in intact Cyp1b1−/−, ovariectomized female, and 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 hours. 2-Methoxyestradiol reduced Ang 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 Ang 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.

Brain Cytosolic Phospholipase A2α Mediates Angiotensin II-Induced Hypertension and Reactive Oxygen Species Production in Male Mice

Abstract BACKGROUND Recently, we reported that angiotensin II (Ang II)-induced hypertension is mediated by group IV cytosolic phospholipase A2α (cPLA2α) via production of prohypertensive eicosanoids. Since Ang II increases blood pressure (BP) via its action in the subfornical organ (SFO), it led us to investigate the expression and possible contribution of cPLA2α to oxidative stress and development of hypertension in this brain area. METHODS Adenovirus (Ad)-green fluorescence protein (GFP) cPLA2α short hairpin (sh) RNA (Ad-cPLA2α shRNA) and its control Ad-scrambled shRNA (Ad-Scr shRNA) or Ad-enhanced cyan fluorescence protein cPLA2α DNA (Ad-cPLA2α DNA) and its control Ad-GFP DNA were transduced into SFO of cPLA2α+/+ and cPLA2α−/− male mice, respectively. Ang II (700 ng/kg/min) was infused for 14 days in these mice, and BP was measured by tail-cuff and radio telemetry. cPLA2 activity, reactive oxygen species production, and endoplasmic reticulum stress were measured in the SFO. RESULTS Transduction of SFO with Ad-cPLA2α shRNA, but not Ad-Scr shRNA in cPLA2α+/+ mice, minimized expression of cPLA2α, Ang II-induced cPLA2α activity and oxidative stress in the SFO, BP, and cardiac and renal fibrosis. In contrast, Ad-cPLA2α DNA, but not its control Ad-GFP DNA in cPLA2α−/− mice, restored the expression of cPLA2α, and Ang II-induced increase in cPLA2 activity and oxidative stress in the SFO, BP, cardiac, and renal fibrosis. CONCLUSIONS These data suggest that cPLA2α in the SFO is crucial in mediating Ang II-induced hypertension and associated pathogenesis. Therefore, development of selective cPLA2α inhibitors could be useful in treating hypertension and its pathogenesis.

Ps 16-05 Angiotensin Ii-induced Hypertension Is Dependent On The Brain Cytosolic Phospholipase A2aα-generated Oxidative Stress

Objective: Angiotensin (Ang) II by activating cytosolic phospholipase A2a (cPLA2a) releases arachidonic acid (AA), and the pro-hypertensive AA metabolites mediate Ang II-induced hypertension and associated pathogenesis. These findings and the demonstration that Ang II increases blood pressure via generation of oxidative stress in subfornical organ (SFO) in the brain, led us to hypothesize that these effects of Ang II are dependent on cPLA2a activation in SFO. Design and method: To test this hypothesis, we investigated the effect of Ang II infusion (700ng/kg/min, s.c.) for 14 days in wild type (cPLA2a+/+) and cPLA2a-/- mice transduced in SFO with enhanced cyan-fluorescence protein (Ad-ECFP)-cPLA2a DNA. Results: Ang II increased mean arterial pressure (MAP) measured by radio-telemetry in cPLA2a+/+ but not cPLA2a-/- mice (100 ± 2 to 161 ± 6 mmHg vs. 104 ± 3 to 112 ± 3 mmHg, respectively, P < 0.05). Ang II increased cPLA2 activity, measured by immunohistochemistry using anti-phospho-Ser505, in SFO of cPLA2a+/+ but not cPLA2a-/- mice. Ang II increased reactive oxygen species (ROS) production measured by dihydroethidium staining (5.09 ± 0.31 to 8.99 ± 0.33 AU, P < 0.05), in SFO of cPLA2a+/+ but not in cPLA2a-/- mice. Ad-ECFP-cPLA2a DNA (1x1012 pfu/0.5 &mgr;L) but not Ad-GFP DNA transduced in SFO of cPLA2a-/- mice, restored Ang II effect on MAP and ROS production (115 ± 4 to 149 ± 2 mmHg and 4.40 ± 0.31 to 7.66 ± 0.47 AU with Ad-ECFP-cPLA2a DNA vs. 109 ± 5 to 116 ± 4 mmHg, and 4.04 ± 0.35 to 4.56 ± 0.19 AU with Ad-GFP, respectively, P < 0.05). In contrast, Ad-cPLA2a shRNA, but not Ad-shRNA transduced in SFO of cPLA2a+/+ mice, inhibited cPLA2 expression and its activity, and abolished Ang II-induced increase in blood pressure. Conclusions: These observations suggest that AA released by cPLA2a via one or more of its metabolites increase oxidative stress in SFO that promotes development of hypertension.