Xiao R. Fang

Angiotensin II–Induced Vascular Smooth Muscle Cell Migration and Growth Are Mediated by Cytochrome P450 1B1–Dependent Superoxide Generation

Cytochrome P450 1B1, expressed in vascular smooth muscle cells, can metabolize arachidonic acid in vitro into several products including 12- and 20-hydroxyeicosatetraenoic acids that stimulate vascular smooth muscle cell growth. This study was conducted to determine whether cytochrome P450 1B1 contributes to angiotensin II–induced rat aortic smooth muscle cell migration, proliferation, and protein synthesis. Angiotensin II stimulated migration of these cells, measured by the wound healing approach, by 1.78-fold; and DNA synthesis, measured by [3H]thymidine incorporation, by 1.44-fold after 24 hours; and protein synthesis, measured by [3H]leucine incorporation, by 1.40-fold after 48 hours. Treatment of vascular smooth muscle cells with the cytochrome P450 1B1 inhibitor 2,4,3′,5′-tetramethoxystilbene or transduction of these cells with adenovirus cytochrome P450 1B1 small hairpin RNA but not its scrambled control reduced the activity of this enzyme and abolished angiotensin II– and arachidonic acid–induced cell migration, as well as [3H]thymidine and [3H]leucine incorporation. Metabolism of arachidonic acid to 5-, 12-, 15-, and 20-hydoxyeicosatetraenoic acids in these cells was not altered, but angiotensin II– and arachidonic acid–induced reactive oxygen species production and extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activity were inhibited by 2,4,3′,5′-tetramethoxystilbene and cytochrome P450 1B1 small hairpin RNA (shRNA) and by Tempol, which inactivates reactive oxygen species. Tempol did not alter cytochrome P450 1B1 activity. These data suggest that angiotensin II–induced vascular smooth muscle cell migration and growth are mediated by reactive oxygen species generated from arachidonic acid by cytochrome P450 1B1 and activation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase.

Cytochrome P450 1B1 Contributes to Angiotensin II–Induced Hypertension and Associated Pathophysiology

Abstract—Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study showing that angiotensin II-induced vascular smooth muscle cell growth depends on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg per minute) or mice (1000 &mgr;g/kg per day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased vascular reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1−/− mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3′,4,5′-tetramethoxystilbene, which prevents both cytochrome P450 1B1-dependent and -independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases.

Cytochrome P450 1B1 Contributes to Renal Dysfunction and Damage Caused by Angiotensin II in Mice

Cytochrome P450 1B1 contributes to the development of angiotensin II–induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of angiotensin II in the kidney, as well as in salt and water homeostasis, and blood pressure regulation, we determined the contribution of cytochrome P450 1B1 to renal dysfunction and injury associated with angiotensin II–induced hypertension in male Cyp1b1+/+ and Cyp1b1−/− mice. Angiotensin II infusion (700 ng/kg per minute) given by miniosmotic pumps for 13 and 28 days increased systolic blood pressure in Cyp1b1+/+ mice; this increase was significantly reduced in Cyp1b1−/− mice. Angiotensin II increased renal Cyp1b1 activity, vascular resistance, and reactivity to vasoconstrictor agents and caused endothelial dysfunction in Cyp1b1+/+ but not Cyp1b1−/− mice. Angiotensin II increased water consumption and urine output, decreased urine osmolality, increased urinary Na+ and K+ excretion, and caused proteinuria and albuminuria in Cyp1b1+/+ mice that was diminished in Cyp1b1−/− mice. Infusion of angiotensin II for 28 but not 13 days caused renal fibrosis, tubular damage, and inflammation in Cyp1b1+/+ mice, which was minimized in Cyp1b1−/− mice. Angiotensin II increased levels of 12- and 20-hydroxyeicosatetraenoic acids; reactive oxygen species; and activity of NADPH oxidase, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src in the kidneys of Cyp1b1+/+ but not Cyp1b1−/− mice. These data suggest that increased thirst, renal dysfunction, and injury and inflammation associated with angiotensin II–induced hypertension in mice depend on cytochrome P450 1B1 activity, thus indicating that cytochrome P450 1B1 could serve as a novel target for treating renal disease and hypertension.

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.

Cytochrome P450 1B1 Gene Disruption Minimizes Deoxycorticosterone Acetate-Salt–Induced Hypertension and Associated Cardiac Dysfunction and Renal Damage in Mice

Previously, we showed that the cytochrome P450 1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene reversed deoxycorticosterone acetate (DOCA)-salt–induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt–induced hypertension, via increased production of reactive oxygen species and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild-type (Cyp1b1+/+) mice that were minimized in Cyp1b1−/− mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1+/+ but not in Cyp1b1−/− mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy and renal fibrosis, increased renal infiltration of macrophages and T lymphocytes, caused proteinuria, increased cardiac and renal nicotinamide adenine dinucleotide phosphate-oxidase activity, caused production of reactive oxygen species, and increased activities of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src; these were all reduced in DOCA-salt–treated Cyp1b1−/− mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that, in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage, and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src independent of eicosanoids.

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.

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.

Disruption of the cytochrome P-450 1B1 gene exacerbates renal dysfunction and damage associated with angiotensin II-induced hypertension in female mice

Recently, we demonstrated in female mice that protection against ANG II-induced hypertension and associated cardiovascular changes depend on cytochrome P-450 (CYP)1B1. The present study was conducted to determine if Cyp1b1 gene disruption ameliorates renal dysfunction and organ damage associated with ANG II-induced hypertension in female mice. ANG II (700 ng·kg(-1)·min(-1)) infused by miniosmotic pumps for 2 wk in female Cyp1b1(+/+) mice did not alter water consumption, urine output, Na(+) excretion, osmolality, or protein excretion. However, in Cyp1b1(-/-) mice, ANG II infusion significantly increased (P < 0.05) water intake (5.50 ± 0.42 ml/24 h with vehicle vs. 8.80 ± 0.60 ml/24 h with ANG II), urine output (1.44 ± 0.37 ml/24 h with vehicle vs. 4.30 ± 0.37 ml/24 h with ANG II), and urinary Na(+) excretion (0.031 ± 0.016 mmol/24 h with vehicle vs. 0.099 ± 0.010 mmol/24 h with ANG II), decreased osmolality (2,630 ± 79 mosM/kg with vehicle vs. 1,280 ± 205 mosM/kg with ANG II), and caused proteinuria (2.60 ± 0.30 mg/24 h with vehicle vs. 6.96 ± 0.55 mg/24 h with ANG II). Infusion of ANG II caused renal fibrosis, as indicated by an accumulation of renal interstitial α-smooth muscle actin, collagen, and transforming growth factor-β in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. ANG II also increased renal production of ROS and urinary excretion of thiobarburic acid-reactive substances and reduced the activity of antioxidants and urinary excretion of nitrite/nitrate and the 17β-estradiol metabolite 2-methoxyestradiol in Cyp1b1(-/-) but not Cyp1b1(+/+) mice. These data suggest that Cyp1b1 plays a critical role in female mice in protecting against renal dysfunction and end-organ damage associated with ANG II-induced hypertension, in preventing oxidative stress, and in increasing activity of antioxidant systems, most likely via generation of 2-methoxyestradiol from 17β-estradiol.

Cytochrome P450 1B1 Gene Disruption Minimizes Deoxycorticosterone Acetate-Salt–Induced Hypertension and Associated Cardiac Dysfunction and Renal Damage in MiceNovelty and Significance

Previously, we showed that the cytochrome P450 1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene reversed deoxycorticosterone acetate (DOCA)-salt–induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt–induced hypertension, via increased production of reactive oxygen species and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild-type (Cyp1b1+/+) mice that were minimized in Cyp1b1−/− mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1+/+ but not in Cyp1b1−/− mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy and renal fibrosis, increased renal infiltration of macrophages and T lymphocytes, caused proteinuria, increased cardiac and renal nicotinamide adenine dinucleotide phosphate-oxidase activity, caused production of reactive oxygen species, and increased activities of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src; these were all reduced in DOCA-salt–treated Cyp1b1−/− mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that, in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage, and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src independent of eicosanoids.

CYTOCHROME P450 1B1 GENE DISRUPTION MINIMIZES DOCA-SALT-INDUCED HYPERTENSION AND ASSOCIATED CARDIAC DYSFUNCTION AND RENAL DAMAGE IN MICE

Previously, we showed that the cytochrome P450 1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene reversed deoxycorticosterone acetate (DOCA)-salt–induced hypertension and minimized endothelial and renal dysfunction in the rat. This study was conducted to test the hypothesis that cytochrome P450 1B1 contributes to cardiac dysfunction, and renal damage and inflammation associated with DOCA-salt–induced hypertension, via increased production of reactive oxygen species and modulation of neurohumoral factors and signaling molecules. DOCA-salt increased systolic blood pressure, cardiac and renal cytochrome P450 1B1 activity, and plasma levels of catecholamines, vasopressin, and endothelin-1 in wild-type (Cyp1b1+/+) mice that were minimized in Cyp1b1−/− mice. Cardiac function, assessed by echocardiography, showed that DOCA-salt increased the thickness of the left ventricular posterior and anterior walls during diastole, the left ventricular internal diameter, and end-diastolic and end-systolic volume in Cyp1b1+/+ but not in Cyp1b1−/− mice; stroke volume was not altered in either genotype. DOCA-salt increased renal vascular resistance and caused vascular hypertrophy and renal fibrosis, increased renal infiltration of macrophages and T lymphocytes, caused proteinuria, increased cardiac and renal nicotinamide adenine dinucleotide phosphate-oxidase activity, caused production of reactive oxygen species, and increased activities of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src; these were all reduced in DOCA-salt–treated Cyp1b1−/− mice. Renal and cardiac levels of eicosanoids were not altered in either genotype of mice. These data suggest that, in DOCA-salt hypertension in mice, cytochrome P450 1B1 plays a pivotal role in cardiovascular dysfunction, renal damage, and inflammation, and increased levels of catecholamines, vasopressin, and endothelin-1, consequent to generation of reactive oxygen species and activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and cellular-Src independent of eicosanoids.