Angiotensin Blood Pressure Research Articles

AT₂ receptor activation induces natriuresis and lowers blood pressure.

Compound 21 (C-21) is a highly selective nonpeptide AT2 receptor (AT2R) agonist. To test the hypothesis that renal proximal tubule AT2Rs induce natriuresis and lower blood pressure in Sprague-Dawley rats and mice. In rats, AT2R activation with intravenous C-21 increased urinary sodium excretion by 10-fold (P<0.0001); this natriuresis was abolished by direct renal interstitial infusion of specific AT2R antagonist PD-123319. C-21 increased fractional excretion of Na(+) (P<0.05) and lithium (P<0.01) without altering renal hemodynamic function. AT2R activation increased renal proximal tubule cell apical membrane AT2R protein (P<0.001) without changing total AT2R expression and internalized/inactivated Na(+)-H(+) exchanger-3 and Na(+)/K(+)ATPase. C-21-induced natriuresis was accompanied by an increase in renal interstitial cGMP (P<0.01); C-21-induced increases in urinary sodium excretion and renal interstitial cGMP were abolished by renal interstitial nitric oxide synthase inhibitor l-N(6)-nitroarginine methyl ester or bradykinin B2 receptor antagonist icatibant. Renal AT2R activation with C-21 prevented Na(+) retention and lowered blood pressure in the angiotensin II infusion model of experimental hypertension. AT2R activation initiates its translocation to the renal proximal tubule cell apical membrane and the internalization of Na(+)-H(+) exchanger-3 and Na(+)/K(+)ATPase, inducing natriuresis in a bradykinin-nitric oxide-cGMP-dependent manner. Intrarenal AT2R activation prevents Na(+) retention and lowers blood pressure in angiotensin II-dependent hypertension. AT2R activation holds promise as a renal proximal tubule natriuretic/diuretic target for the treatment of fluid-retaining states and hypertension.

Role of E2F transcriptional factor family in hydrogen peroxide regulation of dihydrofolate reductase in endothelial cells (855.5)

Endothelial nitric oxide synthase (eNOS) uncoupling consequent to a deficiency in eNOS cofactor tetrahydrobiopterin (H4B) is an important mechanism of vascular dysfunction. Our previous work has shown that endothelial hydrogen peroxide (H2O2) overproduction specifically downregulates H4B salvage enzyme dihydrofolate reductase (DHFR), which mediates persistent H4B deficiency, eNOS uncoupling and elevated blood pressure in Angiotensin II‐infused mice. However, detailed molecular mechanisms underlying H2O2 regulation of DHFR in endothelial cells remain unclear. In this study, we found that hydrogen peroxide dose and time dependently downregulated DHFR protein expression and its enzyme activity in bovine aortic endothelial cells (BAECs). The DHFR mRNA levels were also decreased by H2O2, implicating transcriptional regulation. Based on the fact that DHFR promoter contains cis‐acting elements for Sp1 and E2F, we constructed wildtype DHFR promoter reporter construct (WT‐DHFR), and mutant DHFR promoter constructs that were deleted of either Sp1 or E2F binding sites. The dual‐luciferase reporter assay showed that while the promoter activities of both WT‐DHFR and Sp1 deletion mutant remained inhibited by H2O2, deletion of the E2F binding sites completely abolished H2O2 attenuation of DHFR promoter activity. RT‐PCR analyses indicated that H2O2 treatment resulted in marked downregulation of E2F1, E2F7 and E2F8 by 38%, 43% and 82% respectively. Intriguingly, overexpression of E2F1, E2F7 or E2F8 restored DHFR expression in response to H2O2, and cotransfection with E2F1 also significantly increased DHFR promoter activity. In conclusion, these results suggest that DHFR is downregulated by H2O2 in endothelial cells transcriptionally via a E2F‐dependent manner, and that restoration of E2F family‐transcription factor activity is effective in preserving DHFR function under oxidative stress.Grant Funding Source: Supported by NHLBI awards HL077440 (HC), HL088975 (HC) and an AHA EI award 12EIA8990025 (HC)

Aberrant Endoplasmic Reticulum Stress in Vascular Smooth Muscle Increases Vascular Contractility and Blood Pressure in Mice Deficient of AMP-Activated Protein Kinase-α2 In Vivo

The endoplasmic reticulum (ER) plays a critical role in ensuring proper folding of newly synthesized proteins. Aberrant ER stress is reported to play a causal role in cardiovascular diseases. However, the effects of ER stress on vascular smooth muscle contractility and blood pressure remain unknown. The aim of this study was to investigate whether aberrant ER stress causes abnormal vasoconstriction and consequent high blood pressure in mice. ER stress markers, vascular smooth muscle contractility, and blood pressure were monitored in mice. Incubation of isolated aortic rings with tunicamycin or MG132, 2 structurally unrelated ER stress inducers, significantly increased both phenylephrine-induced vasoconstriction and the phosphorylation of myosin light chain (Thr18/Ser19), both of which were abrogated by pretreatment with chemical chaperones or 5-Aminoimidazole-4-carboxamide ribonucleotide and metformin, 2 potent activators for the AMP-activated protein kinase. Consistently, administration of tauroursodeoxycholic acid or 4-phenyl butyric acid, 2 structurally unrelated chemical chaperones, in AMP-activated protein kinase-α2 knockout mice lowered blood pressure and abolished abnormal vasoconstrictor response of AMP-activated protein kinase-α2 knockout mice to phenylephrine. Consistently, tunicamycin (0.01 μg/g per day) infusion markedly increased both systolic and diastolic blood pressure, both of which were ablated by coadministration of 4-phenyl butyric acid. Furthermore, 4-phenyl butyric acid or tauroursodeoxycholic acid, which suppressed angiotensin II infusion-induced ER stress markers in vivo, markedly lowered blood pressure in angiotensin II-infused mice in vivo. We conclude that ER stress increases vascular smooth muscle contractility resulting in high blood pressure, and AMP-activated protein kinase activation mitigates high blood pressure through the suppression of ER stress in vivo.

MITOCHONDRIA‐TARGETED TREATMENT ATTENUATES VASCULAR OXIDATIVE STRESS, ENDOTHELIAL DYSFUNCTION AND HYPERTENSION

Mitochondrial reactive oxygen species (ROS) have been recently implicated in the development and progression of cardiovascular diseases. We have suggested that mitochondria targeted antioxidants can be beneficial for treatment of endothelial dysfunction, hypetension other parthological conditions. In this work we have studied specific molecular mechanisms responsible for stimulation of mitochondrial O2• and its downstream targets using cultured human aortic endothelial cells and angiotensin II‐induced hypertension. Depletion of cytoplasmic Nox2 isoform of NADPH oxidase inhibited stimulation of mitochondrial ROS by angiotensin II. Meanwhile, treatment with the mitochondria targeted superoxide dismutase mimetic mitoTEMPO or SOD2 overexpression inhibited production of mitochondrial O2•, reduced activity of redox dependent c‐Src and cytoplasmic NADPH oxidase, restored vascular NO in angiotensin II stimulated cells and reduced blood pressure in mice with angiotensin II and DOCA‐salt induced hypertension. We have suggested that mitochondrial ROS stimulates cytoplasmic oxidative stress via release of H2O2. Indeed, scavenging of mitochondrial H2O2 with mitochondria targeted glutathione peroxidase mimetic mitoEbselen inhibited production of cytoplasmic O2• by NADPH oxidase and reduced blood pressure in angiotensin II induced hypertension. Production of angiotensin II‐induced mitochondrial ROS was inhibited by specific PKCε peptide antagonist, ATP‐sensitive potassium channel blocker 5‐hydroxydecanoate, inhibitors of complex I rotenone, inhibitors of complex II malonate or malate. These studies show that mitochondrial ROS play an important role in feed‐forward stimulation of oxidative stress by activating mitochondrial reverse electron transfer which can be treated with mitochondria‐targeted antioxidants.This work has been funded by National Institutes of Health HL094469.

Moderate Hyperbilirubinemia Improves Renal Hemodynamics in Angiotensin II‐Dependent Hypertension

We have previously demonstrated that moderate hyperbilirubinemia decreases blood pressure in Angiotensin (Ang) II‐dependent hypertension through mechanisms that decrease oxidative stress and increase nitric oxide levels. The goal of the present study was to examine the effect of moderate hyperbilirubinemia on glomerular filtration rate (GFR) and renal blood flow (RBF) in a mouse model of Ang II hypertension. Mice were made moderately hyperbilirubinemic by treatment with 1) indinavir or 2) morpholino antisense oligonucleotides directed against hepatic UGT1A1. Levels of unconjugated bilirubin averaged 0.57 ± 0.07, 0.92 ± 0.07, and 1.28 ± 0.1 mg/dL (n=4) in control, indinavir, and antisense treated mice, respectively. GFR and RBF were measured in mice after implantation of an osmotic minipump delivering Ang II at a rate of 1 μg/kg/min. GFR was measured by continuous infusion of I125‐iothalamate on days 7–11 of Ang II infusion in conscious mice. RBF was measured on day 10 of Ang II infusion in anesthetized mice. Treatment with either indinavir or UGT1A1 antisense significantly increased GFR in Ang II infused mice 1.2 ± 0.1, 1.9 ± 0.2, and 1.9 ± 0.1 ml/min/g kidney weight (n=4) in non‐treated, indinavir, and antisense treated mice, respectively. Next, we examined the effect of moderate hyperbilirubinemia on RBF in Ang II infused mice. Ang II infusion significantly decreased RBF and this decrease was normalized by moderate hyperbilirubinemia with RBF averaging 5.2 ± 0.5, 3.8 ± 0.2, and 4.8 ± 0.3 ml/min/g kw (n=4) in control, Ang II, and Ang II+indinavir treated mice, respectively. These results indicate that improvement of renal hemodynamics may be a mechanism by which blood pressure is lowered with moderate hyperbilirubinemia in this model. This study was supported by NIH grant HL‐088421.

Interaction Between P450 Eicosanoids and Nitric Oxide in the Control of Arterial Tone in Mice

Epoxyeicosatrienoic acids (EETs) serve as endothelial-derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) -/- and +/+ mice. EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N-adamantyl-N'-dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin/charybdotoxin (ChTx), but not by iberiotoxin, nor by the cytochrome P450 inhibitor PPOH. All four EETs (order of potency: 8,9-EET >14,15-EET approximately 5,6-EET >11,12-EET) and all 4 dihydroxy derivatives (14,15-DHET approximately 8,9-DHET approximately 11,12-DHET >5,6-DHET) produced dose-dependent vasodilation. Endothelial removal or L-NAME blocked 8,9-EET and 14,15-DHET-dependent dilations. The effects of apamin/ChTx were minimal. 8,9-EET and 14,15-DHET induced NO production in endothelial cells. ADU (100 microg/mL in drinking water) lowered blood pressure in angiotensin II-infused hypertension, but not in L-NAME-induced hypertension. Blood pressure and EDHF responses were similar in L-NAME-treated sEH +/+ and -/- mice. Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO synthase (eNOS) and NO release.

Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension.

Epoxyeicosatrienoic acids (EETs) have antihypertensive properties and play a part in the maintenance of renal microvascular function. A novel approach to increase EET levels is to inhibit epoxide hydrolase enzymes that are responsible for conversion of biologically active EETs to dihydroxyeicosatrienoic acids (DHETs) that are void of effects on the preglomerular vasculature. We hypothesized that inhibition of soluble epoxide hydrolase (sEH) would lower blood pressure in angiotensin II (Ang II) hypertension. Rat renal cortical tissue was harvested and urine collected 2 weeks following implantation of an osmotic minipump containing Ang II (60 ng/min). Renal cortical sEH protein expression was significantly higher in Ang II hypertension compared with normotensive animals. Likewise, urinary 14,15-DHET levels were significantly increased in hypertensive compared with normotensive animals and averaged 8.1 +/- 1.3 and 2.7 +/- 1.1 ng/d; respectively. In additional experiments, the sEH inhibitor N-cyclohexyl-N-dodecyl urea (NCND; 3 mg/d) or vehicle (corn oil, 0.5 mL) was administered daily by intraperitoneal injection starting on day 10. Administration of NCND for 4 days lowered systolic blood pressure by 30 mm Hg in Ang II hypertensive animals, whereas the corn oil vehicle had no effect on blood pressure in normotensive or Ang II hypertensive animals. Measurement of blood pressure by indwelling arterial catheters in conscious animals with free movement in their cages confirmed that NCND had antihypertensive properties. Arterial blood pressure averaged 119 +/- 5 mm Hg in normotensive, 170 +/- 3 mm Hg in hypertensive and 149 +/- 10 mm Hg in NCND-treated, Ang II-infused animals. Administration of the potential metabolite of NCND, N-cyclohexylformamide to Ang II hypertensive rats did not lower the systolic blood pressure. These studies demonstrate that increased sEH expression in the Ang II hypertensive kidney leads to increased EET hydration. Moreover, sEH plays a role in the regulation of blood pressure, and inhibition of sEH during Ang II hypertension is antihypertensive.

Heparin inhibits mesenteric vascular hypertrophy in angiotensin II-infusion hypertension in rats.

Chronic infusion with angiotensin II increases blood pressure and activates growth mechanisms to produce hypertrophy of the heart and vessels. In order to better understand mechanisms of angiotensin II induced vascular hypertrophy, this study aimed to determine whether heparin, a potent inhibitor of smooth muscle proliferation mechanisms, was able to inhibit vascular hypertrophy. Angiotensin II (100, 200 or 300 ng/min/kg s.c.) or a saline vehicle control were infused into rats for 14 days. A separate group of animals were co-infused with heparin (0.3 mg/h/kg i.v.) and angiotensin II (200 ng/min/kg s.c.) to test whether hypertension or hypertrophy were antagonized. Blood pressure was measured by tail cuff method and vessel media cross sectional area was measured by morphometry in aorta and mesenteric arteries. Blood pressure elevation and cardiovascular hypertrophy produced by angiotensin II were strongly dose-dependent. Hypertrophy responses at 14 days of treatment also appeared to be influenced partly by local factors as medial cross sectional area was increased more in mesenteric arteries than in thoracic aorta, and left ventricle weight was least affected. Heparin treatment did not influence the increase of blood pressure in angiotensin II infused animals, but the mesenteric vascular hypertrophy response due to angiotensin II was inhibited by approximately 50%. Inhibition of a modest cardiac hypertrophy and aortic medial hypertrophy did not reach significance. Angiotensin II infusion produced vascular medial hypertrophy and increased blood pressure, however the inhibitory effect of heparin on hypertrophy in mesenteric arteries was not mediated through angiotensin II induced vasoconstriction or blood pressure elevation. These data suggest that heparin interferes directly with the hypertrophy mechanism in mesenteric arteries, and that heparin-sensitive growth mechanisms are important in mediating angiotensin induced mesenteric vascular hypertrophy.

Monocyte chemoattractant protein-1 expression in aortic tissues of hypertensive rats.

Monocyte chemoattractant protein-1 (MCP-1), a potent monocyte chemoattractant synthesized by vascular cells and monocytes, has been proposed to be an important mediator of inflammatory responses in the arterial vasculature. It was recently demonstrated that hypertension is associated with an inflammatory response in the arterial wall. To determine the effect of hypertension on arterial MCP-1 expression, we induced hypertension in Sprague-Dawley rats by infusing angiotensin II (0.75 mg x kg[-1] x d[-1] SC) for 7 days. Using Northern blot analysis, we detected a 3.6-fold increase in MCP-1 mRNA in the aortas of hypertensive rats. When we normalized blood pressure in angiotensin II-treated rats through oral administration of the nonspecific vasodilator hydralazine (15 mg x kg[-1] x d[-1]), aortic MCP-1 mRNA expression was significantly reduced. Similar results were obtained with a norepinephrine model of hypertension. Taken together, these data suggest that mechanical factors may be responsible in part for the upregulation of expression. Consistent with this interpretation, we found that cultured rat aortic vascular smooth muscle cells exposed to mechanical strain (20% peak deformation at 1 Hz) exhibited a marked increase in MCP-1 expression, suggesting the hemodynamic strain imparted onto arterial cells in hypertension is an important stimulus underlying this phenomenon. These results provide important insights into the in vivo regulation of MCP-1 and have potential implications for understanding the influence of hypertension on atherosclerosis.

Endothelium-derived constricting factor in renovascular hypertension.

We have reported that in two-kidney, one clip hypertensive rats, renal perfusion is maintained by a balance between the vasodilator endothelium-derived nitric oxide and the vasoconstrictor angiotensin II. Others have suggested that endothelium-derived constricting factor, reported to be thromboxane A2 and/or endoperoxide, contributes to increased blood pressure in angiotensin II-dependent hypertension. We hypothesized that in angiotensin II-dependent two-kidney, one clip hypertension, endothelium-derived constricting factor contributes to vasoconstriction of the clipped kidney following nitric oxide synthesis inhibition. Using radioactive microspheres, we studied renal blood flow to the stenotic kidney of two-kidney, one clip hypertensive rats 4 weeks after clipping. The influence of nitric oxide on systemic and renal hemodynamics was evaluated by determining the response to nitric oxide synthesis inhibition using 10 mg/kg N omega-nitro-L-arginine methyl ester in these rats, which were either not treated (n = 8) or treated (n = 8) with 4 mg/kg of the constricting factor receptor antagonist BMS 180,291. Mean basal blood pressure in rats was 167 +/- 9 mm Hg (mean +/- SEM). N omega-Nitro-L-arginine methyl ester increased blood pressure by 35 +/- 7 mm Hg (P < .001). In the clipped kidney, N omega-nitro-L-arginine methyl ester decreased renal blood flow by 40% (from 4.5 +/- 0.9 to 2.7 +/- 0.6 mL.min-1.g kidney-1; P < .01) and increased renal vascular resistance by 100% (from 51.9 +/- 9.6 to 105.0 +/- 19.2 mm Hg.mL-1.min-1.g kidney-1; P < .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Salt sensitivity in hypertensive rats with angiotensin II administration

We examined the salt sensitivity of blood pressure in angiotensin II (ANG II)-induced hypertension. Wistar rats, salt loaded (0.66, 2, or 8% salt-containing diet) for 4 or 12 days, were infused intravenously with 15 or 60 ng/min of ANG II. Systolic blood pressure (SBP) was not increased by long-term (12 days) salt loading, and SBP was unchanged with ANG II and normal-salt (0.66%) diet. However, when combined with salt loading, ANG II produced hypertension in a dose-dependent fashion; compared with control (120 +/- 2 mmHg), SBP was increased with 15 ng/min of ANG II and 8% salt diet (145 +/- 5 mmHg, P less than 0.05) and with 60 ng/min of ANG II and either 2 or 8% salt diet (149 +/- 8 and 174 +/- 8 mmHg, P less than 0.05, respectively). Na space (exchangeable Na) was increased in a roughly similar pattern and correlated significantly (r = 0.531, P less than 0.05) with SBP. However, with 15 ng/min of ANG II, Na space was not different among rats on either level of salt loading, although the 8% salt diet elevated SBP. Data obtained with short-term (4 days) treatment indicate that an elevated Na space preceded development of hypertension. With 15 ng/min of ANG II and 8% salt diet for 4 days, Na space was markedly (P less than 0.05) increased, but SBP was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium and blood pressure regulation in normal and hypertensive subjects.

To elucidate the mechanisms involved in calcium-mediated blood pressure (BP) control, plasma norepinephrine (NE), epinephrine, renin activity, and angiotensin II (AII) levels and the cardiovascular pressor responsiveness to NE and AII were assessed before and during acute mild hypercalcemia or short-term calcium (Ca) inhibition with nifedipine in 20 normal and five borderline hypertensive subjects. In normal subjects, systolic BP and plasma NE and epinephrine concentrations were increased significantly (p less than 0.05) during an acute rise in serum Ca of 3.1 mg/dl (intermediate rate Ca infusion, 0.05 mg/kg/min for 3 hours), but not following an increase of 1 mg/dl (low rate Ca infusion, 0.034 mg/kg/min for 2 hours). In the borderline hypertensive group, low-rate Ca infusion elevating serum Ca by 1 mg/dl was associated with a slight increase in systolic BP (p less than 0.05) and plasma catecholamines. In both groups, the pressor responses to infused NE and AII, and plasma renin and AII levels, were unchanged during mild to moderate hypercalcemia. Nifedipine given for 2 weeks (average dose, 48 mg/d) reduced BP significantly (p less than 0.05) in the borderline hypertensive subjects only and NE pressor responses in both groups (p less than 0.025), but had no significant effect on plasma catecholamines, renin, or AII levels. These findings suggest that the adrenergic BP control mechanism may be more dependent on clinical variations in calcium metabolism than the angiotensin BP regulatory mechanism. Acute hypercalcemia may increase BP at least in part by causing an increase in adrenergic activity without an equivalent decrease in cardiovascular reactivity. Calcium inhibition with nifedipine may modify noradrenergic BP control by lowering the NE pressor reactivity without causing an equivalent increase in adrenergic activity.