Angiotensin II Type 1 Receptor Blocker Losartan Research Articles

Losartan impairs HTR-8/SVneo trophoblast migration through inhibition of angiotensin II-induced pro-inflammatory profile in human endometrial stromal cells

A deep interaction between the endometrium and the invading trophoblast occurs during implantation in humans, with the acquisition of uterine receptivity to the invading embryo promoted by an elevation of pro-inflammatory cytokines in the endometrium, and the invasiveness of decidualizing endometrial stromal cells, augmented by trophoblast-derived signals. Considering that usage of angiotensin II type 1 (AT1) receptor blockers, among other renin-angiotensin system (RAS) antagonists, is associated with adverse pregnancy outcomes, here we aim to analyse the involvement of AT1 receptor in the reciprocal dialogue occurring between endometrial stroma and trophoblast cells. In human endometrial stromal cells (T-HESC) pre-incubated with a decidualization cocktail, angiotensin (Ang) II increased protein expression of prolactin and FOXO1, markers of endometrial decidualization, while promoting nuclear translocation of FOXO1. In addition, Ang II treatment increased CXCL8, and matrix metalloprotease (MMP)-2 levels in T-HESC. Incubation with the AT1 receptor blocker losartan or with an NFAT signalling inhibitor, decreased Ang II-induced secretion of prolactin, CXCL8, and MMP-2 in T-HESC. In a wound healing assay, conditioned medium (CM) obtained from Ang II-treated T-HESC, but not CM from losartan-pre-incubated T-HESC, increased migration of HTR-8/SVneo trophoblasts, effect that was inhibited in the presence of a CXCL8-neutralizing antibody. An increased secretion of CXCL8 and MMP-2 was observed after treatment of T-HESC with CM obtained from HTR-8/SVneo cells, which was not observed in T-HESC pre-incubated with losartan or with the NFAT inhibitor. This study evidenced a reciprocal RAS-coded messaging between trophoblast and ESC which is affected by the AT1 receptor blocker losartan.

FC 016SPARSENTAN IMPROVES GLOMERULAR BLOOD FLOW AND AUGMENTS PROTECTIVE TISSUE REMODELING IN MOUSE MODELS OF FOCAL SEGMENTAL GLOMERULOSCLEROSIS (FSGS)

Abstract Background and Aims Preliminary preclinical and emerging clinical evidence indicates strong antiproteinuric actions of dual endothelin type A (ETA) and angiotensin II type 1 (AT1) receptor antagonism with sparsentan. These nephroprotective effects have been more pronounced in different experimental and clinical settings compared to current standard of care using an AT1 receptor blocker (ARB). Considering the broad spectrum of renal actions of endothelin (ET) and angiotensin II (Ang II), inhibition of both pathways using sparsentan is postulated to target multiple renal cell types via a variety of renoprotective mechanisms. The aim of this study was to determine glomerular action of sparsentan as compared to the ARB losartan (Los) by direct visualization of effects on renal hemodynamics and tissue remodeling in the intact living kidney. Method Intravital multiphoton microscopy (MPM) of the glomerular vasculature and filtration barrier structure and function was performed in genetically engineered mice combined with traditional urinalysis and histology-based phenotyping. Glomerular hemodynamic parameters (afferent and efferent arteriole (AA and EA) diameters and single nephron glomerular filtration rate (SNGFR)) and podocyte calcium entry, as a measure of cell injury, were quantitatively visualized in the FSGS model Pod-GCaMP5/Tomato TRPC6 transgenic mice (1.5 years of age), in which TRPC6 is overexpressed together with the calcium reporter GCaMP5 in podocytes. Single cell identification and fate tracking of cells of the renin lineage (CoRL) was performed over time using a second physiologic control mouse model, Ren1d-Confetti mice that feature a multicolor CFP/GFP/YFP/FP reporter. Three groups of mice in each model received treatment with either vehicle (CTRL), the ARB losartan (Los; 10 mg/kg/day), or sparsentan (120 mg/kg/day) for 6 weeks (FSGS model) or 2 weeks (control physiology model). Results Both Los and sparsentan treatment attenuated the acute ET + Ang II-induced elevation of podocyte calcium by ∼80%, and the development of albuminuria, and glomerulosclerosis and tissue fibrosis in the FSGS model. Notably, sparsentan prevented the ET + Ang II increases in podocyte calcium more than Los and was significantly more effective in dilating both AA and EA (Fig. 1A, B), increasing SNGFR (Fig. 1C), increasing capillary blood flow (2-fold; p<0.0001 vs. CTRL), and decreasing albuminuria (20%; p<0.05 vs. CTRL). Sparsentan also preserved p57+ podocyte number by 50% compared to Los (p<0.0001 vs. Los). Similarly, pretreatment with sparsentan was more effective in preventing glomerular arteriolar vasoconstriction induced by acute ET + Ang II iv injection compared to Los (p<0.05 vs. Los). Following a 2-week treatment in control healthy Ren1d-Confetti mice, sparsentan resulted in a more robust increase compared to Los in the number of Confetti+ cells, clones, and individual cells per clone in the glomeruli and AA (Fig. 1D-F). Renal tubule segments also showed active cellular remodeling in response to sparsentan. Conclusion Serial MPM imaging directly visualized several mechanisms underlying beneficial antiproteinuric and structural effects of sparsentan in both FSGS and in the normal mouse kidney and differences between dual ETA/AT1 receptor antagonism of sparsentan and a mono-selective ARB. The sparsentan-induced glomerular hemodynamic pattern was driven by both AA and EA dilation resulting in an increase in capillary blood flow. Compared to Los, sparsentan was more effective in attenuating ET/Ang II-induced podocyte injury and in activation of resident progenitor cells and tissue remodeling. These findings suggest multiple layers of renal protective actions by dual ETA and AT1 receptor antagonism.

Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg−1 day−1) (CONL), 7 days of tail‐traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading‐induced loss of muscle fiber cross‐sectional area (CSA) and fiber‐type shift. Losartan mitigated unloading‐induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

Mechanical activation of the angiotensin II type 1 receptor contributes to abdominal aortic aneurysm formation

ObjectiveThe angiotensin II type 1 receptor (AT1R) can be activated under conditions of mechanical stretch in some cellular systems. Whether this activity influences signaling within the abdominal aorta to promote to abdominal aortic aneurysm (AAA) development remains unknown. We evaluated the hypothesis that mechanical AT1R activation can occur under conditions of hypertension (HTN) and contribute to AAA formation. MethodsBPH/2 mice, which demonstrate spontaneous neurogenic, low-renin HTN, and normotensive BPN/3 mice underwent AAA induction via the calcium chloride model, with or without an osmotic minipump delivering 30 mg/kg/d of the AT1R blocker Losartan. Systolic blood pressure (SBP) was measured at baseline and weekly via a tail cuff. The aortic diameter (AoD) was measured at baseline and terminal surgery at 21 days by digital microscopy. Aortic tissue was harvested for immunoblotting (phosphorylated extracellular signal-regulated kinase-1 and -2 [pERK1/2] to ERK1/2 ratio) and expressed as the fold-change from the BPN/3 control mice. Aortic vascular smooth muscle cells (VSMCs) underwent stretch with or without Losartan (1 μM) treatment to assess the mechanical stimulation of ERK1/2 activity. Statistical analysis of the blood pressure, AoD, and VSMC ERK1/2 activity was performed using analysis of variance. However, the data distribution was determined to be log-normal (Shapiro-Wilk test) for ERK1/2 activity. Therefore, it was logarithmically transformed before analysis of variance. ResultsAt baseline, the SBP was elevated in the BPH/2 mice relative to the BPN/3 mice (P < .05). Losartan treatment significantly reduced the SBP in both mouse strains (P < .05). AAA induction did not affect the SBP. At 21 days after induction, the percentage of increase in the AoD from baseline was significantly greater in the BPH/2 mice than in the BPN/3 mice (101.28% ± 4.19% vs 75.59% ± 1.67% above baseline; P < .05). Losartan treatment significantly attenuated AAA growth in both BPH/2 and BPN/3 mice (33.88% ± 2.97% and 43.96% ± 3.05% above baseline, respectively; P < .05). ERK1/2 activity was increased approximately fivefold in the BPH/2 control mice relative to the BPN/3 control mice (P < .05). In the BPH/2 and BPN/3 mice with AAA, ERK1/2 activity was significantly increased relative to the respective baseline control (P < .05) and effectively reduced by concomitant Losartan therapy (P < .05). Biaxial stretch of the VSMCs in the absence of angiotensin II demonstrated increased ERK1/2 activation (P < .05 vs static control), which was significantly inhibited by Losartan. ConclusionsIn BPH/2 mice with spontaneous neurogenic, low-renin HTN, AAA growth was amplified compared with the normotensive control and was effectively attenuated using Losartan. ERK1/2 activity was significantly elevated in the BPH/2 mice and after AAA induction in the normotensive and hypertensive mice but was attenuated by Losartan treatment. These data suggest that AT1R activation contributes to AAA development. Therefore, further investigation into this signaling pathway could establish targets for pharmacotherapeutic engineering to slow AAA growth. (JVS–Vascular Science 2021;2:194-206.) Clinical RelevanceHypertension (HTN) and abdominal aortic aneurysm (AAA) have been epidemiologically linked for decades; however, a biomechanical link has not yet been identified. Using a murine model of spontaneous neurogenic HTN experimentally demonstrated to have low circulating renin, mechanical activation of the angiotensin II type 1 receptor (AT1R) was identified with elevated blood pressure and AAA induction. HTN amplified AAA growth. However, more importantly, blocking the activation of AT1R with the angiotensin receptor blocker Losartan effectively abrogated AAA development. Although inhibiting the production of angiotensin II has previously been unsuccessful in altering AAA growth, the results from the present study suggest that blocking the activation of AT1R through direct ligand binding or mechanical stimulation might alter aortic wall signaling and warrants further investigation.

Angiotensin II Type I Receptor Blockade Is Associated with Decreased Cutaneous Scar Formation in a Rat Model

Angiotensin II engagement of angiotensin II type 1 receptor (AT1R) is implicated in fibrogenesis, with AT1R blockers used clinically to attenuate cardiac and renal fibrosis. The authors tested the hypothesis that the AT1R blocker losartan could reduce postsurgical cutaneous scarring in rats. Human dermal fibroblasts were treated with losartan and assessed for viability, contractile activity, migration, and profibrotic gene transcription by means of calcein, collagen gel, scratch, and quantitative reverse transcriptase polymerase chain reaction assays, respectively. Monocyte migration and adhesion to losartan-treated and control fibroblasts were examined. Losartan effects in vivo were assessed using a mechanical distraction hypertrophic scar model. Three days after incisions were made and closed on their backs, rats were assigned randomly to receive drinking water with or without losartan (1 mg/kg per day; n = 6 per group). Distraction devices were applied and activated up to day 14. On day 28, scars underwent cross-sectional area and elevation index analyses, and α-SMA (alpha-smooth muscle actin) and CD68 (monocyte/macrophage marker) immunostaining. Losartan-treated human dermal fibroblasts displayed decreased contractile activity, migration, and gene expression of transforming growth factor-β1, collagen I, and monocyte chemoattractant protein-1 relative to controls (p < 0.05). Monocyte migration and adhesion to losartan-treated fibroblasts were reduced (p < 0.01). Compared to controls, scars from losartan-treated rats demonstrated decreased cross-sectional area (19.4 ± 3.1 mm versus 45.0 ± 5.2 mm; p = 0.002), elevation index (1.5 ± 0.1 versus 2.6 ± 0.3; p = 0.003), and α-SMA and CD68 immunostaining (p < 0.001). Losartan decreases myofibroblast activity and reduces monocyte trafficking to cutaneous scar. These findings support losartan as a potential novel therapy for the prevention of hypertrophic scars.

Blockade of Mas Receptor or Mas-Related G-Protein Coupled Receptor Type D Reduces Portal Pressure in Cirrhotic but Not in Non-cirrhotic Portal Hypertensive Rats.

Portal hypertension (PHT) resulting from splanchnic vasodilatation is a major cause of morbidity and mortality in patients with cirrhosis. The renin-angiotensin system (RAS) plays an important role in splanchnic vasodilatation in cirrhosis. This study investigated whether acute blockade of the vasodilatory receptors of the alternate RAS, Mas (MasR), Mas-related G-protein coupled receptor type D (MrgD), and angiotensin II type-2 receptor (AT2R) improves PHT in cirrhotic and non-cirrhotic portal hypertensive rats and counteracts systemic hypotension associated with angiotensin II type 1 receptor (AT1R) blockade. Cirrhotic bile duct ligated (BDL) or carbon tetrachloride (CCl4) injected and non-cirrhotic partial portal vein ligated (PPVL) rats were used for measurement of portal pressure (PP) and mean arterial pressure before and after an intravenous bolus injection of the MasR, MrgD, and AT2R blockers, A779, D-Pro7-Ang-(1-7) (D-Pro) and PD123319, respectively. Separate groups of rats received a combined treatment with A779 or D-Pro given 20 min after AT1R blocker losartan. Mesenteric expression of MasR, MrgD, and AT2R and circulating levels of peptide blockers were also measured. Treatment with A779 and D-Pro significantly reduced PP in cirrhotic rat models. Despite rapid degradation of A779 and D-Pro in the rat circulation, the PP lowering effect of the blockers lasted for up to 25 min. We also found that PD123319 reduced PP in CCl4 rats, possibly by blocking the MasR and/or MrgD since AT2R expression in cirrhotic mesenteric vessels was undetectable, whereas the expression of MasR and MrgD was markedly elevated. While losartan resulted in a marked reduction in PP, its profound systemic hypotensive effect was not counteracted by the combination therapy with A779 or D-Pro. In marked contrast, none of the receptor blockers had any effect on PP in non-cirrhotic PPVL rats whose mesenteric expression of MasR and MrgD was unchanged. We conclude that in addition to MasR, MrgD, a newly discovered receptor for Angiotensin-(1-7), plays a key role in splanchnic vasodilatation in cirrhosis. This implies that both MasR and MrgD are potential therapeutic targets to treat PHT in cirrhotic patients. We also conclude that the alternate RAS may not contribute to the development of splanchnic vasodilatation in non-cirrhotic PHT.

Transforming growth factor-β1 induces cerebrovascular dysfunction and astrogliosis through angiotensin II type 1 receptor-mediated signaling pathways.

Transgenic mice constitutively overexpressing the cytokine transforming growth factor-β1 (TGF-β1) (TGF mice) display cerebrovascular alterations as seen in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID), but no or only subtle cognitive deficits. TGF-β1 may exert part of its deleterious effects through interactions with angiotensin II (AngII) type 1 receptor (AT1R) signaling pathways. We test such interactions in the brain and cerebral vessels of TGF mice by measuring cerebrovascular reactivity, levels of protein markers of vascular fibrosis, nitric oxide synthase activity, astrogliosis, and mnemonic performance in mice treated (6 months) with the AT1R blocker losartan (10 mg/kg per day) or the angiotensin converting enzyme inhibitor enalapril (3 mg/kg per day). Both treatments restored the severely impaired cerebrovascular reactivity to acetylcholine, calcitonin gene-related peptide, endothelin-1, and the baseline availability of nitric oxide in aged TGF mice. Losartan, but not enalapril, significantly reduced astrogliosis and cerebrovascular levels of profibrotic protein connective tissue growth factor while raising levels of antifibrotic enzyme matrix metallopeptidase-9. Memory was unaffected by aging and treatments. The results suggest a pivotal role for AngII in TGF-β1-induced cerebrovascular dysfunction and neuroinflammation through AT1R-mediated mechanisms. Further, they suggest that AngII blockers could be appropriate against vasculopathies and astrogliosis associated with AD and VCID.

β-Arrestin mediates the Frank–Starling mechanism of cardiac contractility

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein β-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking β-arrestin 1, β-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a β-arrestin-biased AT1R ligand to selectively activate β-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in β-arrestin 1, β-arrestin 2, and AT1R knockout mice. Our data reveal β-arrestin 1, β-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with β-arrestin-biased AT1R ligands.

AT1R blocker losartan attenuates intestinal epithelial cell apoptosis in a mouse model of Crohn's disease.

Angiotensin II, which is the main effector of the renin-angiotensin system, has an important role in intestinal inflammation via the angiotensin II type 1 receptor (AT1R). The present study aimed to investigate the protective effects of the AT1R blocker losartan on 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis. Losartan was administered to male adult C57BL/6 J mice 2 weeks prior to the induction of colitis, and images of the whole colon were captured to record changes, scored according to a microscopic scoring system, and reverse transcription-quantitative polymerase chain reaction were performed in order to investigate colonic inflammation. In addition, intestinal epithelial barrier permeability was evaluated, and intestinal epithelial cell (IEC) apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and apoptosis-related protein expression levels were detected by western blotting. Losartan was able to attenuate TNBS-induced body weight loss and colonic damage. Furthermore, T helper 1-mediated proin-flammatory cytokines were suppressed by losartan, and gut permeability was largely preserved. TUNEL staining revealed reduced IEC apoptosis in the losartan-treated mice. Losartan also increased the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) ratio and suppressed caspase-3 induction. These results suggested that the AT1R blocker losartan may attenuate TNBS-induced colitis by inhibiting the apoptosis of IECs. The effects of losartan were partially mediated through increasing the Bcl-2/Bax ratio and subsequently suppressing the induction of the proapoptotic mediator caspase-3.

Evaluation of [11C]methyl-losartan and [11C]methyl-EXP3174 for PET imaging of renal AT1receptor in rats

IntroductionThe angiotensin II type 1 receptor (AT1R) is responsible for the main effects of the renin–angiotensin system (RAS), and its expression pattern is altered in several diseases. The [11C]methylated derivatives of the clinically used AT1R blocker (ARB) losartan and its active metabolite EXP3174, that binds with higher affinity to AT1R, were evaluated as potential PET imaging tracers in rat kidneys. Methods[11C]Methyl-losartan and [11C]methyl-EXP3174 were synthesized by [11C]methylation of the tetrazole-protected analogs using [11C]methyl iodide. Tissue uptake and binding selectivity of [11C]methyl-losartan were assessed by ex-vivo biodistribution and in-vitro autoradiography. Radiolabeled metabolites in rat plasma and kidneys were analysed by column-switch HPLC. Both tracers were evaluated with small animal PET imaging. Due to better pharmacokinetics, [11C]methyl-EXP3174 was further investigated via PET by co-injection with AT1R antagonist candesartan or the AT2R antagonist PD123,319. ResultsBinding selectivity to renal AT1 over AT2 and Mas receptors was demonstrated for [11C]methyl-losartan. Plasma metabolite analysis at 10min revealed stability of [11C]methyl-losartan and [11C]methyl-EXP3174 with the presence of unchanged tracer at 70.8±9.9% and 81.4±6.0%, of total radioactivity, respectively. Contrary to [11C]methyl-losartan, co-injection of candesartan with [11C]methyl-EXP3174 reduced the proportion of unchanged tracer (but not metabolites), indicating that these metabolites do not bind to AT1R in rat kidneys. MicroPET images for both radiotracers displayed high kidney-to-background contrast. Candesartan significantly reduced [11C]methyl-EXP3174 uptake in the kidney, whereas no difference was observed following PD123,319 indicating binding selectivity for AT1R. Conclusions[11C]Methyl-EXP3174 displayed a favorable binding profile compared to [11C]methyl-losartan for imaging renal AT1Rs supporting further studies to assess its full potential as a quantitative probe for AT1R via PET.

Loss of nNOS Sarcolemmal Localization and Activity During Hindlimb Unloading is Mitigated by Losartan Administration

Spaceflight and immobilization lead to weakening and atrophy of antigravity skeletal muscles. Recently we observed that angiotensin II type 1 receptor (AT1R) blockade partially prevented the decrease in muscle fiber cross sectional area (CSA) and the type I to II fiber type shift that occurs in solei during 7 days of hindlimb unloading. Recent studies reveal that translocation of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the sarcoplasm is a causal event to atrophy during disuse. PURPOSE: Determine whether the AT1R blocker Losartan prevents the translocation of nNOS from the sarcolemma to the sarcoplasm in soleus muscle fibers during 7 days of hindlimb unloading. METHODS: Rats were divided into 4 groups: ambulatory control (CON), ambulatory + Losartan (40 mg/kg/day) (CON-L), 7 days hindlimb unloaded (HU), and HU + Losartan (HU-L). Solei were collected and analyzed. Localization of nNOS was assessed through immunohistochemistry. We measured protein content and localization via subcellular fractionation and immunoblotting. nNOS activity was observed through NADPH-diaphorase histochemistry. RESULTS: Hindlimb unloading decreased sarcolemmal-localized nNOS compared to CON muscles, an effect ameliorated by Losartan. Subcellular fractionization and immunoblotting revealed significantly elevated levels of sarcoplasmic nNOS in the HU group compared to CON (p < 0.05). Sarcoplasmic nNOS content in HU-L was not significantly different from CON. nNOS activity in the CON solei displayed activity primarily near the sarcolemma, while sarcoplasmic nNOS activity and lack of sarcolemmal activity was detected in HU muscle fibers. Losartan administration during hindlimb unloading partially preserved nNOS activity at the sarcolemma. CON-L findings were no different than CON findings in any of the experiments. CONCLUSIONS: AT1R blockade with Losartan during 7 days of hindlimb unloading aided in the retention of sarcolemmal nNOS, which partially maintained sarcolemmal nNOS activity, thus, limiting the reduction in fiber CSA and the fiber type shift that occurs during disuse-induced atrophy. Supported by NASA (NNX13AE45G), Predoctoral Space Physiology Grant from ACSM / NASA, and the Sydney and J.L. Huffines Institute.

Angiotensin II Triggers Apoptosis Via Enhancement of NADPH Oxidase-Dependent Oxidative Stress in a Dopaminergic Neuronal Cell Line

Numerous studies reveal that Angiotensin II (Ang II), the main effector of renin-angiotensin system, contributes to the pathogenesis of Parkinson's disease (PD) via triggering dopaminergic cell loss. However, the underlying mechanisms remain largely unclear. In the current study, by using CATH.a cell, a dopaminergic neuronal cell line stably expressing Angiotensin II type 1 receptor (AT1R) and Angiotensin II type 2 receptor (AT2R), we showed that Ang II treatment triggered cell apoptosis in a dose-dependent manner, providing the first evidence that apoptotic cell death contributed to the dopaminergic cell loss induced by Ang II. Ang II treatment also led to a significant increment in intracellular reactive oxygen species generation, which could be fully abolished by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors apocynin or diphenylene iodonium, indicating that Ang II enhanced oxidative stress via a NADPH oxidase-dependent manner. More importantly, inhibition of oxidative stress by NADPH oxidase inhibitors partially attenuated cell apoptosis caused by Ang II, implying that the enhancement of NADPH oxidase-dependent oxidative stress contributed to the cell apoptosis triggered by Ang II. Furthermore, the Ang II-induced oxidative stress and subsequent apoptosis could be completely abolished by AT1R blocker losartan rather than AT2R blocker PD1223319, suggesting that the aforementioned detrimental effects of Ang II are mediated by AT1R. In summary, these findings have deepened our understanding on the role of Ang II in PD pathogenesis, and support the use of AT1R blockers in the treatment of this devastating disease.

Prevalence of angiotensin II type 1 receptor (AT1R)–activating autoantibodies in primary aldosteronism

Autoantibodies to the angiotensin II type 1 receptor (AT1R) have been reported in patients with primary aldosteronism, including aldosterone producing adenoma (APA) and idiopathic adrenal hyperplasia (IAH). Sera from 25 primary aldosteronism subjects (12 with IAH and 13 with APA) and 15 normotensive control subjects were assayed for AT1R autoantibodies by enzyme–linked immunosorbent assay and an AT1R–transfected cell–based bioassay. Nine of 12 IAH subjects (75%) and six of 13 APA subjects (46%) were positive for AT1R autoantibodies in the bioactivity assay. The mean AT1R autoantibody activity for the IAH and APA subjects was significantly greater than controls (P < .001 and P < .01, respectively), and this in vitro activity was suppressed by the AT1R blocker losartan. None of the controls had significant AT1R autoantibody activity. Enzyme–linked immunosorbent assay values were less sensitive but were positive in some subjects with IAH and APA. The mean arterial pressure of these primary aldosteronism subjects correlated modestly with AT1R autoantibody activity. These data confirm the presence of active AT1R autoantibodies in a high percentage of subjects with primary aldosteronism irrespective of their underlying etiology. These observations have both pathophysiological and clinical implications.

Abstract 305: Activation of Central Angiotensin II type 2 Receptors Attenuates Neurogenic Hypertension.

Aim: The role of the angiotensin II type-2 receptor (AT2R) in hypertension is under debate and the expression can be modulated in different pathological states. In hypertension AT2R are upregulated, therefore we hypothesize that central AT2R stimulation will lower blood pressure in conscious spontaneously hypertensive rats (SHR). Methods: SHR were implanted with a radio-telemetry device and an icv cannula connected to a miniosmotic pump delivering saline vehicle, AT2R agonist Compound 21 (C21) (0.002μg/μl/hr) alone or in combination with AT2R antagonist PD123319. MAP was assessed for 21 days: 7 days baseline (saline), 14 days treatment (e.g. C21). To assess the role of the angiotensin II type-1 receptor (AT1R) and NO in these responses, AT1R blocker losartan or NO synthase inhibitor L-NAME were administered centrally with C21. (n=5-7/group). Results: Icv C21-infusion blocked the increase in MAP seen in the vehicle group (MAP (mmHg): baseline: vehicle 153±5 vs C21 157±4; day 21 (D21): vehicle 164±5 vs C21 155±5;p&lt;0.05). PD123319 abolished this blood pressure lowering effect (MAP (mmHg): baseline 154±5; D21 163±5;p&lt;0.05). Co-infusion of C21 and losartan did not reinforce the MAP lowering effect seen with C21 alone (MAP (mmHg): baseline 157±2; D21 153±1;p&lt;0.05). Simultaneous L-NAME administration abolished the effects of C21 (MAP (mmHg): baseline 156±4; D21 190±5;p&lt;0.05). Mechanistically, an improved parasympathetic control of HR was seen in the C21-treated group (change in HR after i.p. propranolol (bpm): vehicle -68.2±2.4 vs C21 -36.3±4.1;p&lt;0.05). Impaired baroreflex sensitivity (BRS) also improved under C21-infusion (BRS (ms/mmHg) D21: vehicle 1.81±0.16 vs C21 3.15±0.07;p&lt;0.05) Conclusion: Selective central AT2R stimulation with C21 attenuates hypertension and corrects autonomic dysfunction in SHR. These effects are mediated through a NO-dependent mechanism. In contrast to an enhanced peripheral AT2R effect following AT1R blockade, concomitant central AT1R blockade did not enhance these responses to central AT2R stimulation. These findings suggest activation of the central AT2R represent a possible new therapeutic target for the treatment of neurogenic hypertension.

Angiotensin II receptor blockade protects against the slow to fast fiber type shift and type I fiber atrophy in the rat soleus with 7 days of hindlimb unloading (LB804)

Reduced mechanical loading during spaceflight and immobilization results in weakening and atrophy of antigravity skeletal muscles. Data indicates that oxidative stress contributes to inactivity‐induced muscle atrophy. Recently we observed that the Nox2 isoform of NADPH oxidase is upregulated during unloading‐induced soleus muscle atrophy. Clues from other tissue types indicate activation of angiotensin II type 1 receptor (AT1R) as an upstream event leading to Nox2 complex formation and activity. Here, we used an AT1R blocker (Losartan) to test the hypothesis that AT1R activation is an upstream signaling event driving Nox2 complex activity, and thus atrophy in the mechanically unloaded soleus. Rats were divided into 4 groups: ambulatory control (CON), ambulatory + Losartan (40 mg/kd/day) (CON‐L), hindlimb unloaded for 7 days (HU), and HU + Losartan (HU‐L). Losartan treatment did not protect against the reduction in soleus mass during 7 days of unloading. However, the increase in Type II muscle fibers seen with unloading was mitigated by Losartan administration. Losartan also protected against atrophy of Type I muscle fibers. In addition, Losartan reduced the signal intensity and membrane localization of the Nox2 subunit p67phox in the unloaded soleus. Our findings suggest a potential role of AT1R activation as an upstream signaling event leading to Nox2 complex formation, activation, and slow to fast fiber‐type shift during mechanical unloading.Grant Funding Source: Supported by NASA (NNX13AE45G), Predoctoral Space Physiology Grant from ACSM/NASA, and the Sydney and J.L. Huffines Institute

Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice

Epigenetic mechanisms such as chromatin histone H3 lysine methylation and acetylation have been implicated in diabetic vascular complications. However, histone modification profiles at pathologic genes associated with diabetic nephropathy in vivo and their regulation by the angiotensin II type 1 receptor (AT1R) are not clear. Here we tested whether treatment of type 2 diabetic db/db mice with the AT1R blocker Losartan not only ameliorates diabetic nephropathy, but also reverses epigenetic changes. As expected, the db/db mice had increased blood pressure, mesangial hypertrophy, proteinuria and glomerular expression of RAGE and PAI-1 versus control db/+ mice. This was associated with increased RNA Polymerase II recruitment and permissive histone marks as well as decreased repressive histone marks at these genes, and altered expression of relevant histone modification enzymes. Increased MCP-1 mRNA levels were not associated with such epigenetic changes, suggesting post-transcriptional regulation. Losartan attenuated key parameters of diabetic nephropathy and gene expression, and reversed some but not all the epigenetic changes in db/db mice. Losartan also attenuated increased H3K9/14Ac at RAGE, PAI-1 and MCP-1 promoters in mesangial cells cultured under diabetic conditions. Our results provide novel information about the chromatin state at key pathologic genes in vivo in diabetic nephropathy mediated in part by AT1R. Thus combination therapies targeting epigenetic regulators and AT1R could be evaluated for more effective treatment of diabetic nephropathy.

Renal inflammatory markers during the onset of hypertension in spontaneously hypertensive rats

Early blockade of the renin-angiotensin system is successful in delaying the development of hypertension in spontaneously hypertensive rats (SHRs) and ameliorating organ damage by inhibition of the inflammatory response. In this study, we investigated the role of the angiotensin II type 1 receptor (AT1R) in the early renal inflammatory response in SHR. Blood pressure development and renal inflammatory markers were measured in 4-, 8- and 12-week-old SHR and age-matched Wistar Kyoto (WKY) rats. Separate groups of SHRs were transiently treated with the AT1R blocker losartan between 4 and 8 weeks of age. Urinary excretion of the renal injury markers osteopontin and neutrophil gelatinase-associated lipocalin increased in young SHR. Further, renal expression of inflammatory genes was also increased in young SHR. Losartan inhibited the increase of these inflammatory markers. In contrast, gene expression of the renal injury marker and T-cell inducer kidney injury molecule-1 (KIM-1) was reduced in 4-week-old SHR when compared with WKY. Similarly, the T-cell marker CD3 was significantly decreased in 4-week-old SHR. These effects were not antagonized by AT1R blockade. This study confirms the presence of an early renal inflammatory response in SHR that can be blocked by AT1R antagonism. In addition, it demonstrates that KIM-1 does not behave as a pure kidney injury marker in young SHR, but may reflect kidney maturation.

Abstract 2628: The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s

Abstract Staphylococcal nuclease domain containing-1 (SND1) is a multifunctional protein that modulates transcription, mRNA splicing and stability, and miRNA function. We previously documented that SND1 is markedly overexpressed in human hepatocellular carcinoma (HCC) patients in a stage-specific manner and inhibition of SND1 in human HCC cells significantly abrogates in vitro proliferation and in vivo tumorigenesis in nude mice. In the present study we document that migration and invasion of human HCC cells are significantly augmented in SND1-overexpressing clones and significantly inhibited in SND1 shRNA-expressing clones. Differential gene expression analysis between control clone and SND1 shRNA expressing clones by Affymetrix microarray revealed downregulation of TGF-β downstream genes upon SND1 inhibition. Additionally, a human angiogenesis array identified plasminogen activator inhibitor-1 (PAI-1), a TGF-β downstream gene, to be upregulated by SND1 and this upregulation was confirmed by ELISA. TGF-β expression is regulated by angiotensin II type 1 receptors (AT1R) and previous studies have documented that SND1 increases AT1R expression by increasing AT1R mRNA stability. We hypothesized that increased AT1R expression, conferred by SND1 overexpression, might facilitate activation of TGF-β signaling and subsequently PAI-1-mediated increase in migration and invasion. Indeed, we observed that AT1R and TGF-β expression was significantly increased in SND1-overexpressing clones and decreased in SND1 knockdown clones. The half-life of AT1R mRNA was significantly longer in SND1-overexpressing clones compared to the control clone confirming that SND1 modulates AT1R post-transcriptionally. The AT1R blocker Losartan Potassium and AT1R siRNA significantly inhibited SND1-induced TGF-β and PAI-1 expression with associated inhibition in migration and invasion. As a corollary, inhibition of PAI-1 by siRNA significantly abrogated migration and invasion of SND1-overexpressing cells. TGF-β induces epithelial-mesenchymal transition (EMT) and we observed increased expression of EMT markers vimentin, snail and slug and decreased expression of E-cadherin in SND1-overexpressing clones and vice versa in SND1-knockdown clones. Our studies thus unravel a novel mechanism in which post-transcriptional regulation of AT1R by SND1, with subsequent activation of TGF-β signaling, promotes invasion of human HCC cells thereby conferring an aggressive phenotype. Small molecule inhibitors of SND1, thus, might be effective therapeutic strategy for late-stage aggressive HCC. Citation Format: Prasanna K. Santhekadur, Rachel Gredler, Maaged Akiel, Paul Dent, Paul B. Fisher, Devanand Sarkar. The multifunctional protein staphylococcal nuclease domain containing-1 (SND1) promotes migration and invasion of hepatocellular carcinoma (HCC) cells by modulating angiotensin II type 1 receptor (AT1R) and transforming growth factor-β (TGF-β) s [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2013-2628

Angiotensin II Type 1 Receptor-Dependent Oxidative Stress Mediates Endothelial Dysfunction in Type 2 Diabetic Mice

The mechanisms underlying the effect of the renin-angiotensin-aldosterone system (RAAS) inhibition on endothelial dysfunction in type 2 diabetes are incompletely understood. This study explored a causal relationship between RAAS activation and oxidative stress involved in diabetes-associated endothelial dysfunction. Daily oral administration of valsartan or enalapril at 10 mg/kg/day to db/db mice for 6 weeks reversed the blunted acetylcholine-induced endothelium-dependent dilatations, suppressed the upregulated expression of angiotensin II type 1 receptor (AT(1)R) and NAD(P)H oxidase subunits (p22(phox) and p47(phox)), and reduced reactive oxygen species (ROS) production. Acute exposure to AT(1)R blocker losartan restored the impaired endothelium-dependent dilatations in aortas of db/db mice and also in renal arteries of diabetic patients (fasting plasma glucose level > or =7.0 mmol/l). Similar observations were also made with apocynin, diphenyliodonium, or tempol treatment in db/db mouse aortas. DHE fluorescence revealed an overproduction of ROS in db/db aortas which was sensitive to inhibition by losartan or ROS scavengers. Losartan also prevented the impairment of endothelium-dependent dilatations under hyperglycemic conditions that were accompanied by high ROS production. The present study has identified an initiative role of AT(1)R activation in mediating endothelial dysfunction of arteries from db/db mice and diabetic patients.

Mechanical stress-evoked but angiotensin II-independent activation of angiotensin II type 1 receptor induces cardiac hypertrophy through calcineurin pathway

Mechanical stress can induce cardiac hypertrophy through angiotensin II (AngII) type 1 (AT1) receptor independently of AngII, however, the intracellular mechanisms remain largely indeterminate. Since calcineurin, a Ca2+-dependent phosphatase, plays a critical role in pressure overload-induced cardiac hypertrophy, we therefore, asked whether calcineurin is involved in the AT1 receptor-mediated but AngII-independent cardiac hypertrophy. Mechanical stretch failed to elicit hypertrophic responses in COS7 cells co-transfected with plasmid of AT1 receptor and siRNA of calcineurin. Mechanical stresses for 2weeks in vivo and for 24h in vitro significantly induced upregulation of calcineurin expression and hypertrophic responses, such as the increases in cardiomyocytes size and specific gene expressions, in cardiomyocytes of angiotensinogen gene knockout (ATG−/−) mice, both of which were significantly suppressed by a specific calcineurin inhibitor FK506, suggesting a critical role of calcineurin in mechanical stress-induced cardiac hypertrophy in the ATG−/− mice. Furthermore, an AT1 receptor blocker Losartan not only attenuated cardiac hypertrophy but also abrogated upregulation of cardiac calcineurin expression induced by mechanical stresses in the AngII-lacking mice, indicating that calcineurin expression is regulated by AT1 receptor without the involvement of AngII after mechanical stress. These findings collectively suggest that mechanical stress-evoked but AngII-independent activation of AT1 receptor induces cardiac hypertrophy through calcineurin pathway.