AT1 Receptor Protein Expression Research Articles

Abstract P114: Differential Expression Of Angiotensin Ii Receptors In Aorta Of Ela2ko Mice With Abdominal Aortic Aneurysm.

Elastase-2 (ELA-2) enzyme generates angiotensin-II (AngII) in arteries and is implicated in abnormal vascular remodeling in animal models.Our group demonstrated previously that ELA-2 knockout (ELA-2KO) mice were less susceptible to develop an aorta dilation in a response to AngII infusion. In this study, we hypothesize that susceptibility to an aorta dilation and abdominal aortic aneurysm (AAA) is mediated by AngII receptors (AT1 and AT2). Methods: Male wild type (C57bl/6, WT) and ELA-2KO (CELA-2aTm1Bdr) mice were treated with either saline or AngII (2.28g/g/min) for twenty-eight days by subcutaneously mini-pumps infusion. Mice were divided into four groups: WT treated with saline (WT+SAL, n=5), ELA-2KO treated with saline (ELA-2KO+SAL, n=5), WT treated with AngII (WT+AngII,n=5), and ELA-2KO treated with AngII (ELA-2KO+AngII, n=5). Protein expression of AT1 and AT2 receptor, CD68, and α-actin was measured by immunofluorescence in formalin-fixed, paraffin-embedded aorta tissue. ANOVA and Kruskal Wallis tests were used to compare the average fluorescent intensity between groups. Results: AngII treatment was associated with increased AT1 receptor expression in a WT group, but not with AT2 receptor expression (vs. WT+SAL,p=0.06 and p=0.31, respectively). The ELA-2KO group showed no difference in AT1 receptor expression in a response to AngII infusion compared to a ELA-2KO+SAL group (p=0.81). While ELA-2KO+Ang II mice had lower AT2 expression compared to ELA-2KO+SAL (p=0.05). The α-actin expression was similar in all groups. WT+AngII group expressed more CD68 expression in a response to an AngII treatment that was increased in a WT group (p=0.03), but not in a ELA-2KO group. Conclusion: Our data demonstrate that Ang II treatment did not increase an expression of AT1 receptor in ELA-2KO, but was associated with a downregulation of AT2 receptors. Taken together, this suggests That ELA-2KO mice resistance to developing an aorta dilation/AAA is related to downregulation of Ang II receptors. Our results also highlight the importance of the local/tissue response in the pathophysiology of AAA.

The renal excretory responses to acute renal interstitial angiotensin (1-7) infusion in anaesthetised spontaneously hypertensive rats.

This study investigated the impact of intrarenal angiotensin 1-7 (Ang [1-7]) infusion on renal excretory function in a rat model of hypertension. Eleven-week-old spontaneously hypertensive rats (SHRs, n=7) and Han Wistar controls (NCR, n=7) were anaesthetised with sodium pentobarbital (60mg/kg i.p.) and prepared for the measurement of mean arterial pressure (MAP) and left renal function during renal interstitial infusion of Ang (1-7) (50ng/min). The kidneys were harvested, the renal cortex and medulla separated, prepared for measurement of Ang II and Ang (1-7) and Western blot determination of AT1 and Mas receptor protein expression. MAP, glomerular filtration rate (GFR), urine flow (UF) and absolute sodium excretion (UNaV) were 109±16mmHg, 4.4±1.0mL/min/kg, 102±16µL/min/kg and 16±3µmol/min/kg, respectively in the NCR and 172±24mmHg, 3.4±0.7mL/min/kg, 58±30μL/min/kg and 8.6±4.8μmol/min/kg respectively in the SHR. Ang (1-7) increased UF (31%), UNa V (50%) and fractional sodium excretion (FENa+ ) (22%) in the NCR group (all p<0.05) but had no effect on GFR in either group. The magnitudes of the Ang (1-7)-induced increases in UF and UNa V were significantly blunted in the SHR group (model×drug p<0.05). The renal cortical AT1: Mas receptor expression ratio was significantly higher in the SHR group (p<0.05) but renal Ang II and Ang (1-7) levels were not statistically different between groups. The Ang (1-7)-induced increases in sodium and water excretion were impaired in the SHR group in the context of an unstimulated RAS. The decrease in responsiveness of the SHR kidney to Ang (1-7) appears to be associated with higher levels of AT1 receptor expression in the renal cortex.

Sacubitril/valsartan treatment relieved the progression of established pulmonary hypertension in rat model and its mechanism

AimsPulmonary hypertension (PH) is a fatal disease identified by progressive elevated pulmonary arterial pressure, which neurohormonal activation is a notable contributor to its development. Sacubitril/valsartan is a complex of sacubitril [via enhancing the natriuretic peptide (NP) system] and valsartan [via blocking the renin-angiotensin-aldosterone system (RAAS)]. Regulation of the two neurohormonal system had been shown to attenuate PH. This study was to explore the role of sacubitril/valsartan in both monocrotaline (MCT)-induced and hypoxia-induced rat models and the underlying mechanism. Main methodsThe rats were treated with MCT or hypoxic environment for 14 days, after that sacubitril/valsartan were given for another 14 days. Hemodynamic measurements and histological assessments were performed. The expression of NPs was measured using RT-PCR and ELISA, while the protein level of natriuretic peptide receptors (NPRs) and AT1 receptor were detected by western blot, the concentrations of cGMP, IL-1β, IL-6, TNF-α and TGF-β1 were tested by ELISA. Key findingsWe found that sacubitril/valsartan significantly improved the hemodynamic and histological data of two PH models. Sacubitril/valsartan suppressed the protein expression of AT1 receptor (P < 0.05). The intervention increased the expression of ANP and CNP (P< 0.05) and therefore upregulated the protein expression of NPRs (P < 0.05), raised the concentration of cGMP (P < 0.05). In addition, the treatment reduced the concentration of IL-1β, IL-6 and TNF-α (P < 0.05) but have no effects on TGF-β1. SignificanceSacubitril/valsartan alleviated PH in MCT-induced and hypoxia-induced rat models by inhibiting the activated RAAS, promoting ANP/NPR-A/cGMP and CNP/NPR-B/cGMP pathway, restoring the NPR-C signaling and the anti-inflammatory effects.

Perinatal nicotine exposure alters miR‐181a‐mediated signaling leading to gender‐dependent development of cardiac ischemia‐sensitive phenotype in adult male offspring

Back groundCigarette smoking/nicotine exposure during pregnancy is one of the major perinatal insults resulting in an increased risk of cardiovascular diseases in offspring. However, the fetal nicotine exposure‐induced programming of adult cardiovascular dysfunction remains largely elusive. The present study tested the hypothesis that perinatal nicotine exposure caused gender‐dependent increase in heart vulnerability to ischemia‐reperfusion injury and cardiac dysfunction in adult male rat offspring through up‐regulation of miRNA‐181a (miR‐181a)‐mediated signaling pathway.MethodsNicotine or saline was administered to pregnant rats via subcutaneous osmotic minipumps from gestational day 4 until postnatal day 10. The adult (~ 3 month old) offspring were treated with miR‐181a antisense (LNA‐miR‐181a) (10 mg/kg × 2 every 5 day, i.p.) or saline. After 10 days of treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the left ventricle (LV) tissues were isolated for molecular biological studies.ResultsPerinatal nicotine exposure enhanced I/R‐induced cardiac infraction and dysfunction in adult male but not in female offspring. The miR‐181a levels in LV tissues were significantly higher in the nicotine‐treated group than the saline control group. In addition, nicotine exposure decreased global DNA methylation levels but enhanced AT1 and AT2 receptor protein expression, and ROS production in LV tissues in the male offspring. Furthermore, the nicotine exposure up‐regulated autophagy‐related proteins (Atg‐5, beclin‐1, LC3 II) expressions as compared to controls in the male offspring. The most importance, treatment with LNA‐miR‐181a reversed nicotine‐mediated I/R‐induced cardiac infarction and dysfunction in male offspring, which was associated with an elimination of the nicotine’s effects on miR‐181a expression, DNA methylation, AT1R/AT2R protein, ROS, and autophagy‐related proteins expressions in the male offspring.ConclusionOur data demonstrated that perinatal nicotine exposure could cause a gender‐dependent fetal programing of adult heart ischemia‐sensitive phenotype. Our findings further suggest that miR‐181a plays a casual role in nicotine‐mediated up‐regulation of oxidative stress, renin‐angiotensin system (RAS) and autophagic flux, which may contribute to the development of ischemia‐sensitive phenotype in adult male rats.Support or Funding InformationSupported in part by the NIH grants: R01HL135623, R03DA041492, and R01HD088039; TRDRP grants: 29IR0437 and T30FT0936

Renal protective effects of the flavonoid (−)‐Epicatechin in a chronic kidney disease (nephrectomy 5/6) model

BackgroundChronic kidney disease (CKD) is a worldwide public health problem affecting 10% of the adult population. One of the most important factors in the progression of CKD from early stages is oxidative stress. Currently there is limited efficacy to treat this problem so the objective of this study was to test Epicatechin (Epi), effects in a mice model of CKDMethodsA nephrectomy 5/6 (Nf) in C‐57BL6 mice model was implemented and Epi treatment (14 days doses doses of 0.01, 0.1 and 1 mg/kg) was provided by gavage. Changes in Systolic blood pressure (SBP), serum urea and creatinine, renal cortex morphology, serum tetrahydrobiopterin (BH4), in 3‐nitrotyrosine as oxidative damage marker and of Angiotensin II AT1 receptor (AGTR1) and NOX‐4 protein expression were evaluatedResultsSBP increased in the Nf group and decrease significantly in Nf+ Epi groups. Serum urea and creatinine levels increased in the Nf group Epi treatment induced a significant decrease. Epi treatment induced a dose dependent increase in serum BH4 in Nf animals. Normal renal cortex morphology was conserved at 1 m/kg Epi dose. The expression of AGTR1 and NOX‐4 was increased in the Nf group, Epi treatment in a dose dependent manner decrease the AGTR1 and NOX‐4 expression.ConclusionThe present findings suggest that Epi could prevent the progression of CKD and may be used as complement for the modulation of blood pressure and f renal dysfunction.Support or Funding InformationConacyt 253769This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hesperidin Suppresses Renin-Angiotensin System Mediated NOX2 Over-Expression and Sympathoexcitation in 2K-1C Hypertensive Rats.

Hesperidin, a flavonoid derived from citrus fruits, possesses several beneficial effects including anti-oxidation and anti-inflammation. The aim of this study was to investigate the effects of hesperidin on the renin-angiotensin system (RAS) cascade that mediated oxidative stress and sympathoexcitation in two-kidney, one-clipped (2K-1C) hypertensive rats. 2K-1C hypertension was induced in male Sprague-Dawley rats. Hypertensive rats were treated with hesperidin at 20[Formula: see text]mg/kg or 40[Formula: see text]mg/kg or losartan at 10[Formula: see text]mg/kg beginning at three weeks after surgery and then continued for four weeks ([Formula: see text]/group). Hesperidin reduced blood pressure in a dose-dependent manner in hypertensive rats compared to untreated rats ([Formula: see text]). Increased plasma angiotensin converting enzyme (ACE) activity and angiotensin II levels, as well as, upregulated AT1 receptor protein expression in aortic tissues were attenuated in hypertensive rats treated with hesperidin. Hesperidin suppressed oxidative stress markers and NADPH oxidase over-expression, and restored plasma nitric oxide metabolites in 2K-1C rats. This was associated with improvement of the vascular response to acetylcholine in isolated mesenteric vascular beds and aortic rings from 2K-1C rats treated with hesperidin ([Formula: see text]). Enhancement of nerve-mediated vasoconstriction related to high plasma noradrenaline in the 2K-1C group was alleviated by hesperidin treatment ([Formula: see text]). Furthermore, losartan exhibited antihypertensive effects by suppressing the RAS cascade and oxidative stress and improved vascular dysfunction observed in 2K-1C rats ([Formula: see text]). Based on these results, it can be presumed that hesperidin is an antihypertensive agent. Its antihypertensive action might be associated with reducing RAS cascade-induced NOX2 over-expression and sympathoexcitation in 2K-1C hypertensive rats.

Losartan suppresses the kainate-induced changes of angiotensin AT1 receptor expression in a model of comorbid hypertension and epilepsy

AimsExperimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (AT1 receptor) in limbic structures, as well as the effect of the AT1 receptor antagonist losartan in a model of comorbid hypertension and epilepsy. Main methodsThe expression of AT1 receptors was compared between spontaneously hypertensive rats (SHRs) and Wistar rats by using immunohistochemistry in the kainate (KA) model of temporal lobe epilepsy (TLE). The effect of losartan was studied on AT1 receptor expression in epileptic rats that were treated for a period of 4weeks after status epilepticus. Key findingsThe naive and epileptic SHRs were characterized by stronger protein expression of AT1 receptor than normotensive Wistar rats in the CA1, CA3a, CA3b, CA3c field and the hilus of the dentate gyrus of the dorsal hippocampus but fewer cells were immunostained in the piriform cortex. Increased AT1 immunostaining was observed in the basolateral amygdala of epileptic SHRs but not of epileptic Wistar rats. Losartan exerted stronger and structure-dependent suppression of AT1 receptor expression in SHRs compared to Wistar rats. SignificanceOur results confirm the important role of AT1 receptor in epilepsy and suggest that the AT1receptor antagonists could be used as a therapeutic strategy for treatment of comorbid hypertension and epilepsy.

Molecular mechanisms in H2O2-induced increase in AT1 receptor gene expression in cardiac fibroblasts: A role for endogenously generated Angiotensin II

The AT1 receptor (AT1R) mediates the manifold actions of angiotensin II in the cardiovascular system. This study probed the molecular mechanisms that link altered redox status to AT1R expression in cardiac fibroblasts. Real-time PCR and western blot analysis showed that H2O2 enhances AT1R mRNA and protein expression via NADPH oxidase-dependent reactive oxygen species induction. Activation of NF-κB and AP-1, demonstrated by electrophoretic mobility shift assay, abolition of AT1R expression by their inhibitors, Bay-11-7085 and SR11302, respectively, and luciferase and chromatin immunoprecipitation assays confirmed transcriptional control of AT1R by NF-κB and AP-1 in H2O2-treated cells. Further, inhibition of ERK1/2, p38 MAPK and c-Jun N-terminal kinase (JNK) using chemical inhibitors or by RNA interference attenuated AT1R expression. Inhibition of the MAPKs showed that while ERK1/2 and p38 MAPK suffice for NF-κB activation, all three kinases are required for AP-1 activation. H2O2 also increased collagen type I mRNA and protein expression. Interestingly, the AT1R antagonist, candesartan, attenuated H2O2-stimulated AT1R and collagen mRNA and protein expression, suggesting that H2O2 up-regulates AT1R and collagen expression via local Angiotensin II generation, which was confirmed by real-time PCR and ELISA. To conclude, oxidative stress enhances AT1R gene expression in cardiac fibroblasts by a complex mechanism involving the redox-sensitive transcription factors NF-κB and AP-1 that are activated by the co-ordinated action of ERK1/2, p38 MAPK and JNK. Importantly, by causally linking oxidative stress to Angiotensin II and AT1R up-regulation in cardiac fibroblasts, this study offers a novel perspective on the pathogenesis of cardiovascular diseases associated with oxidative stress.

Abstract P086: Superoxide but Not Hydrogen Peroxide Increases Nuclear Translocation of Transcription Factor Sp3 and AT1 Receptor Expression in the Renal Cells

Age-associated oxidative stress causes up-regulation of renal AT1 receptor function (AT1R) and hypertension in aging Fischer Brown Norway (FBN) rats. Here we studied the mechanism of up-regulation of renal AT1R, and further tested superoxide Vs hydrogen peroxide (H2O2) specificity in this phenomenon. We found that transcription factor Sp3 plasmid increased (Control vs Sp3: 0.1165 ± 0.01 vs 0.3810 ± 0.03) while Sp3 siRNA decreased (Control siRNA vs Sp3 siRNA: 1.11 ± 0.25 vs 0.64 ± 0.06) the levels of AT1 receptor protein in human kidney (HK2) cells. Whereas transcription factor NF-kB p-65 plasmid did not affect AT1 receptor protein levels in these cells (Control vs NF-kB: 0.25 ± 0.025 vs 0.31 ± 0.035). DDC, a superoxide prodrug, but not H 2 O 2 treatments increased nuclear levels of Sp3 and NF-kB proteins in HK2 cells [(Control vs DDC, vs H 2 O 2, vs H 2 O 2 + tempol): Sp3 (0.50 ± 0.08 vs 1.28 ± 0.21 vs 0.68 ± 0.14 vs 1.04 ± 0.30 densities); NF-kB (0.4025 ± 0.13 vs 1.808 ± 0.54 vs 0.4950 ± 0.15 vs 0.6375 vs ± 0.18 densities)]. Tempol treatment, a superoxide scavenger, attenuated DDC-mediated nuclear accumulation of Sp3 (DDC vs DDC + tempol: 1.28 ± 0.21 vs 0.52 ± 0.12 densities) and NF-kB (DDC vs DDC + tempol: 1.808 ± .54 vs 0.43 ± 0.18 densities]. In addition, DDC but not H 2 O 2 increased AT1 receptor mRNA expression, measured by RT-qPCR [(Control vs DDC, vs H 2 O 2 ): (1.000 ± 0.0 vs 3.323 ± 0.79, vs 1.218 ± 0.49)]. This effect was attenuated by tempol treatment [(DDC vs DDC + tempol) (3.323 ± 0.79 vs 0.7225 ± 0.16 densities)]. Similarly, DDC treatment increased AT1 receptor protein expression that was attenuated by tempol treatment, measured by immunoblotting [(Control vs DDC vs DDC + Tempol) (0.3535 ± 0.99 vs 0.9975 ± 0.19 vs 0.3250 ± 0.02 densities)]. These results suggest that superoxide but not H 2 O 2 regulates both Sp-3 and NF-kB in the renal cells. However, it is Sp3 but not NF-kB that up-regulates renal AT1 receptor expression. Taken together, this phenomenon may represent a mechanism for hypertension in aging FBNs.

Sympathetic hyperactivity and AT1 receptor overexpression are associated with high‐fat diet‐induced hypertension in young rats

We evaluated the autonomic nervous system (ANS) and components of the renin angiotensin system (RAS) AT1 and MAS receptors in the control of blood pressure (BP) in young rats. Male Wistar rats (n=8/group) were fed during 8 weeks with control diet (C) or high‐fat diet (HF, 30% lipids) since the weaning. The animals were catheterized to record basal BP and heart rate (HR) during 15 minutes. The ANS was evaluated by spectral analysis (SA) in the time and frequency domain. At the end of the protocol, the animals were killed by overload of anesthetic (Ketamine/Xylazine, ip.) and the heart was harvested for left ventricle (LV) isolation. The AT1 and MAS receptors in the LV were performed by Western Blot. The HF group showed increase in the basal BP (12%) and HR (6%).In the SA, systolic BP (10%) and low frequency band were higher in HF group compared with C group as revealed by the increase in the variability of pulse interval (C: 9± 1 vs. HF: 15 ± 2 Hz) and in the variability of systolic BP (C: 34 ± 2 vs. HF: 42 ± 3 Hz). No difference was observed in the high frequency band between groups.The AT1 receptor protein expression was higher in the HF group (52%) compared with C group, however the increase in MAS receptor expression (18%) in the HF group was not significantly different from C group. In conclusion, the results suggest that the sympathetic hyperactivity and the overexpression of AT1 receptor could be involved in the genesis of the hypertension observed in young rats fed a high‐fat diet. Supported by Mackpesquisa.

Aldosterone Contributes to Sympathoexcitation in Renovascular Hypertension

Although angiotensin II (Ang II) is essential to the development of renovascular hypertension, aldosterone plays a role as well. Recent studies have demonstrated a cross-talk between Ang II type 1 and mineralocorticoid receptors in the brain and kidneys. However, the role of aldosterone in the autonomic and renal dysfunction of renovascular hypertension is not well understood. The current study evaluated whether aldosterone contributes to cardiovascular and renal dysfunction in the 2 kidney-1 clip (2K1C) model. Mean arterial pressure (MAP) and baroreceptor reflex for control of the heart rate were evaluated in 2K1C treated or not treated with spironolactone (200mg/kg/day, 7 days). Tonic and reflex control of renal sympathetic nerve activity (rSNA) were assessed in urethane-anaesthetized rats. Plasma renin activity (PRA), kidney renin protein expression, renal injury, and central AT1 receptor protein expression were assessed. Spiro reduced MAP (198±4 vs. 170±9mm Hg; P < 0.05), normalized rSNA (147±9 vs. 96±10 pps; P < 0.05), and increased renal baroreceptor reflex sensitivity in the 2K1C rats. Spiro reduced α-smooth muscle actin expression in the nonclipped kidney in the 2K1C group (5±0.6 vs. 1.1±0.2%; P < 0.05). There was no change in PRA; however, a decrease in renin protein expression in the nonclipped kidney was found in the 2K1C treated group (217±30 vs. 160±19%; P < 0.05). Spiro treatment decreased AT1 receptor in the central nervous system (CNS) only in 2K1C rats (138±10 vs. 84±12%; P < 0.05). Aldosterone contributes to autonomic dysfunction and intrarenal injury in 2K1C, these effects are mediated by the CNS.

Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells.

The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

Influence of aerobic training on the reduced vasoconstriction to angiotensin II in rats exposed to intrauterine growth restriction: possible role of oxidative stress and AT2 receptor of angiotensin II.

Intrauterine growth restriction (IUGR) is associated with impaired vascular function, which contributes to the increased incidence of chronic disease. The aim of this study was to investigate whether aerobic training improves AngII-induced vasoconstriction in IUGR rats. Moreover, we assess the role of superoxide dismutase (SOD) isoforms and NADPH oxidase-derived superoxide anions in this improvement. Female Wistar rats were randomly divided into two groups on day 1 of pregnancy. A control group was fed standard chow ad libitum, and a restricted group was fed 50% of the ad libitum intake throughout gestation. At 8 weeks of age, male offspring from both groups were randomly assigned to 4 experimental groups: sedentary control (SC), trained control (TC), sedentary restricted (SRT), and trained restricted (TRT). The training protocol was performed on a treadmill and consisted of a continuous 60-min session 5 days/week for 10 weeks. Following aerobic training, concentration–response curves to AngII were obtained in endothelium-intact aortic rings. Protein expression of SOD isoforms, AngII receptors and the NADPH oxidase component p47phox was assessed by Western blot analysis. The dihydroethidium was used to evaluate the in situ superoxide levels under basal conditions or in the presence of apocynin, losartan or PD 123,319. Our results indicate that aerobic training can prevent IUGR-associated increases in AngII-dependent vasoconstriction and can restore basal superoxide levels in the aortic rings of TRT rats. Moreover, we observed that aerobic training normalized the increased p47phox protein expression and increased MnSOD and AT2 receptor protein expression in thoracic aortas of SRT rats. In summary, aerobic training can result in an upregulation of antioxidant defense by improved of MnSOD expression and attenuation of NADPH oxidase component p47phox. These effects are accompanied by increased expression of AT2 receptor, which provide positive effects against Ang II–induced superoxide generation, resulting in attenuation of AngII-induced vasoconstriction.

Abstract MP03: Intracerebroventricular Administration Of Autologous Bone Marrow-derived Cells Attenuate Inflammation And Increase In AT1 Receptor In The Brain Of Angiotensin II Initiated Hypertensive Rats

Objectives: Bone marrow-derived cells (BMCs) include mesenchymal stem cells (MSC) play critical role in repairing damaged tissue and anti-inflammation. We reported that intracerebroventricular (icv) administration of autologous BMCs attenuate slow pressor Ang II initiated hypertension via attenuating Ang II mediated sympathetic activation in rats. However, mechanisms of this observation have not been elucidated. We hypothesized that BMCs in the brain would attenuate inflammation and modulate overactive renin angiotensin system (RAS) in the brain of Ang II initiated hypertensive rats. Therefore we examined characteristics of BMCs prepared with identical cultural condition to previous experiment and examined expression of inflammation related cytokines and RAS component in the brain of Ang II initiated hypertensive rats. Methods: Aspirated bone marrow cells underwent red blood cell lysis protocol were seeded in an individual dish in Dulbecco Modified Eagle Media with 10% fetal bovine serum. Floating cells were removed and plastic adherent cells were allowed to glow for 3 weeks at passage 3. The number of BMCs proliferated more than 10 6 were used for immunocytochemical, flowcytometric, BrdU uptake, and Western blot analysis. TGF-beta, IL-10 and AT1 receptor protein expression were examined in brains of Ang II initiated hypertensive rats. Results: Cultured BMCs expressed CD29, a marker of mesenchymal stem cell. Flowcytometric analysis revealed that 38% of BMCs were CD29+ CD34-. Endothelial cell (EC) proliferation assay exhibited 52% higher BrdU uptake with conditioned media from BMCs than the media from ECs. Expression of angiotensin converting enzyme (ACE) was 87% lower in BMCs than in ECs, whereas ACE2 expression was 142% higher in BMCs. Furthermore, Ang II infusion significantly increased expression of TGF-beta and AT1 receptor in the brain by 146% and 135% respectively and icv administration of BMCs attenuated the increases of both expressions (39%, 59% respectively). Ang II infusion with or without BMCs icv did not affect to IL-10 expression in the brain. Conclusion: BMCs in the brain may have protective role against Ang II induced sympathetic activation via attenuation of brain inflammation and increase in AT1 receptors.

Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II‐mediated BMP4‐oxidative stress cascade

Boldine is a potent natural antioxidant present in the leaves and bark of the Chilean boldo tree. Here we assessed the protective effects of boldine on endothelium in a range of models of diabetes, ex vivo and in vitro. Vascular reactivity was studied in mouse aortas from db/db diabetic and normal mice. Reactive oxygen species (ROS) production, angiotensin AT1 receptor localization and protein expression of oxidative stress markers in the vascular wall were evaluated by dihydroethidium fluorescence, lucigenin enhanced-chemiluminescence, immunohistochemistry and Western blot respectively. Primary cultures of mouse aortic endothelial cells, exposed to high concentrations of glucose (30 mmol L(-1) ) were also used. Oral treatment (20 mg kg(-1) day(-1) , 7 days) or incubation in vitro with boldine (1 μmol L(-1) , 12 h) enhanced endothelium-dependent aortic relaxations of db/db mice. Boldine reversed impaired relaxations induced by high glucose or angiotensin II (Ang II) in non-diabetic mouse aortas while it reduced the overproduction of ROS and increased phosphorylation of eNOS in db/db mouse aortas. Elevated expression of oxidative stress markers (bone morphogenic protein 4 (BMP4), nitrotyrosine and AT1 receptors) were reduced in boldine-treated db/db mouse aortas. Ang II-stimulated BMP4 expression was inhibited by boldine, tempol, noggin or losartan. Boldine inhibited high glucose-stimulated ROS production and restored the decreased phosphorylation of eNOS in mouse aortic endothelial cells in culture. Boldine reduced oxidative stress and improved endothelium-dependent relaxation in aortas of diabetic mice largely through inhibiting ROS overproduction associated with Ang II-mediated BMP4-dependent mechanisms.

GW24-e1171 Effect of different ARB-eluting stents on coronary intimal hyperplasia in vivo

ObjectivesInvestigate the difference of inhibiting intima proliferation between telmisartan, valsartan and sirolimus eluting stent.MethodsTelmisartan eluting stent (TES), valsartan eluting stent (VES), sirolimus eluting stent (SES) and polymer eluting stent (PES)...

Angiotensin II and angiotensin-(1-7) in paraventricular nucleus modulate cardiac sympathetic afferent reflex in renovascular hypertensive rats.

BackgroundThe enhanced cardiac sympathetic afferent reflex (CSAR) is involved in the sympathetic activation that contributes to the pathogenesis and progression of hypertension. Activation of AT1 receptors by angiotension (Ang) II in the paraventricular nucleus (PVN) augments the enhanced CSAR and sympathetic outflow in hypertension. The present study is designed to determine whether Ang-(1-7) in PVN plays the similar roles as Ang II and the interaction between Ang-(1-7) and Ang II on CSAR in renovascular hypertension.Methodology/Principal FindingsThe two-kidney, one-clip (2K1C) method was used to induce renovascular hypertension. The CSAR was evaluated by the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to epicardial application of capsaicin in sinoaortic-denervated and cervical-vagotomized rats with urethane and α-chloralose anesthesia. Either Ang II or Ang-(1-7) in PVN caused greater increases in RSNA and MAP, and enhancement in CSAR in 2K1C rats than in sham-operated (Sham) rats. Mas receptor antagonist A-779 and AT1 receptor antagonist losartan induced opposite effects to Ang-(1-7) or Ang II respectively in 2K1C rats, but losartan had no effects in Sham rats. Losartan but not the A-779 abolished the effects of Ang II, while A-779 but not the losartan blocked the effects of Ang-(1-7). PVN pretreatment with Ang-(1-7) dose-dependently augmented the RSNA, MAP, and CSAR responses to the Ang II in 2K1C rats. Ang II level, AT1 receptor and Mas receptor protein expression in PVN increased in 2K1C rats compared with Sham rats but Ang-(1-7) level did not.ConclusionsAng-(1-7) in PVN is as effective as Ang II in enhancing the CSAR and increasing sympathetic outflow and both endogenous Ang-(1-7) and Ang II in PVN contribute to the enhanced CSAR and sympathetic outflow in renovascular hypertension. Ang-(1-7) in PVN potentiates the effects of Ang II in renovascular hypertension.

Abstract 312: Activation of GPR30 Attenuates Cardiac Injury in the Ovariectomized Diabetic mRen2.Lewis Rat

Our previous study demonstrated that the exacerbation of hypertension as well as alterations in the angiotensin II type 1 receptor (AT1) and endothelial nitric oxide synthase (eNOS) pathways were associated with the development of cardiac remodeling in the diabetic, ovariectomized (OVX-D) mRen2.Lewis rats. The present study evaluated the cardiovascular effects of G-1, a selective agonist of the estrogen receptor GPR30, in insulin- and estrogen-depleted mRen.Lewis rats. Hemizygous littermates were divided into 3 groups: intact controls (C); OVX-D, and OVX-D treated with G1 (G-1). Ovariectomy was performed at 10 weeks of age and diabetes subsequently induced at 11 weeks with streptozotocin (65 mg/kg). G-1 was given by osmotic minipump implanted 2 days after STZ injection (0.2 mg/kg/day; ip). After four weeks blood pressure was measured and heart removed for analysis of AT1 receptor and eNOS protein expression as well as immunohistochemical identification of GPR30. Left ventricle (LV) wet weight was measured and cardiac tissue fibrosis was analyzed from the LV sections stained with picrosirius red. Immunohistochemical staining for GPR30 was evident in both coronary vessels and cardiac myocyte of the OVX-D rats. Chronic treatment with G1 decreased the systolic blood pressure (C: 142 ± 3; OVX-D: 184 ± 3*; G-1: 167 ± 6* # mmHg; *p&lt;0.05 vs C; # p&lt;0.05 vs OVX-D) and the incidence of microinfractions but did not reduce LV weight of OVX-D mRen2.Lewis rats (C: 0.57 ± 0.01; OVX-D: 0.62 ± 0.02*; G-1: 0.63 ± 0.01*g). G-1 treatment was associated with an increase in the cardiac expression of eNOS (C: 100 ± 6%; OVX-D: 59 ± 2%*; G-1: 86 ± 2% # ) but the agonist did not alter cardiac AT1 receptor expression (C: 100 ± 14%; OVX-D: 163 ± 11%*; G-1: 174 ± 9%*). We detected no change in uterine weight of the OVX-D rats treated with G-1 suggesting no crossover of the agonist with the steroid receptors ERα or ERβ (C: 0.68 ± 0.07; OVX-D: 0.14 ± 0.01*; G-1: 0.14 ± 0.01*g). In conclusion, the present study suggests that selective activation of a novel GPR30 receptor may attenuate cardiac injury in estrogen- and insulin-depleted mRen2.Lewis rats by reducing blood pressure and preserving eNOS pathway.

Downregulation of AT1 receptor in the RVLM induced by mesenchymal stem cells treatment prevent the hypertension and sympathetic hyperactivity in 2K‐1C Wistar rats

Current approaches to pharmacological treatment of renovascular hypertension are problematic and thus, alternative therapies are needed. We investigated the effects of mesenchymal stem cells (MSC) treatment on systolic arterial pressure (SAP), renal sympathetic nervous activity (rSNA) and AT1 receptor (AT1R) expression in the rostral ventrolateral medulla (RVLM) in an experimental model of renovascular hypertension, 2 Kidney‐1 clip (2K‐1C). Fluorescently tagged MSC (2×105cells/animal, IV) were injected weekly after the 3rd week of renal artery occlusion and the experiments were performed at the end of 6 weeks after clipping. The rats were divided in control group (n=5), control group treated with MSC (n=2), 2K‐1C (n=8) and 2K‐1C treated (n=7). Bilateral punches were taken from the RVLM to perform AT1R protein expression analyses by western blotting. Tracking assays by citometry showed MSC in the medulla oblongata. SAP in 2K‐1C‐treated rats was lower compared to untreated animals (2K‐1C, 224 ± 8 vs 173 ± 6 mmHg, p&lt;0,05) at 6th week followed by a significant decrease in rSNA (27%). MSC treatment reduced brainstem AT1R expression only 2K‐1C rats (28%, p&lt;0,05). Therefore, the results suggest MCS treatment can both prevent the increase in SAP as well as reduce the sympathetic hyperactivity and the expression of AT1R in the RVLM, constituting a future alternative for the treatment of this type of hypertension.Supported by CAPES

Antenatal maternal hypoxic stress: Adaptations of the placental renin-angiotensin system in the mouse

AbstractThe stress of Antenatal Maternal Hypoxia (AMH) can lead to a number of physiological and pathological changes in both mother and fetus, changes which can be linked to alterations in placental morphology and gene regulation. Recently, in the Brown Norway rat “model” of placental insufficiency, we reported alterations in placental renin-angiotensin system (RAS) genes. Moreover, AMH can lead to reduced oxygen availability to the fetus, similar to a state of placental insufficiency. Thus, in pregnant mice dams we tested the hypothesis that antenatal maternal hypoxic stress leads to alterations in the placental RAS. These alterations may, in part, account for the phenotypic changes in both pregnant mice dams as well as fetus and adult offspring. MethodsPregnant FVB/NJ mice dams were either maintained as controls, or exposed to 10.5% O2 for 48 h from 15.5 to 17.5 day post coitum. We then measured placental mRNA and protein expression of several RAS genes (n = 4 to 5; P < 0.05 was considered significant). ResultIn murine placenta: (1) angiotensinogen (AGT) mRNA was undetectable; however, AGT protein was detectable and increased significantly with AMH. (2) In AMH, although renin mRNA was reduced protein expression increased, in association with decreased microRNA (miRNA) 199b, which can lead to increased renin translation. (3) Also in AMH placenta, angiotensin converting enzyme (ACE) -1 mRNA was unaltered; however, protein expression increased significantly, in association with decreased miRNA 27a, which can result in increased ACE-1 translation. (4) In AMH placenta, ACE-2 mRNA was reduced significantly, whereas protein expression was significantly greater, in association with reduced miRNA 429. (5) In AMH placenta, angiotensin II type (AT) -1a receptor mRNA expression was unaltered while AT-1b receptor mRNA was undetectable in both groups. Moreover, AT-1 receptor protein expression was unchanged in response to AMH. (6) AT-2 receptor mRNA and proteins were undetectable in both groups. ConclusionThe normal murine placenta possesses several components of RAS, and in response to AMH several of these elements undergo important changes. In addition, differential expression of RAS mRNA, miRNA and protein, indicate post-transcriptional regulatory mechanisms involved with hypoxic stress, and necessitate further investigation.