Anne M. Estes

Angiotensin II-Induced Hypertension Contribution of Ras GTPase/Mitogen-Activated Protein Kinase and Cytochrome P450 Metabolites

We reported that norepinephrine and angiotensin II (Ang II) activate the Ras/mitogen-activated protein (MAP) kinase pathway primarily through the generation of cytochrome P450 (CYP450) metabolites. The purpose of the present study was to determine the contribution of Ras and CYP450 to Ang II–dependent hypertension in rats. Infusion of Ang II (350 ng/min for 6 days) elevated mean arterial blood pressure (MABP) (171±3 mm Hg for Ang II versus 94±5 for vehicle group, P <0.05). Ras is activated on farnesylation by farnesyl protein transferase (FPT). When Ang II was infused in combination with FPT inhibitor FPT III (232 ng/min) or BMS-191563 (578 ng/min), the development of hypertension was attenuated (171±3 mm Hg for Ang II plus vehicle versus 134±5 mm Hg for Ang II plus FPT III and 116±6 mm Hg for Ang II plus BMS-191563, P <0.05). Treatment with the MAP kinase kinase inhibitor PD-98059 (5 mg SC) reduced MABP. The CYP450 inhibitor aminobenzotriazole (50 mg/kg) also diminished the development of Ang II–induced hypertension to 113±8 mm Hg. The activities of Ras, MAP kinase, and CYP450 measured in the kidney were elevated in hypertensive animals. The infusion of FPT III, BMS-191563, or aminobenzotriazole reduced the elevation in Ras and MAP kinase activity. Morphological studies of the kidney showed that FPT III treatment ameliorated the arterial injury, vascular lesions, fibrinoid necrosis, focal hemorrhage, and hypertrophy of muscle walls observed in hypertensive animals. These data suggest that the activation of Ras and CYP450 contributes to the development of Ang II–dependent hypertension and associated vascular pathology.

Mechanism of High Glucose–Induced Angiotensin II Production in Rat Vascular Smooth Muscle Cells

Angiotensin II (Ang II), a circulating hormone that can be synthesized locally in the vasculature, has been implicated in diabetes-associated vascular complications. This study was conducted to determine whether high glucose (HG) (≈23.1 mmol/L), a diabetic-like condition, stimulates Ang II generation and the underlying mechanism of its production in rat vascular smooth muscle cells. The contribution of various enzymes involved in Ang II generation was investigated by silencing their expression with small interfering RNA in cells exposed to normal glucose (≈4.1 mmol/L) and HG. Angiotensin I (Ang I) was generated from angiotensinogen by cathepsin D in the presence of normal glucose or HG. Although HG did not affect the rate of angiotensinogen conversion, it decreased expression of angiotensin-converting enzyme (ACE), downregulated ACE-dependent Ang II generation, and upregulated rat vascular chymase–dependent Ang II generation. The ACE inhibitor captopril reduced Ang II levels in the media by 90% in the presence of normal glucose and 19% in HG, whereas rat vascular chymase silencing reduced Ang II production in cells exposed to HG but not normal glucose. The glucose transporter inhibitor cytochalasin B, the aldose reductase inhibitor alrestatin, and the advanced glycation end product formation inhibitor aminoguanidine attenuated HG-induced Ang II generation. HG caused a transient increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and ERK1/2 inhibitors reduced Ang II accumulation by HG. These data suggest that polyol pathway metabolites and AGE can stimulate rat vascular chymase activity via ERK1/2 activation and increase Ang II production. In addition, decreased Ang II degradation, which, in part, could be attributable to a decrease in angiotensin-converting enzyme 2 expression observed in HG, contributes to increased accumulation of Ang II in vascular smooth muscle cells by HG.

Contribution of Ras GTPase/MAP Kinase and Cytochrome P450 Metabolites to Deoxycorticosterone-Salt–Induced Hypertension

We recently reported that norepinephrine and angiotensin II activate the Ras/mitogen-activated protein (MAP) kinase pathway through generation of a cytochrome P450 (CYP450) and lipoxygenase metabolites. The purpose of this study was to determine the contribution of Ras/MAP kinase to deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats. Administration of DOCA and 1% saline drinking water to uninephrectomized rats for 6 weeks significantly elevated mean arterial blood pressure (MABP) (166+/-5 mm Hg, n=19) compared with that of normotensive controls (95+/-5 mm Hg, n=7) (P<0.05). The activity of Ras and MAP kinase measured in the heart was increased in DOCA-salt hypertensive rats. Infusion of the Ras farnesyl transferase inhibitors FPT III (138 ng/min) and BMS-191563 (694 ng/min) significantly (P<0.05) attenuated MABP to 139+/-4 mm Hg (n=14) and 126+/-1 mm Hg (n=4), respectively. Moreover, infusion of MAP kinase kinase inhibitor PD-98059 (694 ng/min) also reduced MABP in hypertensive rats. Morphological studies of the kidney showed that treatment of rats with FPT III, which reduced Ras activity, minimized the hyperplastic occlusive arteriosclerosis and fibrinoid vasculitis observed in untreated hypertensive rats. In addition, the rise in CYP450 activity and MABP in hypertensive rats was prevented by the CYP450 inhibitor aminobenzotriazole (50 mg/kg) and was associated with a decrease in Ras and MAP kinase activity in the heart. These data suggest that the Ras/MAP kinase pathway contributes to DOCA-salt-induced hypertension and associated vascular pathology consequent to activation of CYP450.

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.

PKC-ζ Mediates Norepinephrine-Induced Phospholipase D Activation and Cell Proliferation in VSMC

Abstract—Norepinephrine (NE) stimulates phospholipase D (PLD) activity and cell proliferation in vascular smooth muscle cells (VSMCs). The objective of this study was to determine the contribution of PKC-&zgr; to NE-induced PLD activation and cell proliferation in VSMCs. PLD activity was measured by the formation of [3H]phosphatidylethanol in VSMCs labeled with [3H]oleic acid and exposed to ethanol. A high basal PLD activity was detected, and NE increased PLD activity over basal by 70%. This increase was abolished by the broad-range PKC inhibitor Ro 31-8220 (1 &mgr;mol/L, 30 minutes) and myristoylated PKC-&zgr; pseudosubstrate peptide inhibitor (25 &mgr;mol/L, 1 hour). Transfection of VSMCs with PKC-&zgr; antisense, but not sense, oligonucleotides, which reduced PKC-&zgr; protein level and basal PLD activity, caused a 92% decrease in NE-induced PLD activation. NE-induced increase in PLD activity was also reduced by 61% in cells transfected with kinase-deficient FLAG-T410A-PKC-&zgr; plasmid but not in those transfected with wild-type PKC-&zgr;. NE increased immunoprecipitable PKC-&zgr; activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC-&zgr; activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC-&zgr; antisense, but not sense, oligonucleotides also inhibited basal and NE-stimulated cell proliferation by 54% and 57%, respectively, as measured by [3H]thymidine incorporation. The inhibitor of PLD activity n-butanol, but not its inactive analog tert-butanol, also reduced the basal and blocked NE-induced cell proliferation. These data suggest that PKC-&zgr; mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.

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.

Contribution of Arachidonic Acid Metabolites Derived Via Cytochrome P4504A to Angiotensin II–Induced Neointimal Growth

Angiotensin II and the arachidonic acid metabolite derived via cytochrome P450 20-hydroxyeicostetraenoic acid promote vasoconstriction and vascular smooth muscle cell (VSMC) proliferation. This study was conducted to determine if 20-hydroxyeicostetraenoic acid contributes to angiotensin II-induced neointimal formation in balloon-injured rat carotid artery. In anesthetized rats, the drugs were infused into the clamped segment of the injured right common carotid artery for 60 minutes. The drug solution and catheter were withdrawn, the common carotid artery was ligated, and blood flow was restored. Exposure of the injured artery to angiotensin II (200 nmol/L) or arachidonic acid (10 &mgr;mol/L) increased neointimal thickening at day 14 (intima/media ratio 0.71±0.14 with vehicle versus 1.65±0.10 with angiotensin II or 1.31±0.13 with arachidonic acid; P<0.05). Cytochrome P450 4A1 antisense, but not scrambled, oligodeoxynucleotide (100 nmol/L) reduced angiotensin II-induced or arachidonic acid-induced neointimal thickening (intima/media ratio 0.90±0.07 for angiotensin II and 0.95±0.06 for arachidonic acid). 20-hydroxyeicostetraenoic acid (0.5 &mgr;mol/L) also increased neointimal thickening of injured artery (intima/media ratio 1.15±0.03); this was not altered by cytochrome P450 4A1 antisense oligodeoxynucleotide. Angiotensin II, arachidonic acid, and 20-hydroxyeicostetraenoic acid also induced the expression of cytochrome P450 4A and increased the number of CD45-positive cells; the latter effect of angiotensin II and arachidonic acid, but not 20-hydroxyeicostetraenoic acid, was diminished by cytochrome P450 4A1 antisense oligodeoxynucleotide. These data suggest that arachidonic acid metabolites derived via cytochrome P450 4A, most likely 20-hydroxyeicostetraenoic acid, mediate angiotensin II-induced neointimal thickening in injured rat carotid artery.

Angiotensin II-induced process of angiogenesis is mediated by spleen tyrosine kinase via VEGF receptor-1 phosphorylation

Spleen tyrosine kinase (Syk), expressed in endothelial cells, has been implicated in migration and proliferation and in vasculogenesis. This study was conducted to determine the contribution of Syk and the underlying mechanism to the angiogenic effect of ANG II and VEGF. Angiogenesis was determined by tube formation from the endothelial cell line EA.hy926 (EA) and human umbilical vein endothelial cells (HUVECs) and microvessel sprouting in rat aortic rings. ANG II (10 nM), EGF (30 ng/ml), and VEGF (50 ng/ml) stimulated EA cells and HUVECs to form tubular networks and increased aortic sprouting; these effects were blocked by VEGF receptor-1 and Flt-1 antibody (Flt-1/Fc) but not by the VEGF receptor-2 (Flk-1) antagonist SU-1498. ANG II increased the phosphorylation of Flt-1 but not Flk-1, whereas VEGF increased the phosphorylation of both receptors in EA cells and HUVECs. VEGF expression elicited by ANG II was not altered by Flt-1/Fc or SU-1498. EGF stimulated tube formation from EA cells and HUVECs and Flt-1 phosphorylation and aortic sprouting, which were blocked by the EGF receptor antagonist AG-1478 and Flt-1/Fc but not by SU-1498. ANG II-, EGF-, and VEGF-induced tube formation and aortic sprouting were attenuated by the Syk inhibitor piceatannol and by Syk short hairpin interfering (sh)RNA and small interfering RNA, respectively. ANG II, EGF, and VEGF increased Syk phosphorylation, which was inhibited by piceatannol and Syk shRNA in EA cells and HUVECs. Neither piceatannol nor Syk shRNA altered ANG II-, EGF-, or VEGF-induced phosphorylation of Flt-1. These data suggest that ANG II stimulates angiogenesis via transactivation of the EGF receptor, which promotes the phosphorylation of Flt-1 and activation of Syk independent of VEGF expression.