Ang II-infused Research Articles

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

CCN2 deficiency in smooth muscle cells triggers cell reprogramming and aggravates aneurysm development.

Vascular smooth muscle cell (SMC) phenotypic switching is widely recognized as a key mechanism responsible for the pathogenesis of several aortic diseases, such as aortic aneurysm. Cellular communication network factor 2 (CCN2), often upregulated in human pathologies and animal disease models, exerts myriad context-dependent biological functions. However, current understanding of the role of SMC-CCN2 in SMC phenotypic switching and its function in the pathology of abdominal aortic aneurysm (AAA) is lacking. Here, we show that SMC-restricted CCN2 deficiency causes AAA in the infrarenal aorta of angiotensin II-infused (Ang II-infused) hypercholesterolemic mice at a similar anatomic location to human AAA. Notably, the resistance of naive C57BL/6 WT mice to Ang II-induced AAA formation is lost upon silencing of CCN2 in SMC. Furthermore, the pro-AAA phenotype of SMC-CCN2-KO mice is recapitulated in a different model that involves the application of elastase-β-aminopropionitrile. Mechanistically, our findings reveal that CCN2 intersects with TGF-β signaling and regulates SMC marker expression. Deficiency of CCN2 triggers SMC reprograming associated with alterations in Krüppel-like factor 4 and contractile marker expression, and this reprograming likely contributes to the development of AAA in mice. These results identify SMC-CCN2 as potentially a novel regulator of SMC phenotypic switching and AA biology.

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces hypertension and vascular inflammation

Abstract Background Arterial hypertension is one of the most important modifiable risk factors for all-cause morbidity and mortality worldwide. Angiotensin II (Ang II) plays a pathogenic role in the development of hypertension, vascular dysfunction, inflammation and tissue damage. In the context of hypertension, heme-oxygenase 1 (HO-1) gene expression is upregulated as an antioxidant defense system in response to AngII through its action on heme catabolism, which generates carbon monoxide (CO), ferritin and biliverdin, which is reduced to bilirubin by biliverdin reductase A (BLVRA). Previous studies have shown the important role of HO-1 in the maturation and migration of immune cells. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion damage. However, the role of myeloid cell specific HO-1 in the detrimental effects of AngII induced vascular dysfunction has not yet been explored. Objectives To investigate the potential vascular protection of myeloid cell specific overexpression of HO-1 in AngII-induced arterial hypertension. Methods Hypertension was induced in 8–13 weeks old male mice with selective over-expression of HO-1 (HO-1indLysMcre) in myelomonocytic cells versus LysMCre/wt by AngII infusion (1mg/kg/d). Blood pressure was recorded by tail-cuff. Bilirubin levels were quantified in plasma by High Performance Liquid Chromatography (HPLC). The quantification of adherent and rolling leukocytes in carotid arteries was detected by intravital video microscopy (IVM). Endogenuos Thrombin potential (ETP) was measured in platelet rich plasma (PRP) and platelets poor plasma (PPP) by Calibrated Automated Thrombogram (CAT) assay. Endothelium-dependent vasodilation was assessed in isolated aortic rings by concentration-relaxation curves in response to acetylcholine (ACh). Results AngII-infused HO-1indLysMcre had decreased blood pressure values and improved endothelial function as compared to LysMcre controls. IVM revealed reduced leukocyte rolling and adhesion to the vascular endothelium in AngII infused HO-1indLysMcre mice compared to controls, paralleled by reduced expression of NOX-2 mediated oxidative stress and vascular inflammation in AngII-induced arterial hypertension by decreasing VCAM-1, CCR2 and MCP-1 expression. mRNA analysis revealed an increased expression of BLVRA in liver, spleen, heart and aortic tissues in response to AngII, which was higher in HO-1indLysMcre mice than in controls. By CAT assay we registered a decrease ETP in PPP and PRP in HO-1ind mice infused with AngII compatible with less abundance of inflammatory platelets, potentially regulated by BLVRA activity. This was supported by increased in bilirubin levels in response to AngII, which was higher in HO-1indLysMcre mice than in controls. Conclusion Myeloid cell specific overexpression of HO-1 confers anti-inflammatory protection to the vasculature in AngII induced hypertension. This effect is, at least in part, mediated by BLVRA. Funding Acknowledgement Type of funding sources: None.

Asiatic acid alleviates Ang-II induced cardiac hypertrophy and fibrosis via miR-126/PIK3R2 signaling

BackgroundCardiac hypertrophy is an independent risk factor of many cardiovascular diseases. Studies have demonstrated that microRNA-126 (miR-126) was involved in angiogenesis during physiological and pathological process. However, its role in cardiac hypertrophy has not been known clearly. Our previous study demonstrated that asiatic acid (AA) has obvious protective effect on cardiac hypertrophy. Here, this study aimed to discover the regulatory role of miR-126 and its mechanism in cardiac hypertrophy, and to determine whether AA’s anti-hypertrophy effect is partially miR-126 dependent.MethodsMale Sprague Dawley rats were AngII infused via osmotic minipumps for 4 weeks and were treated with AA (20 mg/kg/day) by oral gavage. Cardiac hypertrophy was assessed using the echocardiography and histological analysis. In vitro studies,cardiomyocyte and cardiac fibroblasts (CF) were treted with AngII and AngII plus AA. And, the effect of AA on miR-126 and PI3K/AKT signaling pathway was investigated.ResultsTreatment of rats with AA decreased the ratio of heart weight to tibia length and hypertrophy markers. In vitro exprements demonstrated that AA significantly attenuated AngII-induced cardiac growth and cardiac fibroblast collagen expression. Moreover, our results found downregulation of miR-126 and activation of PI3K/AKT signaling pathway in AngII infusion induced cardiac hypertrophy model. It was also determined that miR-126 targets PIK3R2 directly.ConclusionsAA supplementation upregulated the expression of miR-126 and conferred cardio-protection effect against AngII induced cardiac hypertrophy.

Central Ang II (Angiotensin II)-Mediated Sympathoexcitation: Role for HIF-1α (Hypoxia-Inducible Factor-1α) Facilitated Glutamatergic Tone in the Paraventricular Nucleus of the Hypothalamus.

Central infusion of Ang II (angiotensin II) has been associated with increased sympathetic outflow resulting in neurogenic hypertension. In the present study, we appraised whether the chronic increase in central Ang II activates the paraventricular nucleus of the hypothalamus (PVN) resulting in elevated sympathetic tone and altered baro- and chemoreflexes. Further, we evaluated the contribution of HIF-1α (hypoxia-inducible factor-1α), a transcription factor involved in enhancing the expression of N-methyl-D-aspartate receptors and thus glutamatergic-mediated sympathetic tone from the PVN. Ang II infusion (20 ng/minute, intracerebroventricular, 14 days) increased mean arterial pressure (126±9 versus 84±4 mm Hg), cardiac sympathetic tone (96±7 versus 75±6 bpm), and decreased cardiac parasympathetic tone (16±2 versus 36±3 versus bpm) compared with saline-infused controls in conscious rats. The Ang II-infused group also showed an impaired baroreflex control of heart rate (-1.50±0.1 versus -2.50±0.3 bpm/mm Hg), potentiation of the chemoreflex pressor response (53±7 versus 30±7 mm Hg) and increased number of FosB-labeled cells (53±3 versus 19±4) in the PVN. Concomitant with the activation of the PVN, there was an increased expression of HIF-1α and N-Methyl-D-Aspartate-type1 receptors in the PVN. Further, Ang II-infusion showed increased renal sympathetic nerve activity (20.5±2.3% versus 6.4±1.9% of Max) and 3-fold enhanced renal sympathetic nerve activity responses to microinjection of N-methyl-D-aspartate (200 pmol) into the PVN of anesthetized rats. Further, silencing of HIF-1α in NG108 cells abrogated the expression of N-methyl-D-aspartate-N-methyl-D-aspartate-type1 induced by Ang II. Taken together, our studies suggest a novel Ang II-HIF-1α-N-methyl-D-aspartate receptor-mediated activation of preautonomic neurons in the PVN, resulting in increased sympathetic outflow and alterations in baro- and chemoreflexes.

Angiotensin II activates mTORC1 pathway in the kidneys through a pressor‐independent mechanism.

BackgroundAngiotensin II (AngII) has been found in vitro to stimulate the mTORC1 pathway and we have observed that AngII treatment enhanced mTORC1 phosphorylation of the ribosomal protein S6 kinase (pS6/S6) in a dose related manner in the NRK cell line. However, the in vivo effects of chronic elevations of AngII upon kidney activity of mTORC1 or upon mTORC2 (pAKT/AKT) have not been determined.GoalTo determine the effects of chronic hypertensive elevations of AngII upon the kidney expression of mTORC1 and mTORC2 in Sprague‐Dawley (SD) rats. Since it would normally not be possible to separate the direct effects of AngII upon mTORC1 activation from the effects of arterial pressure, studies were carried out using a unique servo‐control system that enabled the renal perfusion pressure (RPP) to the left kidney to be maintained at normal levels while the contralateral right kidney was subjected to the elevated systemic pressure.MethodsMale SD rats (n = 15) were maintained on a 0.4% NaCl diet throughout the study. At 9 weeks of age, rats were instrumented with carotid and femoral arterial catheters for measuring blood pressure, a femoral venous catheter for AngII infusion and a balloon occluder cuff was placed around the abdominal aorta between the renal arteries. The carotid and the femoral mean arterial pressures were used as surrogates to the RPP of the right and left kidneys respectively. After 7 days of recovery, baseline blood pressure was measured for 3 days, then the n = 9 of rats were infused with AngII (50 ng/kg/min) for 7 days to induce hypertension. N = 6 of rats were infused with only saline (vehicle control) throughout the study. A computer‐driven servo‐control system was used to inflate the aortic cuff to maintain left RPP to baseline levels while right RPP was left uncontrolled. Following 7 days of Ang II infusion, kidneys were removed for molecular analysis.ResultsPrior to infusion of AngII, baseline RPP values to the right and left kidneys were similar (117.3±3.0 vs 112.2±3.4 mmHg). Following 7 days of AngII infusion, the right RPP was significantly higher than left RPP (162.4±9.0 vs 113.2±2.7 mmHg; p<0.05). Average 24 hr mean arterial pressures (MAP) remained unchanged in saline infused rats throughout the study. Western blot analysis of renal cortical tissue at day 7 found pS6/S6 was higher in both pressure‐uncontrolled and pressure‐controlled kidneys of AngII infused rats (2.25±0.21 vs 2.00±0.23) compared to saline infused rat kidneys (1.42±0.21; p<0.05). In contrast, pAKT/AKT was unchanged by either AngII or differences in RPP.ConclusionChronic elevations of circulating AngII activates mTORC1 pathway in the kidneys through a pressor‐independent mechanism but has little effect on activation of mTORC2.Support or Funding InformationP01 HL116264, RO1 HL137748

Soluble RAGE attenuates AngII-induced endothelial hyperpermeability by disrupting HMGB1-mediated crosstalk between AT1R and RAGE

Increased endothelial permeability, one of the earliest signs of endothelial dysfunction, is associated with the development of cardiovascular diseases such as hypertension and atherosclerosis. Recent studies suggest that the receptor for advanced glycation end products (RAGE) regulates endothelial permeability in inflammation. In the present study, we investigated the regulatory mechanism of RAGE in endothelial hyperpermeability induced by angiotensin II (Ang II), a well-known inflammatory mediator, and the potential therapeutic effect of soluble RAGE (sRAGE), a decoy receptor for RAGE ligands. For in vitro studies, Ang II-treated human umbilical vein endothelial cells (HUVECs) were treated with siRNA specific to either RAGE or sRAGE to disrupt RAGE-mediated signaling. Endothelial permeability was estimated using FITC-labeled dextran 40 and a resistance meter. To evaluate intercellular junction disruption, VE-cadherin expression was examined by western blotting and immunocytochemistry. Ang II increased the expression of the Ang II type 1 receptor (AT1R) and RAGE, and this increase was inhibited by sRAGE. sRAGE prevented Ang II-induced VE-cadherin disruption in HUVECs. For in vivo studies, Ang II-infused, atherosclerosis-prone apolipoprotein E knockout mice were utilized. Endothelial permeability was assessed by Evans blue staining of the aorta. Ang II increased endothelial barrier permeability, and this effect was significantly attenuated by sRAGE. Our data demonstrate that blockade of RAGE signaling using sRAGE attenuates Ang II-induced endothelial barrier permeability in vitro and in vivo and indicate the therapeutic potential of sRAGE in controlling vascular permeability under pathological conditions.

ANGIOTENSIN II INDUCES UCP1 IN ADIPOSE TISSUE OF AN EXPERIMENTAL MODEL OF CARDIO-RENAL CACHEXIA

Objective: Cachexia is a severe complication of advanced heart failure and chronic kidney disease. We previously reported that high dose Angiotensin II infusion in rats determined body weight reduction, prevented by an AT-1 receptor blocker treatment and beta-adrenergic non selective antagonism. The possible role of Ang II on UCP1 in white and brown adipose tissue, as a possible mechanism, was investigated in this study. Design and method: Male Sprague-Dawley rats, were implanted subcutaneously in the dorsal region with osmotic minipumps delivering Ang-II at high doses (525 ng/Kg/min) for 12 days to mimic heart failure and the development of cachexia. Five concurrent groups of rats were evaluated: 1) vehicle–infused rats (CTR-rat, n = 9); 2) Ang-II infused rats (n = 7); Ang- II rats receiving by oral gavage 2 days before minipump implantation until the 12th day of Ang-II infusion, either 3) bisoprolol (BIS,β1-adrenergic antagonist, 35 mg/Kg/day, n = 5), or 4) carvedilol (CVD,α1-non-selective-β1,2,3-adrenergic blocker, 90 mg/kg/day, n = 5) or 5) bupranolol (BPR,β3-antagonist, 200 mg/day/kg, n = 4). White adipose tissue (WATs) from retroperitoneal (RWAT), epidydimal (EWAT), dorsal (DWAT) and brown interscapular (IBAT) depot were withdrawn in order to determine UCP1 mRNA expression, norepinephrine tissue level (TNE) and tyrosine hydroxylase activity (THA) Results: THA and TNE were higher in Ang-II infused rats in all the white adipose tissue depots analyzed. CVD prevented TNE increase in all WATs, while BPR and BIS were effective in RWAT and EWAT but not DWAT. All the three beta adrenergic blocker treatments significantly reduced THA increased activity related to Ang-II infusion in all the WATs. UCP1 mRNA was increased in IBAT (+39%, p < 0.05) of Ang II-infused rat, significantly prevented by CVD and BPR but not by BIS. UCP1 was also increased in all the three WATs of Ang-II infused rats, with all the three beta blockers able to reduce the effect in RWAT and DWAT. No effect of beta-blockers treatment was observed in EWAT. Conclusions: High dose angiotensin II infusion determined an increase in UCP1 trascription in brown and white adipose tissue in rats. CVD and BPR were able to prevent UCP1 increase in brown and white adipose tissue more than beta-1 selective antagonist BIS.

Abstract 462: Reductions of ApoB-containing Lipoproteins Prevented the Progression of Established Abdominal Aortic Aneurysms in AngII Infused Mice

Background and Objective: We have demonstrated that increased plasma apolipoprotein B (apoB)-containing lipoproteins augment development of AngII-induced abdominal aortic aneurysms (AAAs) in male mice. The purpose of this study was to determine whether plasma apoB-containing lipoproteins also influenced progression of established AAAs during chronic AngII-infusion. Methods and Results: Male LDL receptor -/- mice fed either a normal (Group 1) or Western (42% calories from saturated fat) diet were infused with AngII (1,000 ng/kg/min) for 28 days to induced AAAs. Maximal luminal diameters of the suprarenal region were compared with baseline after 4 weeks of AngII infusion to determine AAA presence. Mice fed Western diet were stratified by aortic lumen diameter into either a group maintained on Western diet (Group 2) or switched to a normal laboratory diet (Group 3). AngII infusion was continued for another 8 weeks in all 3 groups. Plasma cholesterol concentrations were determined at 4, 6, and 12 weeks of AngII infusion. Mice fed a Western diet for the initial 4 weeks were hypercholesterolemic (260 ± 13 to 1,469 ± 59 mg/dl). Two weeks after Group 3 were returned to normal laboratory diet, plasma cholesterol concentrations were profoundly reduced to 382 ± 22 mg/dl which was maintained for the remaining interval. These reductions were due to decreases in plasma cholesterol concentrations apoB-containing lipoproteins. Luminal diameters of suprarenal aortas were measured at 6 and 12 weeks of AngII infusion by ultrasonography. While luminal expansion progressed in mice fed Western diet, no continuous expansion was detected in mice with established AAAs that were fed normal laboratory diet. Ultrasonic measurements were confirmed at study termination by ex vivo measurements of maximal widths of suprarenal aortas (Group 1 verus 2 versus 3: 1.35 ± 0.04 mm, 3.32 ±0.34 mm, 1.91 ± 0.11 mm, respectively). Conclusions: Plasma apoB-containing lipoproteins contribute to the progressive expansion of established AAAs in AngII-infused mice.

Abstract 418: Microfibrillar-Associated Protein 4 Deficiency Reduces Size and Incidence Rate of Angiotensin II Induced Abdominal Aortic Aneurysms in Mice

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein primarily located in elastic arteries. It can bind elastin and collagen, and furthermore activate vascular cells through cellular integrin binding and modulate matrix metalloprotinase (MMP) activity. We hypothesized that lack of MFAP4 would decrease vascular inflammation and abdominal aortic aneurysm (AAA) formation. AAA was induced in 9-11 week old mice using two experimental mouse models: 1) Male Mfap4 -/- /ApoE -/- double knock-out (dKO) and ApoE -/- littermate control mice were feed western diet and subjected to continuously angiotensin II (AngII, 1000 ng/kg/min) infusion for 9-28 days via subcutaneous osmotic mini-pumps. Arterial blood pressure was measured in the femoral artery. 2) 1.5 U/mL elastase was infused into the infrarenal aorta in Mfap4 -/- and littermate Mfap4 +/+ mice for 5 minutes. Aortic blood flow was restored and the mice recovered for 9-16 days. Aortic diameter was measured in mice subjected to AngII or elastase infusion at day 28 and 16 respectively. MMP activity was detected by zymography. No difference in AAA formation was observed between genotypes after elastase perfusion. In response to AngII infusion dKO mice showed a significantly decrease in AAA diameter and incidence rate compared to ApoE -/- mice. AngII-induced increase in blood pressure was not dependent of MFAP4. However, there was decreased aortic arch atherosclerotic plaque formation, MMP2 and MMP9 activity in aortic tissue from dKO mice compared to ApoE -/- mice. Furthermore there was a non-significant tendency of decreased elastin degradation score in the AngII infused dKO mice, however this was not observed in the elastase perfused mice. Activity of MMP12 and extent of infiltrating leukocytes in aneurysmal tissue from both models will be further investigated. In conclusion we observed a decreased AAA formation and MMP activity in Mfap4 -/- /ApoE -/- mice which was not explained by variation in blood pressure or altered elastin degradation. The data suggest that MFAP4 induces MMP2-activity and thus the propensity for AAA formation.

SCD40L ‐CD40/VLA‐ 5 Signaling Contributes to AngII‐induced Thrombosis and Inflammation.

AngII‐induced hypertension is associated with accelerated thrombus formation and leukocyte recruitment. The objective of this study was to evaluate the mechanisms underlying the prothrombogenic‐proinflammatory responses associated with chronic AngII infusion. We assessed the contribution of CD40/CD40L to these AngII‐mediated responses and whether/how the generation of soluble CD40L(sCD40L) contributes to the exacerbated thrombosis and leukocyte recruitment. Thrombus formation in mouse arterioles was induced using the light/dye injury model, while firm adhesion of leukocytes was quantified in adjacent venules. Wild type (WT), CD40‐/‐, CD40L‐/‐ , bone marrow chimeric CD40‐/‐ and CD40L‐/‐ mice (CD40‐/‐→WT or CD40L‐/‐→WT) were infused with AngII (1 µg/kg/min for 14 days) via osmotic pumps. Some WT mice and AngII infused WT mice were treated with either recombinant sCD40L, a P‐selectin blocking antibody, or a VLA5 antagonist (ATN‐161). The studies revealed that both AngII‐infused CD40‐/‐ and CD40L‐/‐ exhibited reduced leukocyte adhesion and oxidative stress in venules, and a blunted thrombosis response in arterioles. WT‐AngII mice treated with ATN‐161 were completely protected against the enhanced thrombosis and leukocyte adhesion responses to AngII, while P‐selectin immunoblockade offered only partial protection. Infusion of recombinant sCD40L in either WT, CD40‐/‐, or CD40L‐/‐ mice restored the accelerated thrombus formation elicited by AngII infusion. These findings implicate sCD40‐CD40L/VLA‐5‐dependent signaling in the prothrombotic‐proinflammatory responses elicited by chronically elevated AngII levels (Postdoctoral Fellowship from Malcolm Feist Endowment).

Abstract 043: Acute K+ Intake Does Not Reduce Renal Na-Cl Cotransporter Phosphorylation during Chronic AngII Hypertension

In Sprague Dawley (SD) rats, a single K + rich meal provokes a 50% decrease in DCT Na-Cl cotransporter phosphorylation (NCCp) which shifts Na + reabsorption downstream for reabsorption by CCD ENaC which drives a homeostatic increase in K + secretion. During Ang II dependent hypertension, DCT NCCp increases 2-4 fold as does NCC mediated Na + reabsorption. Motivated by these two recent findings, we tested the hypothesis that NCCp elevated by AngII hypertension (400 ng AngII/kg/min x 14 d in SD rats) could be reduced by a single K + rich meal (3 hr 2% K meal). Three groups (n=5-6) of male SD rats were compared: 1) Untreated fed 0%K meal, 2) AngII infused fed 0%K meal, and 3) AngII infused fed 2%K meal. By immunoblot of renal cortical homogenates, AngII infusion increased the following 2-5 fold compared to the untreated group: NCC, NCCpS71, NCCpT53, NCCpS89, SPAK, SPAKpS373, and cleaved ENaC-gamma. A single 2%K meal did not reduce abundance or phosphorylation of NCC, SPAK or ENaC cleavage. Interestingly, the AngII infused rats exhibited signs of kaliuresis and K + depletion compared to the untreated group: overnight urine K + (UKV) was 4.3 +/- 0.1 vs. 2.3 +/- 0.3 mM, fasting plasma [K + ] was 3.3 +/- 0.1 vs. 3.9 +/- 0.1 mEq in AngII vs. untreated, and while plasma [K + ] climbed to 4.6 +/- 0.1 after the 2% K meal in AngII infused rats, UKV was not elevated compared to AngII rats fed a 0%K meal. The results indicate that a single K + rich meal does not reduce NCCp in AngII infused rats, likely due to our finding that the AngII hypertension protocol provokes kaliuresis and K + deficiency. Thus, the ingested K + replenishes K + stores and is not excreted. The results also suggest that the rise in NCCp during AngII hypertension may be, at least in part, a response aimed to reduce K + excretion.

Abstract 610: Cardiac and Vascular Hypertrophy Produced by Angiotensin II is Associated with Increases of mTOR/P70S6 Kinases Expression

It is fairly well-accepted now that Angiotensin II (Ang II)-infusion produces hypertension and cardiac as well as vascular hypertrophy. In cell culture, the mammalian target of rapamycin (mTOR) and its effector kinase (P70S6) have been shown to increase in cardiomyocytes and smooth muscle cells in response to Ang II and have been suggested to contribute to hypertrophy in these two cell types. The goal of this study was to test the Ang II infusion is associated with increased mTOR and P70S6 expression in vivo . C57BL/6 mice (3 mo of age) were subjected to either sham (control) surgery or surgically implanted with osmotic containing Ang II (1000ng/mit/kg). In mice implanted with Ang II, systolic blood pressure (SBP) was 134±10 mmHg versus 120±5 in control mice. The heart hypertrophy was indicated by heart weight in Ang II group with 30% heavier than the sham controls (mean ± SEM 165.1 ± 5.5 mg versus 126.4 ± 2.9 mg, P&lt;0.01). Quantitative densitometry evaluation of Western Blot showed the ratio 2447 phosphorylated mTOR / total mTOR was significantly elevated in the heart of AngII group with 54% greater than controls (0.51 ± 0.06 versus 0.33 ± 0.01, P &lt; 0.05); and p389-P70S6/total P70S6 was increased by ~75% in Ang II group versus controls (0.07 ± 0.009 to 0.04 ± 0.002 in Ang II-infused and sham mice, respectively, P&lt;0.05). Similarly, p-mTOR/t-mTOR expression increased by ~17% with Ang II treatment (e.g., p-mTOR/t-mTOR expression was 0.69 ± 0.14 to 0.59 ± 0.14 in Ang II-infused and sham mice, respectively, P&lt;0.05); and p-P70S6 / t-P70S6 in Ang II - infused mice was over 50% greater than controls (0.73 ± 0.15 versus 0.46 ± 0.14, P=0.055). Conclusion: mTOR/P70S6 upregulation in the aorta and the heart induced by AngII stimulation indeed happened in vivo and indicates mTOR/P70S6 may play important roles in cardiovascular hypertrophy. Also, they may serve as biomarkers or therapeutics indications. The mechanism of mTOR/P70S6 signaling pathway causing hypertrophy should be further explored.

Angiotensin II-induced hypertension increases plasma membrane Na pump activity by enhancing Na entry in rat thick ascending limbs

Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na-K-2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (ANG II) infusion increases net TAL Na transport and Na apical entry; however, little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by ANG II-infusion, a model of ANG II-induced hypertension. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 7 days, and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from ANG II-infused rats was 12.8 ± 0.4 arbitrary fluorescent units (AFU)·mg(-1)·min(-1) compared with only 9.9 ± 1.1 AFU·mg(-1)·min(-1) in controls (P < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (P < 0.043) greater in tubules from ANG II-treated than vehicle rats. ANG II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane, or the affinity for Na. When furosemide (1.1 mg·kg(-1)·day(-1)) was coinfused with ANG II, no increase in plasma membrane Na pump activity was observed. We concluded that in ANG II-induced hypertension Na pump activity is increased in the plasma membrane of TALs and that this increase is caused by the chronically enhanced Na entry occurring in this model.

Abstract 282: Pattern of Development of Abdominal Aortic Aneurysms Induced by Angiotensin In Hypercholesterolemic Mice

Background Understanding the pattern of development of abdominal aortic aneurysms (AAA) may help optimize translational research strategies. Thus we sought mechanistic insight on the pattern of AAA evolution in apolipoprotein E -/- mice. Methods Fifty-nine mice underwent a 4-week pump-mediated infusion of saline (controls; n=13) or 1500 ng/kg/min of AngII (n=46), and AAA development was tracked via in vivo serial ultrasound imaging. We adjusted for hemodynamic covariates in the mixed logistic regression model for AAA occurrence in relation to time. Results The incidence of intact AAA in AngII infused mice was 59% (27/46) of which 55.6% (15/27) were small AAA and 44.4% were large AAA (1.2-1.5 vs. &gt;1.5x baseline diameter, respectively). The incidence of rupture was 22% (10/46). All large aneurysms and most ruptures occurred within the first week of AngII exposure. Subsequent aneurysms were small and evolved in the second week of AngII infusion. The overall effect of time was statistically significant (p&lt;0.001) (Figure). When compared to day 7 of the study period, there was no decrease in the log odds of AAA occurrence by day 14 (-0.234, p = 0.65). However, compared to 21 and 28 days into AngII infusion, the log odds of an AAA occurrence decreased by -9.07 (p&lt; 0.001) and -2.35 (p= 0.04), respectively (Figure). Conclusions These are the first formal estimates of the odds of AAA occurrence vis-à-vis time in the context of a consistent pattern of AAA development in this mouse model of AngII-induced AAA. They occur in the first (large AAA) and second (small AAA) weeks of AngII infusion, and the odds of occurrence diminish thereafter. These findings provide guidance for experimental strategies.

Abstract 533: MAS Receptor Activation Contributes to the Improvement of Vascular Remodeling During Chronic Angiotensin II Type 1 Receptor Blockade in Angiotensin II Type 2 Receptor Knockout Mice.

Angiotensin (Ang)-(1-7) through MAS receptor may counteract the Ang II-induced actions. We previously demonstrated that the Ang receptor blocker olmesartan (OLM) improved vascular remodeling in SHR in part through the activation of MAS receptor. Here, we hypothesized that such an effect is independent of Ang II type 2 (AT2) receptor activation. AT2-knockout mice (AT2-K/O, 12 weeks old, n=6 per group) were treated with vehicle (control) or Ang (1-7) (15,5 pmol/kg/min) or Ang II (400 ng/kg/min) ± OLM (10 mg/kg/day) ± the MAS antagonist A-779 (11 pmol/min) for 14 days. BP was measured by tail-cuff method. In isolated mesenteric arteries pre-contracted with norepinephrine (10 μM) endothelium -dependent and -independent relaxation was assessed by dose-response curves to acetylcholine (1 nM to 100 μM) and SNP (10 nM to 1 mM) respectively. Mesenteric arteries media-to-lumen ratio (M/L) and cross sectional area (CSA) were evaluated on pressurized preparations. MAS expression in aorta was evaluated by immunoblotting. Ang II infusion increased BP in AT2-K/O (+20% vs control mice, P&lt;0.05). OLM reduced BP in Ang II-infused mice (104.8±4.4 mmHg vs 121.3±2.8 mmHg, respectively, P&lt;0.05). A-779 had no effect on BP in Ang II-infused mice treated with OLM. Ang (1-7) did not lower BP in AT2-K/O. Endothelium -dependent and -independent relaxations were similar in treated and control mice. M/L was similar in Ang II-infused and control mice. OLM significantly reduced M/L in Ang II-infused AT2-K/O (5.45±0.12 % vs 7.01±0.4%, respectively, -23%, P&lt;0.05). A-779 increased M/L in Ang II-infused AT2-K/O treated with OLM (+18%, P&lt;0.05). Ang (1-7) reduced M/L in AT2-K/O (-21%, P&lt;0.05). CSA was similar in all the groups. MAS expression was similar in Ang II-infused and control mice. MAS expression was increased by OLM in Ang II-infused mice (+108%, P&lt;0.05). A-779 prevented MAS increase in Ang II-infused mice treated with OLM. Ang (1-7) increased MAS expression in AT2-K/O (+81%, P&lt;0.05). In conclusion OLM reduced M/L and improved vascular remodeling in AT2-K/O in part through the increased expression and activation of MAS, independently of BP reduction. Such an effect is indeed independent of AT2 activation, as MAS is functionally active in the vasculature of AT2-K/O.

Abstract 427: Mammalian Target of Rapamycin (mTOR) Inhibition Attenuates Angiotensin II-Induced Hypertension and Endothelial Dysfunction

Angiotensin (Ang) II produces hypertension, endothelial dysfunction as well as vascular hypertrophy. mTOR is a key regulator of cell growth and protein synthesis, however the contribution of mTOR to hypertension and endothelial dysfunction produced by Ang II has not been previously elucidated. The goal of this study was to test the hypothesis that mTOR contributes to the hypertension and endothelial dysfunction produced by Ang II. C57Bl/6 mice were infused (via osmotic minipump) with vehicle or rapamycin (an inhibitor of mTOR; 2 mg/kg/d) for 10 days following which mice were then infused with Ang II (1000 ng/kg/min) plus vehicle or rapamycin for 14 subsequent days. Systolic blood pressure (SBP) was measured on Days -10, 0, and 14 using tail-cuff plethysmography and vascular function was examined in carotid arteries (in vitro) on Day 14. Ang II infusion produced a significant degree (P&lt;0.05) of hypertension in wild-type mice (eg, SBP: 114±4 vs. 145±3 mmHg on Day 0 and Day 14 of Ang II infusion, respectively). In rapamycin-treated mice, Ang II infusion had no effect (P&gt;0.05) on SBP (eg, SBP: 110±6 vs. 116±9 mmHg, on Day 0 and Day 14 of co-infusion of rapamycin plus Ang II, respectively). Rapamycin alone or vehicle infusion had no effect (P&gt;0.05) on blood pressure during Day -10 through Day 0. In terms of endothelial responses, acetylcholine-induced relaxation was impaired (P&lt;0.05) in Ang II-infused mice by approximately 50%. In contrast, rapamycin was associated with a significant degree of protection against Ang II-induced endothelial dysfunction (eg, 100 μM acetylcholine produced 47±10 vs. 90±4% relaxation in carotid arteries from Ang II- and Ang II plus rapamycin-infused mice, respectively). Tempol, a scavenger of superoxide was very effective in improving endothelial responses in Ang II-infused mice. Molecular analysis revealed that Ang II-infusion was associated with increased mTOR expression as well as mTOR phosphorylation. Taken together, these data provide strong pharmacologic and molecular evidence that mTOR activity contributes to the hypertension and endothelial dysfunction produced by Ang II.

Adenosine A1-receptor knockout mice have a decreased blood pressure response to low-dose ANG II infusion

Adenosine, acting on A(1)-receptors (A(1)-AR) in the nephron, increases sodium reabsorption, and also increases renal vascular resistance (RVR), via A(1)-ARs in the afferent arteriole. ANG II increases blood pressure and RVR, and it stimulates adenosine release in the kidney. We tested the hypothesis that ANG II-infused hypertension is potentiated by A(1)-ARs' influence on Na(+) reabsorption. Mean arterial pressure (MAP) was measured by radiotelemetry in A(1)-AR knockout mice (KO) and their wild-type (WT) controls, before and during ANG II (400 ng·kg(-1)·min(-1)) infusion. Baseline MAP was not different between groups. ANG II increased MAP in both groups, but on day 12, MAP was lower in A(1)-AR KO mice (KO: 128 ± 3 vs. 139 ± 3 mmHg, P < 0.01). Heart rates were significantly different during days 11-14 of ANG II. Basal sodium excretion was not different (KO: 0.15 ± 0.03 vs. WT: 0.13 ± 0.04 mmol/day, not significant) but was higher in KO mice 12 days after ANG II despite a lower MAP (KO: 0.22 ± 0.03 vs. WT: 0.11 ± 0.02 mmol/day, P < 0.05). Phosphate excretion was also higher in A(1)-AR KO mice on day 12. Renal expression of the sodium-dependent phosphate transporter and the Na(+)/glucose cotransporter were lower in the KO mice during ANG II treatment, but the expression of the sodium hydrogen exchanger isoform 3 was not different. These results indicate that the increase in blood pressure seen in A(1)-AR KO mice is lower than that seen in WT mice but was increased by ANG II nonetheless. The presence of A(1)-ARs during a low dose of ANG II-infusion limits Na(+) and phosphate excretion. This study suggests that A(1)-AR antagonists might be an effective antihypertensive agent during ANG II and volume-dependent hypertension.

Sexual Dimorphism in Urinary Angiotensinogen Excretion During Chronic Angiotensin II−Salt Hypertension

The intrarenal renin-angiotensin system contributes to hypertension by regulating sodium and water reabsorption throughout the nephron. Sex differences in the intrarenal components of the renin-angiotensin system have been involved in the greater incidence of high blood pressure and progression to kidney damage in males than females. This study investigated whether there is a sex difference in the intrarenal gene expression and urinary excretion of angiotensinogen (AGT) during angiotensin II (Ang II)-dependent hypertension and high-salt (HS) diet. Male and female Sprague-Dawley rats were divided into 5 groups for each sex: Normal-salt control, HS diet (8% NaCl), Ang II-infused (80 ng/min), Ang II-infused plus HS diet, and Ang II-infused plus HS diet and treatment with the Ang II receptor blocker, candesartan (25 mg/L in the drinking water). Rats were evaluated for systolic blood pressure (SBP), kidney AGT mRNA expression, urinary AGT excretion, and proteinuria at different time points during a 14-day protocol. Both male and female rats exhibited similar increases in urinary AGT, with increases in SBP during chronic Ang II infusion. HS diet greatly exacerbated the urinary AGT excretion in Ang II-infused rats; males had a 9-fold increase over Ang II alone and females had a 2.5-fold increase. Male rats displayed salt-sensitive SBP increases during Ang II infusion and HS diet, and female rats did not. In the kidney cortex, males displayed greater AGT gene expression than females during all treatments. During Ang II infusion, both sexes exhibited increases in AGT gene message compared with same-sex controls. In addition, HS diet combined with Ang II infusion exacerbated the proteinuria in both sexes. Concomitant Ang II receptor blocker treatment during Ang II infusion and HS diet decreased SBP and urinary AGT similarly in both sexes; however, the decrease in proteinuria was greater in the females. During Ang II-dependent hypertension and HS diet, higher intrarenal renin-angiotensin system activation in males, as reflected by higher AGT gene expression and urinary excretion, indicates a mechanism for greater progression of high blood pressure and might explain the sex disparity in development of salt-sensitive hypertension.

Toll‐Like Receptor 4 Contributes to Hypertension and Endothelial Dysfunction Produced By Angiotensin II

We have previously found that C3H/He mice are a strain of mice that is resistant to endothelial dysfunction produced by Angiotensin II (Ang II). Interestingly, C3H/He mice carry a point mutation in the Toll‐Like Receptor 4 (TLR4) gene that results in defective TLR4‐mediated signaling. The goal of this study was to further explore the potential role of TLR4 in the hypertension and endothelial dysfunction produced by Ang II. C57Bl/6 (TLR4+/+ strain) and C57Bl/10ScN mice (a strain that carries a null‐mutation for the TLR4 gene) were infused with either vehicle or Ang II (1000 ng/kg/min) for 14 days. Baseline blood pressure was similar in C57Bl/6 and C57Bl/10ScN and on Day 14 of vehicle‐infusion. Ang II‐infusion produced hypertension in both C57Bl/6 and C57Bl/10ScN mice, however the degree of hypertension was significantly greater in C57Bl/6 than C57Bl/10ScN mice (164±6 vs. 147±9 mmHg, respectively). Acetylcholine produced concentration‐dependent relaxation that was similar in carotid arteries from vehicle‐infused C57Bl/6 and C57Bl/10ScN mice. In contrast, responses to acetylcholine were markedly impaired in Ang II‐infused C57Bl/6 mice. Responses to acetylcholine in Ang II‐infused C57Bl/10ScN mice were similar to their vehicle‐infused counterparts. Taken together, these findings demonstrate that TLR4 expression contributes to the hypertension and endothelial dysfunction produced by Ang II.