AT1 Receptor Expression Research Articles

Change of concept about the regulation of angiotensin II-induced monocyte chemoattractant protein-1 production in human endothelial cells

AimsSome intriguing clinical observations about the anti-inflammatory effects of angiotensin type 1 (AT1) receptor blockers and angiotensin converting enzyme inhibitors in cardiovascular patients brought us to study the signalling pathways which lead to angiotensin II (ANG)-induced monocyte chemoattractant protein-1 (MCP-1) production in human endothelial cells. MethodsMCP-1 production in human umbilical vein endothelial cells (HUVECs) under treatments with ANG, AT1 and angiotensin type 2 (AT2) receptor blockers and pravastatin was measured by ELISA. The expression of AT1 and AT2 receptors and NADPH oxidase catalytic subunits (NOX 1–5) was analysed at mRNA and protein levels. Nuclear factor-kappa B (NF-κB) activation was studied by p65 subunit translocation to the cellular nucleus. Cell viability was tested by the MTT method. Nox4 subcellular distribution was analysed by subcellular protein fractionation and by immunoprecipitation followed by matrix-assisted laser desorption/ionization mass spectrometry analysis. ResultsANG-induced MCP-1 production was mediated by AT2 receptor, but not AT1 receptor in HUVECs in culture, which in turn activated NF-κB, promoting p65 subunit translocation to the nucleus. Reactive oxygen species produced by NADPH oxidase participated in this activation, mainly by the Nox4 subunit, ubiquitously expressed in all the compartments of HUVECs. Pravastatin inhibited ANG-induced MCP-1 production. ConclusionsOur results support that ANG-induced MCP-1 production in HUVECs is mediated by AT2 instead AT1 receptor activation, which in turn activates NF-κB involving reactive oxygen species produced by the NADPH oxidase complex. Statins can also block ANG-induced MCP-1 production, probably by their inhibitory effects on NADPH oxidase activity.

Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner.

Angiotensin Receptor Blockade Modulates NFκB and STAT3 Signaling and Inhibits Glial Activation and Neuroinflammation Better than Angiotensin-Converting Enzyme Inhibition.

Neuroinflammation, sustained by astroglial and microglial activation, is the preceding event in neurodegeneration. Various clinical reports showed better neuroprotection by AT1 receptor blockade (ARB) than angiotensin-converting enzyme inhibition (ACEi), but experimental evidences and associated mechanism for this observation are lacking. Therefore, we investigated the effect of ARB, using Candesartan, and ACEi, using Perindopril, in equimolar concentrations in astroglial (C6) and microglial (BV2) cells employing lipopolysaccharide (LPS) to induce neuroinflammation. Further, Candesartan (0.1mg/kg) and Perindopril (0.1mg/kg) were orally administered in male SD rats for five consecutive days, and on the fifth day, rats were challenged with LPS (i.p.; 250μg/kg) and sacrificed after 24h. LPS-induced neuroinflammation (increased astroglial and microglial activation, IκBα degradation, NFкB nuclear translocation, STAT3 activation, and TNF-α release) was more efficiently prevented by Candesartan (even at lower concentration of 1nM) than by Perindopril (1μM) in both the cell types and in rat model of neuroinflammation. In addition, increased AT1 receptor (AT1R) and decreased AT2 receptor (AT2R) expression was observed in LPS-induced neuroinflammation in both in vitro and in vivo studies. Candesartan, as compared to Perindopril, increased the expression of AT2R in both the experimental conditions. Interestingly, concomitant blockade of AT2R by PD123319 significantly reversed the beneficial effects of Candesartan in both the cell types and in rat model of neuroinflammation. Finally, our data emphasize that superiority of Candesartan as compared to Perindopril is due to better activation of AT2R which results in PP2A activation, IκBα stabilization, and suppression of NFкB and STAT3 inflammatory signaling.

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats.

BackgroundAngiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.MethodsTo examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.ResultsAngiotensin II (Ang II) and angiotensin type 1 receptor (AT1R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.ConclusionAng-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT1R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.

Abstract 12265: Normalization of Renal AT1 Receptor Expression is Observed With Enalapril But Not Diltiazem in a Rat Model of Chronic Kidney Disease

Introduction: Use of ACE inhibitors (ACEi) improves outcomes in patients with proteinuric chronic kidney disease (CKD) and is considered first line of treatment in recent guidelines. In addition to renin angiotensin (Ang) system blockers, other antihypertensive agents such as calcium channel blockers (CCBs) achieve similar therapeutic effectiveness in renoprotection. Our objective was to determine the impact of antihypertensive therapy on renal Ang type 1 receptor (AT 1 R) in 5/6 subtotal nephrectomy (Nx) rat model of CKD using PET imaging. Methods: Ten weeks after Nx, Sprague-Dawley rats (n=6-8/group) were administered 10mg/kg/d enalapril (NxE), 30 mg/kg/d diltiazem (NxD) or left untreated (Nx) for an additional ∼10 weeks. Systolic blood pressure (SBP), plasma creatinine, urine albumin, plasma and tissue Ang II were measured at the end. AT 1 R was assessed using in vivo [ 18 F]FPyKYNE-losartan PET and in vitro autoradiography. Heart function was evaluated with echocardiography. Results: Compared to shams, Nx rats exhibited higher SBP that was reduced by both enalapril and diltiazem (142.7±2, 188.8±17.3, 152±8.7 and 145.8±5.8 mm Hg in sham, Nx, NxE and NxD, respectively, p <0.05). At ∼20 weeks, plasma creatinine and albuminuria were significantly increased in Nx rats, while no changes occurred in NxE rats compared to shams. By contrast, NxD rats demonstrated further increases in these parameters, compared to Nx alone ( p <0.05). Plasma and kidney Ang II were raised in Nx rats, lowered by enalapril and increased significantly in NxD rats ( p <0.05). PET kidney AT 1 R level decreased in Nx rats compared to shams and only enalapril treatment normalized AT 1 R expression (2.8±0.5, 2±0.3, 2.9±0.6, 1.9±0.5 ml/cm 3 in sham, Nx, NxE and NxD respectively, p <0.05) and was confirmed by in vitro results. Enhanced echo LV mass in Nx rats was not reversed by enalapril but was further increased with diltiazem ( p <0.001). Conclusions: In rats with CKD, delayed treatment with the ACEi enalapril normalized AT 1 R expression and reduced progression of the disease, whereas the CCB diltiazem exacerbated renal and cardiac dysfunctions. [ 18 F]FPyKYNE-Losartan PET allows longitudinal determination of AT 1 R abnormalities with progression of cardiorenal failure and monitoring of therapy.

Vinegar decreases blood pressure by down-regulating AT1R expression via the AMPK/PGC-1α/PPARγ pathway in spontaneously hypertensive rats.

Vinegar has been reported to lower blood pressure, but its mechanism is unclear. This study explored whether vinegar plays antihypertensive effect by activating AMP-activated protein kinase (AMPK) pathway. Male spontaneously hypertensive rats (SHRs) were assigned to vinegar, acetic acid, nifedipine, nifedipine + vinegar, or distilled water by oral gavage for 8 weeks. Blood and aortas were analyzed for biochemical indices and protein expression levels. Sv40-transformed aortic rat endothelia cell line (SVAREC) cells were treated with acetate at different doses for 24 h; protein expression levels were assessed. Vinegar and acetic acid decreased blood pressure in SHRs on weeks 6 and 8, and nifedipine + vinegar had a better effect on blood pressure control than vinegar or nifedipine alone. Vinegar and acetic acid could decrease serum renin and angiotensin-converting enzyme (ACE) activities, angiotensin II and aldosterone concentrations in SHRs. Vinegar and acetic acid also increased AMP/ATP ratios and expression levels of pAMPK, PPARγ coactivator-1α (PGC-1α), and PPARγ while inhibited angiotensin II type 1 receptor (AT1R) expression in SHRs. The changes in these protein expressions were also found in SVAREC cells treated with 200 or 400 μmol/L acetate. In the presence of AMPK inhibitor or PGC-1α small interfering RNA, the effects of acetate on their downstream protein expression in SVAREC cells were abolished, respectively. Vinegar activates AMPK by increasing AMP/ATP ratios, thereby increases PGC-1α and PPARγ expressions, and inhibits AT1R expression in SHRs. Acetic acid is responsible for the antihypertensive effects of vinegar. There is a joint effect between vinegar and nifedipine in blood pressure control.

Angiotensin II stimulation alters vasomotor response to adenosine in mouse mesenteric artery: role for A1 and A2B adenosine receptors.

Stimulation of the A1 adenosine receptor and angiotensin II receptor type-1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non-selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs). We used a Danish Myo Technology wire myograph to measure muscle tension in isolated MAs from wild type (WT), A1 receptor and A2B receptor knockout (KO) mice. Western blots were performed to determine the expression of AT1 receptors and CYP4A. Acute exposure (15 min) to angiotensin II attenuated the NECA-dependent vasodilatation and enhanced vasoconstriction. This vasoconstrictor effect of angiotensin II in NECA-treated MAs was abolished in A1 receptor KO mice and in WT mice treated with the A1 receptor antagonist DPCPX, CYP4A inhibitor HET0016 and ERK1/2 inhibitor PD98059. In MAs from A2B receptor KO mice, the vasoconstrictor effect of angiotensin II on the NECA-induced response was shown to be dependent on A1 receptors. Furthermore, in A2B receptor KO mice, the expression of AT1 receptors and CYP4A was increased and the angiotensin II-induced vasoconstriction enhanced. In addition, inhibition of KATP channels with glibenclamide significantly reduced NECA-induced vasodilatation in WT mice. Acute angiotensin II stimulation enhanced A1 receptor-dependent vasoconstriction and inhibited A2B receptor-dependent vasodilatation, leading to a net vasoconstriction and altered vasomotor response to NECA in MAs. This interaction may be important in the regulation of BP.

GW26-e3962 Activation of D4 Dopamine Receptor Decreases AT1 Angiotensin II 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 AT1 receptor expression. We hypothesize that D4 receptor can inhibit AT1 receptor expression and

Regulation of cardiac miR-208a, an inducer of obesity, by rapamycin and nebivolol.

ObjectiveResistance to obesity is observed in rodents and humans treated with Rapamycin (Rap) or Nebivolol (Neb). Since cardiac miR-208a promotes obesity, we tested whether the modes of actions of Rap and Neb involve inhibition of miR-208a.MethodsMouse cardiomyocyte HL-1 cells and Zucker obese (ZO) rats were used to investigate regulation of cardiac miR-208a.ResultsAngiotensin II (Ang II) increased miR-208a expression in HL-1 cells. Pre-treatment with an AT1 receptor (AT1R) antagonist, losartan (1µM), antagonized this effect, whereas a phospholipase C inhibitor, U73122 (10µM) and an NADPH oxidase inhibitor, apocynin (0.5mM) did not. Ang II-induced increase in miR-208a was suppressed by Rap (10nM), an inhibitor of nutrient sensor kinase mTORC1, and Neb (1µM), a 3rd generation β-blocker that suppressed bioavailable AT1R binding of 125I-Ang II. Thus, suppression of AT1R expression by Neb, inhibition of AT1R activation by losartan, and inhibition of AT1R-induced activation of mTORC1 by Rap attenuated the Ang II-induced increase in miR-208a. In ZO rats, Rap treatment (750µg/kg/day; 12 weeks) reduced obesity despite similar food intake, suppressed cardiac miR-208a, and increased cardiac MED13, a suppresser of obesity.ConclusionRap and Neb suppress cardiac miR-208a. MiR-208a suppression and increase in MED13 correlated with attenuated weight gain despite leptin resistance.

Early Training-Induced Reduction of Angiotensinogen in Autonomic Areas-The Main Effect of Exercise on Brain Renin-Angiotensin System in Hypertensive Rats.

BackgroundExercise training (T) blunts functional deficits and renin-angiotensin system (RAS) hyperactivity in hypertensive individuals. There is no information on T-induced temporal changes of brain RAS. We evaluate now the simultaneous effects of T on functional responses and time course changes in the expression/activity of brain RAS components in autonomic cardiovascular-controlling areas.Methods and ResultsSpontaneously hypertensive rats (SHR) and age-matched normotensive controls (WKY) were trained for 0, 1, 2, 4, 8 and 12 weeks. Sedentary (S) groups served as time-controls. After arterial pressure (AP) and heart rate (HR) recordings at rest, fresh and fixed brains were harvested for qPCR and immunofluorescence assays. SHR-S vs. WKY-S exhibited higher mean AP (MAP) and HR, increased pressure variability and sympathetic activity, elevated AT1 receptor (AT1) expression in nucleus tractus solitarii (NTS) and higher Mas receptor expression in the rostroventrolateral medulla (RVLM). In SHR, T promptly (T2 on) reduced sympathetic variability to heart/vessels and largely decreased angiotensinogen expression in the paraventricular hypothalamic nucleus (PVN) and NTS, with a late RVLM reduction (T4). AT1 expression was only reduced at T12 (PVN and NTS) with transient, not maintained Mas receptor changes in PVN and RVLM. These responses were accompanied by baseline MAP and HR reduction in the SHR-T (from T4 on). In the SHR group, PVN angiotensinogen expression correlated positively with sympathetic activity, resting MAP and HR. In WKY-T, a precocious (T2-T12) RVLM AT1 decrease preceded the appearance of resting bradycardia (from T8 on).ConclusionsEarly and maintained reduction of angiotensinogen content in autonomic areas of the SHR is the most prominent effect of training on brain RAS. Down-regulation of PVN RAS expression is an essential factor to drive cardiovascular benefits in SHR-T, while resting bradycardia in WKY-T is correlated to RVLM AT1 reduction.

Reciprocal regulation between sirtuin-1 and angiotensin-II in the substantia nigra: implications for aging and neurodegeneration.

Local angiotensin II (AII) and sirtuin 1 (SIRT1) play a major role in the modulation of neuroinflammation, oxidative stress and aging-related dopaminergic vulnerability to damage. However, it is not known whether the modulation is related to reciprocal regulation between SIRT1 and AII. In the present study, a single intraventricular injection of AII increased nigral SIRT1 levels in young adult rats. Although AII activity is known to be increased in aged rats, levels of SIRT1 were significantly lower than in young controls. Treatment with the SIRT1-activating compound resveratrol increased nigral SIRT1 levels in aged rats. Levels of SIRT1 were significantly higher in aged wild type mice than in AII type-1 receptor (AT1) deficient mice. In cell culture studies, treatment with AII also induced a transitory increase in levels of SIRT1 in the MES 23.5 dopaminergic neuron and the N9 microglial cell lines. In aged rats, treatment with resveratrol induced a significant decrease in the expression of AT1 receptors and markers of NADPH-oxidase activation (p47phox). In aged transgenic mice over-expressing SIRT1, levels of AT1 and p47 phox were lower than in aged wild type controls. In vitro, the inhibitory effects of resveratrol on AII/AT1/NADPH-oxidase activity were confirmed in primary mesencephalic cultures, the N9 microglial cell line, and the dopaminergic neuron cell line MES 23.5, and they were blocked by the SIRT1 specific inhibitor EX527. The present findings show that SIRT1 and the axis AII/AT1/NADPH-oxidase regulate each other. This is impaired in aged animals and may be mitigated with sirtuin-activating compounds.

Abstract P636: Sex Differences in Cardioprotective AT2R Expression in Diabetic Rats and Its Correlation with Myocardial Damage

Diabetes mellitus (DM) is an independent risk factor for cardiovascular disease (CVD). Healthy, young women are protected from CVD, while diabetic women are more susceptible to CVD compared to age-matched diabetic men and non-diabetic women. Underlying mechanisms for this sex difference in CVD are not fully elucidated. The angiotensin II type 2 receptor (AT2R) is a member of the protective, vasodilative arm of the renin angiotensin system. The Agtr2 gene that codes for AT2R is X-linked, and increased Agtr2 expression is reported in female vasculature of rodent models. We hypothesized that a sex difference might exist in DM-associated regulation of cardiac AT2R expression. To test this, we used hyperglycemic, male and female Zucker diabetic fatty (ZDF) rats and age- and sex-matched normoglycemic Zucker lean (ZL) rats. The male ZDF (ZDF-M) rat is an established model of type 2 DM. We have reported previously that hyperglycemic, female ZDF (ZDF-F) rats had the highest body fat and lowest lean muscle mass compared to male and female lean rats (ZL-M, ZL-F) and ZDF-M. Cardiac Agtr2 expression was measured by qRT-PCR at 5-months, cardiac function by echocardiography was compared at 3- and 5-months, and histopathology of cardiac tissue was assessed at 5-months. ZL-F had a nearly 2-fold increase of Agtr2 compared to ZL-M (p<0.01). Relative to lean controls, ZDF-M had no significant change in Agtr2, while ZDF-F exhibited ~60% suppression (Rq=0.42) of Agtr2 (p<0.001). Echocardiography data revealed evidence of compensated systolic function in all groups since fractional shortening was >50% at both ages, while heart rate and stroke volume were similar. However, diastolic dysfunction was observed in both ZDF-F and ZDF-M, relative to their lean counterparts, due to increased isovolumic relaxation time and decreased early:late ventricular filling ratio (E/A). ZDF-F exhibited the highest cardiomyocyte hypertrophy (≥35% over ZL-F, ZL-M and ZDF-M). Both ZDF and ZDF-M showed mitochondrial clustering and disrupted spatial orientation of mitochondria relative to the sarcomere (assessed by TEM). Based on our results, we propose that myocardial remodeling, diastolic dysfunction and loss of cardioprotective AT2R may underlie greater susceptibility of diabetic females to CVD.

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.

Abstract P102: Type I Diabetes Mellitus is More Sensitive to Ang II Due to Increased AT1 Receptor Expression in Afferent Arteriole

The prevalence of hypertension is about twice in diabetic subjects than that in non-diabetics. In the presence of hypertension, the progress of diabetic nephropathy has been shown to be more severe and rapid. But the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that angiotensin II receptor type 1 (AT1 receptor) expression level in renal afferent arteriole is elevated, which enhances the responses to angiotensin II (Ang II) stimulation and contributes to the development of hypertension in diabetes. First, we measured mean arterial pressure (MAP) with telemetry in diabetic and non-diabetic mice with Ang II infusion for 8 weeks. MAP was 26.7 mmHg higher in diabetic mice (157.4±11.3 mmHg) than in non-diabetic mice (130.7±6.4 mmHg). Next, we induced 2-kidney-1-clip Goldblatt hypertension in diabetic mice and non-diabetic mice and measured MAP with telemetry for 8 weeks. MAP was 17.6 mmHg higher in diabetic mice (151.1±9.5 mmHg) than in non-diabetic mice (133.5±4.2 mmHg), indicating blood pressure of diabetic mice is also more responsive to both exogenous and endogenous Ang II than no-diabetic mice. To investigate the mechanisms of the enhanced sensitivity to Ang II, we dissected afferent arterioles from non-diabetic mice and diabetic mice and measured the expression of AT1 receptor mRNA by real-time PCR. AT1 receptor mRNA level was 6 times higher in diabetic mice that in non-diabetic control. AT1 receptor protein abundance in renal cortex was also 5 times higher in diabetic mice than controls. In conclusion, the blood pressure of the diabetic mice are more sensitive to Ang II than non-diabetic controls. Increased AT1 receptor expression in the afferent arterioles may contribute to the development of hypertension in diabetes.

Changes in protein and gene expression of angiotensin II receptors (AT1 and AT2) in aorta of diabetic and hypertensive rats

Diabetes and hypertension have been associated with cardiovascular diseases and stroke. Some reports have related the coexistence of hypertension and diabetes with increase in the risk of developing vascular complications. Recently some studies have shown results suggesting that in the early stages of diabetes and hypertension exist a reduced functional response to vasopressor agents like angiotensin II (Ang II), which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of AT1 and AT2 receptors in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes, the protein expression was obtained by western blot and the mRNA expression by RT-PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1 receptors than normotensive rats while the AT2 expression remained unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion, our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT1 and AT2 receptors. However, the overexpression of AT2 could be associated with the reduction in the response to Ang II in the early stage of diabetes.

Age‐related changes in bladder function with altered angiotensin II receptor mechanisms in rats

To examine alterations in expression of angiotensin II type 1 receptors (AT1R) which induce organ tissue remodeling, angiotensin II type 2 receptors (AT2R) which protect against it, and related molecules in the bladder of matured rats with bladder dysfunction. Female SD rats of three different ages were used: 8 weeks old (8W; n = 5), 9 months old (9M; n = 5), and 15 months old (15M; n = 5). After cystometry, the expression levels of AT1R, connexin43 (Cx43), MAP kinase (MAPK), collagen1, AT2R, PPAR-γ, adiponectin (Adipo), and adiponectin receptor (Adipo-R) were investigated in the bladder. Pressure threshold, post-void residual volume and the number of non-voiding contractions were significantly increased in 15M versus 8W rats (P < 0.01). Maximum voiding pressure was significantly decreased in 15M versus 8W rats (P < 0.05). There was no significant difference in CMG parameters between 8W and 9M rats. In the bladder, the mRNA expression of AT1R, Cx43, MAPK, collagen 1, AT2R, PPAR-γ, Adipo, and Adipo-R were significantly higher in 15M than in 8W rats. The relative expression ratio of AT1R protein against AT2R protein in the mucosa and detrusor was significantly increased in 15M versus 8W rats. These results indicate that matured rats exhibit not only bladder overactivity but also impaired voiding, which are associated with upregulation of AT1R. The upregulation of AT2R also may play a significant role in the suppressing of AT1R induced remodelling. However, because AT1R upregulation is more dominant than AT2R increases, AT2R activation may not be sufficient to suppress AT1R stimulation in matured rats. Neurourol. Urodynam. 35:908-913, 2016. © 2015 Wiley Periodicals, Inc.

Chronic Losartan Treatment Up-Regulates AT1R and Increases the Heart Vulnerability to Acute Onset of Ischemia and Reperfusion Injury in Male Rats.

Inhibition of angiotensin II type 1 receptor (AT1R) is an important therapy in the management of hypertension, particularly in the immediate post-myocardial infarction period. Yet, the role of AT1R in the acute onset of myocardial ischemia and reperfusion injury still remains controversial. Thus, the present study determined the effects of chronic losartan treatment on heart ischemia and reperfusion injury in rats. Losartan (10 mg/kg/day) was administered to six-month-old male rats via an osmotic pump for 14 days and hearts were then isolated and were subjected to ischemia and reperfusion injury in a Langendorff preparation. Losartan significantly decreased mean arterial blood pressure. However, heart weight, left ventricle to body weight ratio and baseline cardiac function were not significantly altered by the losartan treatment. Of interest, chronic in vivo losartan treatment significantly increased ischemia-induced myocardial injury and decreased post-ischemic recovery of left ventricular function. This was associated with significant increases in AT1R and PKCδ expression in the left ventricle. In contrast, AT2R and PKCε were not altered. Furthermore, losartan treatment significantly increased microRNA (miR)-1, -15b, -92a, -133a, -133b, -210, and -499 expression but decreased miR-21 in the left ventricle. Of importance, addition of losartan to isolated heart preparations blocked the effect of increased ischemic-injury induced by in vivo chronic losartan treatment. The results demonstrate that chronic losartan treatment up-regulates AT1R/PKCδ and alters miR expression patterns in the heart, leading to increased cardiac vulnerability to ischemia and reperfusion injury.

Selective C1 Lesioning Slightly Decreases Angiotensin II Type I Receptor Expression in the Rat Rostral Ventrolateral Medulla (RVLM)

Cardiovascular homeostasis is regulated in large part by the rostral ventrolateral medulla (RVLM) in mammals. Projections from the RVLM to the intermediolateral column of the thoracolumbar spinal cord innervate preganglionic neurons of the sympathetic nervous system causing elevation of blood pressure and heart rate. A large proportion, but not all, of the neurons in the RVLM contain the enzymes necessary for the production of epinephrine and are identified as the C1 cell group. Angiotensin II (Ang II) activates the RVLM acting upon AT1 receptors. To assess the proportion of AT1 receptors that are located on C1 neurons in the rat RVLM this study employed an antibody to dopamine-beta-hydroxylase conjugated to saporin, to selectively destroy C1 neurons in the RVLM. Expression of tyrosine hydroxylase immunoreactive neurons in the RVLM was reduced by 57 % in the toxin injected RVLM compared to the contralateral RVLM. In contrast, densitometric analysis of autoradiographic images of (125)I-sarcosine(1), isoleucine(8) Ang II binding to AT1 receptors of the injected side RVLM revealed a small (10 %) reduction in AT1-receptor expression compared to the contralateral RVLM. These results suggest that the majority of AT1 receptors in the rat RVLM are located on non-C1 neurons or glia.

O19. Decreased plasma hemopexin activity in preeclampsia is associated with decreased plasma AT-1 receptor levels and increased placental and monocyte AT-1 receptor expression

Introduction Hemopexin (Hx), apart from being a free heme scavenger, also has protease activity. While during healthy pregnancy, Hx activity was increased as compared with non-pregnant individuals, in preeclampsia, Hx activity was decreased as compared with healthy pregnant women. Since we have previously shown that active Hx was able to shed the angiotensin II type 1 receptor (AT-1R) from various cell types in vitro , we now hypothesized that the increased Hx activity in pregnancy leads to increased shedding of the AT-1R from the vascular wall and subsequently decreased angiotensin II sensitivity. In preeclampsia the decreased Hx activity may lead to decreased shedding of the AT-1R and therefore increased angiotensin II activity. Objective We aimed to evaluate whether decreased Hx activity in preeclampsia was associated with increased AT-1R expression (on placental tissue and monocytes) and reduced presence of the AT-1R in the circulation. Methods We obtained peripheral blood samples from non pregnant ( n =21), healthy pregnant ( n =25) and (early onset) preeclamptic ( n =26) women. We measured plasma Hx activity (colorimetric assay) and plasma AT-1R levels (Western blotting) and monocyte AT-1R expression (flow cytometry). From another group of healthy pregnant ( n =15) and (early onset) preeclamptic women ( n =24), we collected placental biopsies, in which we measured placental AT-1R expression using immunohistochemistry and qPCR. Results The increase in plasma Hx activity in healthy pregnant women vs non-pregnant women ( p p p p p p p Conclusions Our data suggest that also in vivo active Hx sheds the AT-1R from tissues, such as monocytes and placenta. Therefore active Hx may play a role in the development of the decreased responsiveness to angiotensin II in healthy pregnancy, while the decreased Hx activity in preeclampsia may contribute to increased angiotensin II sensitivity in this disease.

Arsenic causes aortic dysfunction and systemic hypertension in rats: Augmentation of angiotensin II signaling

The groundwater pollutant arsenic can cause various cardiovascular disorders. Angiotensin II, a potent vasoconstrictor, plays an important role in vascular dysfunction by promoting changes in endothelial function, vascular reactivity, tissue remodeling and oxidative stress. We investigated whether modulation of angiotensin II signaling and redox homeostasis could be a mechanism contributing to arsenic-induced vascular disorder. Rats were exposed to arsenic at 25, 50 and 100ppm of sodium arsenite through drinking water consecutively for 90days. Blood pressure was recorded weekly. On the 91st day, the rats were sacrificed for blood collection and isolation of thoracic aorta. Angiotensin converting enzyme and angiotensin II levels were assessed in plasma. Aortic reactivity to angiotensin II was assessed in organ-bath system. Western blot of AT1 receptors and G protein (Gαq/11), ELISA of signal transducers of MAP kinase pathway and reactive oxygen species (ROS) generation were assessed in aorta. Arsenic caused concentration-dependent increase in systolic, diastolic and mean arterial blood pressure from the 10th, 8th and 7th week onwards, respectively. Arsenic caused concentration-dependent enhancement of the angiotensin II-induced aortic contractile response. Arsenic also caused concentration-dependent increase in the plasma levels of angiotensin II and angiotensin converting enzyme and the expression of aortic AT1 receptor and Gαq/11 proteins. Arsenic increased aortic protein kinase C activity and the concentrations of protein tyrosine kinase, extracellular signal-regulated kinase-1/2 and vascular endothelial growth factor. Further, arsenic increased aortic mRNA expression of Nox2, Nox4 and p22phox, NADPH oxidase activity and ROS generation. The results suggest that arsenic-mediated enhancement of angiotensin II signaling could be an important mechanism in the arsenic-induced vascular disorder, where ROS could augment the angiotensin II signaling through activation of MAP kinase pathway.