Ang II-infused Research Articles

ACE2 overexpression in the paraventricular nucleus attenuates angiotensin II-induced hypertension

Angiotensin II (Ang II) has been shown to have both central and peripheral effects in mediating hypertension, for which the hypothalamic paraventricular nucleus (PVN) is an important brain cardio-regulatory centre. Angiotensin-converting enzyme 2 (ACE2) has been identified as a negative regulator of the pro-hypertensive actions of Ang II. Recent findings from our laboratory suggest that Ang II infusion decreases ACE2 expression in the PVN. In the present study, we hypothesized that ACE2 overexpression in the PVN will have beneficial effects in counteracting Ang II-induced hypertension. Male Sprague-Dawley rats were used in this study. Bilateral microinjection of an adenovirus encoding hACE2 (Ad-ACE2) into the PVN was used to overexpress ACE2 within this region. Mean arterial pressure measured by radiotelemetry was significantly increased after 14 days in Ang II-infused (200 ng/kg/min) rats vs. saline-infused controls (162.9 ± 3.6 vs. 102.3 ± 1.5 mmHg). Bilateral PVN microinjection of Ad-ACE2 attenuated this Ang II-induced hypertension (130.2 ± 5.7 vs. 162.9 ± 3.6 mmHg). ACE2 overexpression also significantly decreased AT(1)R and ACE expression and increased AT(2)R and Mas expression in the PVN. Additionally, ACE2 overexpression in the PVN attenuated the Ang II-induced increase in the expression of the pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-1β and IL-6 in the PVN. Our findings suggest that attenuation of pro-inflammatory cytokines in the PVN in combination with the shift of the renin-angiotensin system towards the anti-hypertensive axis (ACE2/Ang-(1-7)/Mas) may be responsible for the overall beneficial effects of ACE2 overexpression in the PVN on the Ang II-induced hypertensive response.

Angiotensin II-induced reduction in body mass is Ang II receptor mediated in association with elevated corticosterone

The mechanisms by which elevated glucocorticoids contribute to decreased body mass via angiotensin II (Ang II) infusion are not completely described. This study addressed the hypothesis that chronic Ang II infusion suppresses hepatic growth hormone receptor (GHr) and IGF1 expressions via an Ang II receptor (AT1)-mediated pathway associated with elevated glucocorticoids. Sprague–Dawley rats were assigned to three groups: 1) Control, 2) Ang II-infused (80 ng/min x 28d) and 3) Ang II + angiotensin receptor blocker (ARB; 10 mg losartan/kg/d × 21d). After 28 d, Ang II decreased body mass by 14% (407 ± 8 vs 350 ± 17 g) and hepatic AT1a, GHr, and IGF1 mRNA expressions by 45%, 44%, and 44%, respectively. ARB treatment completely prevented the loss in body mass (409 ± 9 g) and AT1a and GHr expressions and partially recovered the loss of hepatic IGF1. Ang II increased plasma corticosterone (B) 3-fold (173 ± 28 vs 555 ± 42 ng/mL) and ARB treatment prevented the response (150 ± 47 ng/mL). Food consumption did not change suggesting that the decrease in body mass resulted from the catabolic actions of the Ang II-induced increase in systemic B and not from reduced caloric intake. The prevention by ARB treatment of the Ang II-induced decrease in body mass and downregulation of AT1a, GHr and IGF1 coinciding with suppression of plasma B suggests that the Ang II-induced decrease in body mass is AT1 receptor mediated in conjunction with elevated B. These data suggest that alleviating the Ang II-induced cachexia requires targeting AT1 and suppressing glucocorticoid secretion.

AT1 receptor‐ independent oxidative stress in angiotensin II (Ang II) infused Male Spontaneously Hypertensive Rats (SHR)

The AT1‐receptor blocker, Candesartan lowers Ang II induced hypertension and oxidative stress. We hypothesize that Ang II/AT1 receptor induces oxidative stress via NADPH oxidase. 12 week old rats were divided into 4 groups control, Ang II‐infused (200ng/kg/min, 14 days minipump), Ang II+Candesartan (10 mg/kg/day, 14 days in drinking water), Ang II+Tempol (30 mg/kg/day, 14 days in drinking water). The renal cortex was isolated to measure NADPH oxidase and O2.‐ levels via lucigenin. Ang II‐infusion significantly increased NADPH activity compared to control (733±26 vs 260±13, p<0.001). Tempol lowered NADPH activity (401±20, p<0.01) but Candesartan was comparable in AngII SHR (727±38). Ang II increased basal O2.‐ in SHR (10.±1, p<0.05). Co‐treatment with Candesartan and Tempol lowered O2.‐ levels (5.4±0.5,p<0.01; 1.0±0.9,p<0.01). Cortical TBARS levels were comparable in all groups. 8‐isoprostane levels increased with Ang II (68±6 vs 35±4., p<0.01) and was comparable in Candesartan and Tempol groups. Western blot analysis showed Ang II decreased Nox 4 expression (4.6±0.3 rdu vs 3.5±0.2 rdu, p<0.05) whereas rest of the groups were comparable. Subunits of NADPH oxidase, catalase and SOD were comparable in all groups. We conclude that Ang II induces oxidative stress independent of AT1 receptor via NADPH oxidase suggesting an alternate mechanism. Funded by NIH.

Role of the proximal tubule AT1A receptor in sodium transport regulation

Chronic AngII infusion raises blood pressure (BP) in mice which depends on renal AngII receptors (AT1R). AngII is anti‐natriuretic while high BP is natriuretic.Aim 1To determine how AngII infusion affects Na+ transporter abundance and phosphorylation (by immunoblot of homogenates). AngII infusion (1 μg/kg/min x 14d) increases systolic BP ~30 mmHg. Versus sham infused (=1.0), AngII decreased total NHE3 to 0.7±0.1, NHE3‐P (at Ser552) to 0.8±0.1 without altering NHE3 ‐P/total ratio. NaPi2 and myosin VI were unchanged. Distal convoluted tubule (DCT) total NCC increased to 2.2±0.3, NCC‐P (at Thr60) to 5.4 ± 1.6 and NCC‐P/total ratio to 2.2±0.5 fold.Aim 2To determine the role of proximal tubule (PT) AT1R in regulation of Na+ transporters in mice lacking PT AT1R (KO). BP decreased 10mmHg in KO vs. WT and baseline expression of NHE3, NaPi2, and NCC were unchanged in KO vs. WT. AngII infusion increased BP 20mmHg less than in WT. AngII infusion in KO decreased: total NHE3 to 0.55±0.1, NHE3‐P to 0.66±0.1 (no change in ‐P/total NHE3 ratio), myosin VI to 0.56±0.1, NaPi2 to 0.64±0.1. NCC total and NCC‐P were unchanged in the AngII infused KO.Conclusions1) increased NCC contributes to AngII induced high BP while decreased PT NHE3 compensates for high BP in WT, 2) AngII dependent increase in NCC depends on PT AT1R, and 3) the removal of PT AT1R facilitates pressure natriuresis by further decreasing PT transporters during AngII infusion.

Intrarenal RAS expression during Ang II‐infusions and ACE inhibition

ACE inhibition (ACEi) ameliorates the development of hypertension and the intrarenal Ang II augmentation observed during chronic Ang II infusions in mice, the objective of this study was to determine if these effects are associated with changes in the mouse intrarenal RAS. 9‐12 week C57BL/6J male mice were distributed as follows: controls (n=10), Ang II‐infused (Ang II=8, 400 ng/kg/min), controls + ACEi (ACEi=10, lisinopril, 100 mg/L) and Ang II‐infused + ACEi (Ang II + ACEi=11). As previously reported, ACEi attenuated the increases in MAP (monitored by telemetry) and intrarenal Ang II caused by Ang II infusions (measured by RIA). No significant changes were observed in angiotensinogen (AGT), ACE or AT1R mRNA by qRT‐PCR. AGT protein expression [determined by Western blot (WB) and Immunohistochemistry (IHC)] was increased by Ang II infusions (Ang II, 1.41 ± 0.14 by WB, 2.5‐fold increase by IHC) vs. controls (1.00, p<0.05) and this was prevented by ACEi (ACEi + Ang II, 0.80 ± .07 by WB, NS). Tubular renin protein increased in ACEi‐treated mice (ACEi, 2.3 ± 0.36; Ang II + ACEi, 4.01 ± 1.1 by IHC) vs. controls (1.00, p<0.05), but was not altered by Ang II alone. ACE protein was increased by Ang II (Ang II, 1.3 ± 0.18 by IHC) vs. controls (1.00, p<0.05) and ACEi prevented this changes (Ang II + ACEi, 0.85 ± 0.11). AT1R protein was increased by Ang II infusions (Ang II, 1.27 ± 0.06 by WB) vs. controls (1.00 p<0.05) and this was prevented by ACEi (Ang II + ACEi, 1.14 ± 0.06, NS). In summary, Ang II infusions augmented the expression of intrarenal AGT, ACE and the AT1R and did not suppress tubular renin. The findings that these changes are prevented by ACEi suggest that they are mediated by endogenous ACE‐derived Ang II.

Abstract 598: Deletion of Microsomal PGE Synthase-1 Decreases Oxidative Stress and Protects Against Abdominal Aortic Aneurysm Formation in Angiotensin II-Infused Mice

Microsomal (m) prostaglandin (PG) E 2 synthase(S)-1, an enzyme that catalyzes the isomerization of the cyclooxygenase (COX) product, PGH 2 , into PGE 2 , is a major source of PGE 2 in vivo . mPGES-1 deletion in mice was found to modulate experimentally evoked pain and inflammation and atherogenesis is retarded in mPGES-1 knockout (KO) mice. The impact of mPGES-1 deletion on formation of angiotensin II (Ang II)-induced abdominal aortic aneurysms (AAA) was studied in mice lacking the low density lipoprotein receptor (LDLR −/− ). AngII infusion increased aortic macrophage recruitment and nitrotyrosine staining while upregulating both mPGES-1 and COX-2 and urinary excretion of the major metabolite of PGE 2 (PGE-M). Deletion of mPGES-1 decreased both the incidence and severity of AAA and depressed excretion of both PGE-M and 8, 12-iso-iPF 2a -VI, which reflects lipid peroxidation in vivo . While Ang II infusion augmented prostaglandin biosynthesis, deletion of mPGES-1 resulted in rediversion to PGD 2 , reflected by its major urinary metabolite. However, deletion of the PGD 2 receptor, DP1, did not affect AAA in Ang II infused LDLR −/− mice. These observations indicate that deletion of mPGES-1 protects against AAA formation by AngII in hyperlipidemic mice, perhaps by decreasing oxidative stress. Inhibition of mPGES-1 may represent an effective treatment to limit aneurysm occurrence and expansion.

Angiotensin Converting Enzyme-Independent Angiotensin II Production by Chymase is Up-regulated in the Ischemic Kidney in Renovascular Hypertension

Tissue angiotensin II (ANG II) levels are elevated in both kidneys in renovascular hypertension (RVH). It has been demonstrated previously that intrarenal ANG II is augmented by an angiotensin converting enzyme (ACE) dependent mechanism in the non-ischemic kidney, but the role of ACE-independent production of ANG II in the kidney by the enzyme chymase is unknown. This study tested the hypothesis that intrarenal chymase activity is up-regulated in RVH. A two-kidney, one-clip (2K1C) rat model was used to induce RVH (n = 6 rats/group). Regulation of intrarenal chymase activity by plasma ANG II was investigated using an ANG II-infusion model. At sacrifice 14 days post-operatively, steady-state ANG II levels in plasma and kidney were quantified by radioimmunoassay. ANG II production was quantified in kidney homogenates by incubating at 37 degrees C for 60 min with enzyme substrate (200 microm ANG I) alone or substrate containing the chymase inhibitor chymostatin. ANG II was separated and quantitated by HPLC. Chymase activity was defined as the fraction of ANG II production inhibited by Chymostatin. 2K1C and ANG II-infused rats developed significant hypertension, compared to control rats (P = 0.0001 and P = 0.001, respectively). Chymase-dependent ANG II production was increased in the ischemic kidney, but not the non-ischemic kidney, of 2K1C rats compared to control animals (*P < 0.05). Intrarenal chymase activity was unchanged by ANG II infusion (P = NS). Chymase activity is up-regulated in the ischemic kidney of 2K1C rats. Plasma ANG II does not appear to regulate intrarenal chymase activity, suggesting that ischemia per se up-regulates chymase activity in the kidney. ACE-independent ANG II production by chymase may provide a mechanism for augmenting intrarenal ANG II in the ischemic kidney in RVH.

Altered nitric oxide synthase 3 distribution in mesenteric arteries of hypertensive rats.

A high-salt (HS) diet and angiotensin II (Ang II) are both associated with the development of hypertension and impaired endothelial function. We hypothesize that alterations in nitric oxide synthase (NOS) activity or subcellular localization of NOS 3 protein may contribute to endothelial dysfunction in salt-dependent hypertension. To test this hypothesis, two models of salt-dependent hypertension were studied: DOCA-salt and Ang II. For Ang II hypertension, rats were divided into 4 groups: control on normal or HS diet, Ang II-infused on normal or HS diet. The mesenteric arterial bed was isolated and frozen in liquid nitrogen. Frozen arteries were homogenized and separated into cytosolic and particulate fractions. NOS activity was assayed by determining the conversion of (3)H-arginine to (3)H-citrulline in the absence and presence of the NOS inhibitor, Nomega-nitro-L-arginine. NOS 3 protein expression was significantly increased in the cytosol of arteries from DOCA-salt compared with placebo rats and in Ang II-infused and Ang HS rats compared with control. NOS 3 expression in the particulate fraction was comparable among all groups. NOS activity (pmol/30 min/total protein) was significantly increased in the cytosolic fraction of arteries from DOCA-salt rats compared with placebo and in Ang HS rats compared with control. NOS activity was comparable in the particulate fraction in all rat groups. In conclusion, there is an altered subcellular distribution of NOS 3 in salt-dependent hypertension that may contribute to the development of hypertension and endothelial dysfunction.

Expression of Cell Cycle Proteins P21waf1/Cip1, P27kip1, Cyclin D1 and CDK4 in Blood Vessels of Angiotensin II-Infused Rats. Role of AT1 Receptors.

P79 Angiotensin II (Ang II) is an important modulator of cell growth via AT 1 receptors, as demonstrated both in vivo and in vitro . Here, we investigated the role of different proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (cdk4) and cdk inhibitors p21 and p27 in blood vessels of Ang II-infused rats and the effect therein of the AT 1 receptor antagonist losartan. Male Sprague Dawley rats were infused for 7 days with Ang II (120 ng/kg/min s.c.) and/or treated with losartan (10 mg/kg/day orally). DNA synthesis in mesenteric arteries was evaluated by radiolabeled 3 H-thymidine incorporation. The expression of p21, p27, cyclin D1, cdk4 and E2F, which play critical roles during G1-phase of the cell cycle process, was examined by Western blot analysis. Tail cuff systolic blood pressure (mmHg) was elevated (p 3 H-thymidine incorporation (cpm/100 μg DNA) showed that Ang II-infusion significantly increased DNA synthesis (152±5 vs. 102±6, p was significantly decreased in the Ang II group to 23.2±10.4% and 10.3±5.3% of controls, respectively, whereas expression of cyclin D1 and cdk4 was significantly increased in the Ang II group to 213.7±8% and 263.6±37% of controls, respectively. These effects induced by Ang II infusion was normalized in the presence of losartan. Ang II had no effect on the expression of E2F. Thus, when AT 1 receptors are stimulated in vivo , DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT 1 receptor stimulation.

Expression of Cell Cycle Proteins P21 waf1/Cip1 , P27 kip1 , Cyclin D1 and CDK4 in Blood Vessels of Angiotensin II-Infused Rats. Role of AT 1 Receptors.

P79 Angiotensin II (Ang II) is an important modulator of cell growth via AT 1 receptors, as demonstrated both in vivo and in vitro . Here, we investigated the role of different proteins involved in the cell cycle, including cyclin D1, cyclin-dependent kinase 4 (cdk4) and cdk inhibitors p21 and p27 in blood vessels of Ang II-infused rats and the effect therein of the AT 1 receptor antagonist losartan. Male Sprague Dawley rats were infused for 7 days with Ang II (120 ng/kg/min s.c.) and/or treated with losartan (10 mg/kg/day orally). DNA synthesis in mesenteric arteries was evaluated by radiolabeled 3 H-thymidine incorporation. The expression of p21, p27, cyclin D1, cdk4 and E2F, which play critical roles during G1-phase of the cell cycle process, was examined by Western blot analysis. Tail cuff systolic blood pressure (mmHg) was elevated (p&lt;0.05, n=9) in Ang II-infused rats (161.3±8.2) vs. controls (110.1±5.3) and normalized by losartan (104.4±3.2). Radiolabeled 3 H-thymidine incorporation (cpm/100 μg DNA) showed that Ang II-infusion significantly increased DNA synthesis (152±5 vs. 102±6, p&lt;0.05). Expression of p21 and p27 was significantly decreased in the Ang II group to 23.2±10.4% and 10.3±5.3% of controls, respectively, whereas expression of cyclin D1 and cdk4 was significantly increased in the Ang II group to 213.7±8% and 263.6±37% of controls, respectively. These effects induced by Ang II infusion was normalized in the presence of losartan. Ang II had no effect on the expression of E2F. Thus, when AT 1 receptors are stimulated in vivo , DNA synthesis is enhanced in blood vessels by activation of cyclin D1 and cdk4. Reduction in cell cycle kinase inhibitors p21 and p27 may contribute to activation of growth induced by in vivo AT 1 receptor stimulation.

Role of renal nerves in afferent arteriolar reactivity in angiotensin-induced hypertension.

The objective of this study was to determine the contribution of renal nerves to the enhanced afferent arteriolar reactivity observed in angiotensin II (Ang II)-induced hypertension. Uninephrectomized Sprague-Dawley rats were divided into four groups: sham rats, renal-denervated rats, Ang II-infused (at 40 ng/min for 13 days) rats, and Ang II-infused+renal-denervated rats. With the use of an implanted arterial catheter, mean arterial pressure (MAP) was monitored in conscious rats. Ang II infusion resulted in a progressive increase in MAP from 98 +/- 1 (day 0) to 166 +/- 7 mm Hg (day 13). This increase in MAP was attenuated in denervated rats and averaged 136 +/- 3 mm Hg on day 13. Kidneys were harvested on day 13 for microcirculatory experiments or measurement of intrarenal Ang II levels. Basal afferent arteriolar diameter was similar in all groups, and group averages ranged from 19.6 to 20.7 microns. Chronic Ang II infusion increased intrarenal Ang II levels. Renal denervation did not alter this effect. Increasing perfusion pressure from 100 to 160 mm Hg reduced afferent arteriolar diameter significantly by 11.2 +/- 0.6% in the sham group and by a similar degree in the remaining three groups. Superfusion with Ang II (10 nmol/L) reduced afferent arteriolar diameter by 34.3 +/- 2.0% in the sham group. This response was enhanced in Ang II-infused (62.3 +/- 3.4%) but not in renal-denervated or Ang II-infused+renal-denervated rats. Additionally, the enhanced afferent arteriolar reactivity to Ang II was not influenced by adrenergic receptor blockade. The afferent arteriolar response to norepinephrine was enhanced in renal-denervated, Ang II-infused, and Ang II-infused+renal-denervated rats compared with sham controls. Administration of the calcium ionophore A23187 decreased afferent arteriolar diameter similarly in all four groups. These results indicate that renal nerves contribute to the development of hypertension and to the enhanced afferent arteriolar responsiveness to Ang II elicited by chronic Ang II infusion.

Depletion of resistance vessel polyamines attenuates angiotensin II induced blood pressure rise in rats.

Vascular structural changes in hypertension are proposed to contribute to raised peripheral resistance. Endogenous polyamines (putrescine, spermidine and spermine) have an essential role in cellular growth and may be required in vascular restructuring. We have previously shown raised polyamines in resistance vessels in response to angiotensin II (angII) infusion in the rat and here we examined whether polyamine depletion influences the hypertensive process. Wistar-Kyoto rats were infused with either angII or saline by osmotic minipump, and maintained on either 2% difluoromethylornithine (DFMO; polyamine synthesis inhibitor) or water for 12 days. AngII significantly increased tail-cuff blood pressure (bp), resistance vessel spermidine and spermine concentration, and media:lumen ratios. DFMO attenuated both the rise in bp (p < 0.05) and vascular spermidine (p < 0.05) in the angII infused rats but vascular structure was apparently unaffected on day 12. In conclusion, raised concentration of resistance vessel spermidine may be a necessary component of the ang II pressor effect.