Angiotensin II Type 1 Receptor Protein Research Articles

N-Methyl-d-Aspartate-Induced Excitotoxicity and Its Impact on the Renin-Angiotensin System in Retinal Tissue.

Purpose: Renin-angiotensin system (RAS) is expressed in neuronal tissue and plays a role in neurodegenerative diseases involving excitotoxicity as a pathophysiological mechanism. In retina, excessive excitatory neurotransmission via N-methyl-d-aspartate (NMDA) receptors underlies neuronal apoptosis in conditions like glaucoma. However, it is not known if NMDA-mediated excitotoxicity alters retinal RAS expression. Hence, this study investigated the effect of NMDA exposure on the expression of RAS in rat retinas. Methods: Two groups of Sprague-Dawley rats received either phosphate buffer saline or NMDA (160 nmol). On day 7 posttreatment, retinal expression of RAS components including renin, angiotensinogen, angiotensin-converting enzyme (ACE), angiotensin II (Ang II), Ang 1-7, Ang 1-9, MAS receptor, angiotensin II type 1 receptor (AT1R), ACE2, and aldosterone was measured using enzyme-linked immunosorbent assay and polymerase chain reaction. Morphometric studies were done to assess morphological alterations. Results: Following the exposure to NMDA, an upregulation of ACE expression was noted at both the protein (2.03-folds; P < 0.001) and mRNA (1.86-folds; P < 0.01) levels in rat retinas. AT1R protein and mRNA expression were greater by 1.73 (P < 0.0001) and 2.28-folds (P < 0.0001), respectively. However, mRNA expression for ACE2, Ang 1-7, and Ang 1-9, showed a 1.51-(P < 0.05), 2.41-(P < 0.001), and 2.37-(P < 0.0001) fold decrease. Ganglion cell layer (GCL) thickness and linear cell density in GCL were significantly lower in the NMDA-treated group (P < 0.05). Conclusions: NMDA exposure increases expression of the classical RAS and suppresses that of alternate RAS in rat retinas. These alterations are associated with retinal morphological changes indicating significant loss of neuronal cells in the GCL of rat retinas.

PS-B12-10: CLASSIC CARDIOVASCULAR PROTECTIVE SIGNALING IN ANGIOTENSIN II-INDUCED NOVEL PREECLAMPSIA RAT MODEL

Angiotensin II (Ang II)-induces vasoconstriction, in vivo perfusion has been shown to induce preeclampsia (PE) in pregnant rats. In this study, we aimed to examine the protein and mRNA expressions of endothelial and neuronal NOS (eNOS, nNOS) as well as angiotensin II type 2 receptor (AT2R) in normal pregnant and Ang II-induced PE rats. Pregnant (8 weeks) and non-pregnant SD rats (8 weeks, GD-8) were infused with Ang II via an osmotic minipump (1 ug/kg/min), and the rats (GD-21) were sacrificed 13 days later. Pregnant and non-pregnant rats without Ang II were taken as sham controls. The fetal weight, crown-rump length and placental weight of PE rats were significantly lower compared to those without Ang II infusion. Immunohistochemistry results showed an approximately 30% increase in glomerular volume, suggesting morphological changes in the kidney and Ang II infusion exhibits a PE phenotype. Ang II infusion did not affect AT2R, AT1R and nNOS protein or mRNA expression in the uterus of non-pregnant or pregnant rats. However, eNOS mRNA expression was reduced in PE rats (p &lt; 0.0001) although protein expression was not affected by Ang II infusion. However, in the placenta, AT1R and AT2R protein expression were not different in PE, but AT2R mRNA was decreased (p &lt; 0.0001). eNOS protein was only expressed in fetal vascular endothelial cells, and eNOS protein expression was higher in PE rats (P = 0.001) but eNOS mRNA was decreased (p = 0.0096). Notably, strong nNOS expression was observed in placental trophoblast cell groups of sham and PE rats, and both mRNA and protein expression of nNOS was decreased in placental lysates of Ang II rats (respectively p = 0.0287 and p &lt; 0.0001). These results demonstrate that we have recapitulated the PE model of Ang II infusion in rats. The expression of nNOS was reduced, and the localization of nNOS in the trophoblast suggested a new functional remodeling in the fetal vasculature. In the placenta, decreased expression of AT2R and nNOS may be a novel mechanism of impaired fetal development.

Maternal exercise upregulates the DNA methylation of Agtr1a to enhance vascular function in offspring of hypertensive rats.

The angiotensin II signaling system regulates vascular dysfunction and is involved in the programming of hypertension. Maternal exercise has been linked to both short-term and long-term benefits for the mother and fetus. However, the impacts of maternal exercise on the intravascular renin-angiotensin system (RAS) in hypertensive offspring remain unexamined. This study examined whether maternal exercise has an epigenetic effect in repressing angiotensin II type 1 receptor (AT1R) expression, which leads to favorable alterations in the mesenteric artery (MA) function of spontaneously hypertensive offspring. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) pregnant rats were randomly divided into an exercise group and a control group. Blood pressure, vascular tone, AT1R protein and mRNA expression, and AT1R gene (Agtr1a) promoter methylation status were examined in the MAs of 3-month-old male offspring. Maternal exercise significantly reduced the resting blood pressure and cardiovascular reactivity of offspring from SHRs. Furthermore, Ang II-AT1R activity in regulating vascular tone and AT1R expression was decreased in the MAs of the SHR offspring from the exercise groups. Importantly, exercise during gestation suppressed AT1R expression via hypermethylation of the Agtr1a promoter region and upregulated DNA methyltransferase (DNMT) expression in MAs of SHR offspring. These results suggest that maternal exercise upregulates DNMT expression, resulting in hypermethylation and repression of the Agtr1a gene, which may prevent MA dysfunction in the offspring of SHRs. A mechanistic model on the epigenetics of exercise during pregnancy. Maternal exercise during pregnancy triggers hypermethylation and transcriptional suppression of the Agtr1a gene via increased DNMT1 and DNMT3B expression in MAs of SHR offspring. Downregulation of AT1R expression reduces the contribution of Ang II to vascular tone, ultimately improving vascular structure and function. VSMC vascular smooth muscle cell; Ang II angiotensin II; AT1aR angiotensin type 1 receptor (AT1R) alpha subtypes; Agtr1a AT1R alpha isoform gene; MAs mesenteric arteries; BP blood pressure.

CLASSIC CARDIOVASCULAR PROTECTIVE SIGNALING IN ANGIOTENSIN II-INDUCED NOVEL PREECLAMPSIA RAT MODEL

Objective: Angiotensin II (Ang II)-induces vasoconstriction, in vivo perfusion has been shown to induce preeclampsia (PE) in pregnant rats. In this study, we aimed to examine the protein and mRNA expressions of endothelial and neuronal NOS (eNOS, nNOS) as well as angiotensin II type 2 receptor (AT2R) in normal pregnant and Ang II-induced PE rats. Design and method: Pregnant (8 weeks) and non-pregnant SD rats (8 weeks, GD-8) were infused with Ang II via an osmotic minipump (1 ug/kg/min), and the rats (GD-21) were sacrificed 13 days later. Pregnant and non-pregnant rats without Ang II were taken as sham controls. The fetal weight, crown-rump length and placental weight of PE rats were significantly lower compared to those without Ang II infusion. Immunohistochemistry results showed an approximately 30% increase in glomerular volume, suggesting morphological changes in the kidney and Ang II infusion exhibits a PE phenotype. Results: Ang II infusion did not affect AT2R, AT1R and nNOS protein or mRNA expression in the uterus of non-pregnant or pregnant rats. However, eNOS mRNA expression was reduced in PE rats (p &lt; 0.0001) although protein expression was not affected by Ang II infusion. However, in the placenta, AT1R and AT2R protein expression were not different in PE, but AT2R mRNA was decreased (p &lt; 0.0001). eNOS protein was only expressed in fetal vascular endothelial cells, and eNOS protein expression was higher in PE rats (P = 0.001) but eNOS mRNA was decreased (p = 0.0096). Notably, strong nNOS expression was observed in placental trophoblast cell groups of sham and PE rats, and both mRNA and protein expression of nNOS was decreased in placental lysates of Ang II rats (respectively p = 0.0287 and p &lt; 0.0001). Conclusions: These results demonstrate that we have recapitulated the PE model of Ang II infusion in rats. The expression of nNOS was reduced, and the localization of nNOS in the trophoblast suggested a new functional remodeling in the fetal vasculature. In the placenta, decreased expression of AT2R and nNOS may be a novel mechanism of impaired fetal development.

Sexually dimorphic role of smooth muscle cell estrogen receptor alpha in aging‐associated arterial stiffness

BackgroundAging is associated with rising arterial stiffness (AS), an independent risk factor for cardiovascular disease. The development of aging‐associated AS follows a sexually dimorphic pattern with women developing AS later in life. Estrogen and estrogen receptor alpha (ERα) are protective in the premenopausal state, potentially due to the regulation of the vasoprotective angiotensin II type 2 receptor (AT2R), however, the role of estrogen/ERα in aging‐associated AS has not been defined and the mechanisms of the sexually‐dimorphic development of aging‐associated AS are unknown.Methods/ResultsAll studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We generated novel smooth muscle cell‐specific ERα‐KO mice and measured aortic pulse wave velocity (PWV), an index of AS, in 3, 12, and 18 month old male and female intact and KO mice. Male mice develop increases in AS from 3 to 12 months compared to 3 to 18 months in females (p&lt;0.05). SMC‐ERα‐KO protected females from the aging‐associated increase in AS (p&lt;0.05). Circulating estrogens also declined in 18 month old females vs. 3 mo. (p&lt;0.05), suggesting a role for unliganded SMC‐ERα in aging‐associated AS. SMC‐ERα‐KO females were also protected from the aging‐associated decline in aortic AT2R expression observed in intact females (p&lt;0.05). To further investigate the potential role of unliganded SMC‐ERα in AS, we performed ovariectomy (OVX) or sham surgeries in young female SMC‐ERα‐Intact and KO mice and measured PWV. Results demonstrated an OVX‐driven increase in AS that was prevented by SMC‐ERα‐KO (p&lt;0.05). This was also associated with an OVX‐mediated decrease in aortic expression of the AT2R, which was prevented by SMC‐ERα‐KO. To determine the in vivo role of AT2R in the vascular protection observed in 12 month old female mice, we treated 12 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increased AS in 12 month old females (p&lt;0.05), but not males, suggesting that AT2R‐mediated protection contributes to the delay in aging‐associated AS in female mice. In vitro studies also demonstrated that estrogen upregulates AT2R mRNA and protein in female mouse aortic SMC, supporting an estrogen‐mediated mechanism of AT2R driven vascular protection in females.ConclusionThese data indicate sex differences in the time course of the development of aging‐associated arterial stiffness and suggest the presence of unliganded SMC‐ERα contributes specifically to aging‐associated arterial stiffness in females via reductions in vascular AT2R. Further, these results implicate that AT2R agonist therapy may improve cardiovascular outcomes in aging women to retain the beneficial downstream effects of estrogen signaling, without the potential detrimental effects of hormone replacement therapy.

MicroRNAs Regulate Hypothalamic AT2R Expression in Hypercaloric Diet‐Induced Epigenetic Programming of Male Mice Offspring

One in ten adults are living with diabetes worldwide and it is expected to be one in eight adults by the year 2045. Maternal epigenetic programming is one of the determinants of disease susceptibility in offspring. The renin‐angiotensin system contributes to metabolic homeostasis through the balance between Angiotensin II (Ang‐II) binding to Angiotensin II Type 1 receptor (AT1R) and Angiotensin II Type 2 receptor (AT2R). We have shown that maternal hypercaloric (HCD) diet leads to a pre‐diabetic state exclusively in adult male offspring. Additionally, we showed a paradoxical reversal of hyperglycemia in programmed males challenged with Ang‐II. Here, we hypothesize that this hypoglycemic response in programmed males is due to a shift in Ang‐II binding from AT1R towards the protective AT2R. To test this hypothesis, C57BL6/J dams were fed HCD or regular diet (RD) for 1 month and mated with RD‐fed males. After weaning, offspring were maintained on RD for 3 months resulting in two groups: HCD‐RD (programmed) and RD‐RD (control). Half of each group was implanted with a subcutaneous Ang‐II osmotic pump (200 ng/kg/min/2 weeks). Another osmotic pump filled with AT2R blocker, PD123319 (10 mg/kg/day) was implanted in a subgroup of Ang‐II programmed mice resulting in 5 groups: HCD‐RD, RD‐RD, HCD‐RD+Ang‐II, RD‐RD+Ang‐II and HCD‐RD+Ang‐II+PD123319 (n=7‐10/group). A glucose tolerance test was performed before and after drugs infusion. Following euthanasia, half of each hypothalamus was harvested for protein quantification using simple western while the other halves were processed for microRNA (miRNA) sequencing. Results show that HCD‐RD+Ang‐II+PD123319 had elevated glucose levels compared to HCD‐RD+Ang‐II mice at 15 min (355.7 ± 32 vs. 279.8 ± 19.66 mg/dL, p=0.04), 30 min (405 ± 18.54 vs. 263.5 ± 22.68mg/dL, p=0.0013) and 60 min (298.3 ± 19.75 vs.189.8 ± 21.35 mg/dL, p=0.0036). On the other hand, there was no difference in the glycemic levels of HCD‐RD+Ang‐II+PD123319 compared to RD‐RD+Ang‐II groups at 15 min (355.7 ± 32.97 vs. 349.0 ± 32.06 mg/dL, p=0.8), 30 min (405.0 ± 18.54 vs. 405.8 ± 44.28 mg/dL, p=0.9) and 60 min (298.3 ± 19.75 vs. 230.0 ± 35.52 mg/dL, p=0.1). AT2R protein expression was higher in the HCD‐RD compared to RD‐RD (p=0.04) and in HCD‐RD+Ang‐II compared to RD‐RD+Ang‐II (p=0.03). For miRNA sequencing, miRNA 124‐3p, targeting AT2R was differentially expressed in the HCD‐RD compared to RD‐RD (FC=1.4, p=0.04) as well as HCD‐RD compared to HCD‐RD+Ang‐II (FC=0.74, p=0.0001). Whereas, no change in the miRNA expression was depicted in the non‐programmed groups; RD‐RD vs RD‐RD+Ang‐II (FC=0.97, p=0.85). In conclusion, maternal hypercaloric diet epigenetic programming takes place probably through regulating the gene expression of AT2R via changing miRNA ‐124 levels that works on the post‐transcriptional modification level.

Losartan Mitigates Oxidative Stress in the Brains of Aged and Inflamed IL-10-/- Mice.

Chronic inflammation, oxidative stress, and dysregulation of the renin-angiotensin system are closely linked, and their crosstalk commonly contributes to age-related physical and cognitive decline. The primary dementia-protective benefits of Angiotensin II type 1 receptor (AT1R) blockers are believed to arise from systemic effects on blood pressure. However, there is an independently regulated brain-specific renin-angiotensin system. Here, we examined the impact of 4 weeks of oral Losartan treatment on the brains of aged (100 weeks old) IL-10-/- mice, an animal model of chronic inflammation and frailty. Our data show that aged IL-10-/- mice have higher AT1R and Nitrotyrosine (oxidative stress marker) levels in their frontal cortex tissue but not in cerebellar or hippocampal tissue compared to age- and sex-matched wild type mice. Losartan treatment for 4 weeks is associated with lower AT1R protein level, Nitrotyrosine, and Tau protein in the frontal cortex of aged IL-10-/- mice. Our results highlight the impact of Losartan, an AT1R blocker commonly prescribed for treating high blood pressure, on the brain-specific angiotensin system and AT1R-linked downstream effects such as brain oxidative stress damage and Tau burden in a frailty mouse model.

Study on the mechanism of rhynchophylline in improving obese hypertension through JAK2 / STAT3 pathway

Objective The paper studied the effects of Rhynchophylline on the leptin-mediated JAK2/STAT3 pathway in hypothalamus. Using the leptin / leptin receptor and leptin-mediated JAK2/STAT3 pathway as an entry point, the research evaluated the effect of the hypotensive drug, and clarified the preliminary mechanism, expecting to provide new therapeutic ideas for essentially improving obese hypertension, balancing the body's neuro-endocrine function, and preventing the occurrence of cardiovascular diseases. Methods: A model of obese hypertensive rats was established, and the serum lipids, Lep, AngII, NPY and NO contents in each group of rats were measured. Immunohistochemical staining was used to observe the distribution of leptin receptor (LepR) and angiotensin II type 1 receptor (AT1R) in hypothalamus and aorta of rats. Western Blot method was employed to detect JAK2, STAT3, SOCS3, LepR and AT1R protein expressions in the hypothalamus of rats. After the third ventricle injection of AG490, Western blot was applied to detect the expressions of various proteins. Results: Rhynchophylline can significantly reduce AngII, Lep, and NPY levels in serum circulation. Immunohistochemistry showed that the hypothalamus and aorta AT1R distribution was significantly reduced, the hypothalamic LepR distribution increased, and the aortic LepR distribution decreased. The expressions of JAK2, LepR, and STAT3 proteins in the hypothalamus increased significantly, and the expressions of SOCS3 and AT1R proteins significantly decreased. After injection of AG490 in the third ventricle of Rhynchophylline rats, the expressions of JAK2 and STAT3 in the hypothalamus decreased significantly, while the SOCS3expression increased remarkably. Conclusion: Rhynchophylline can reduce blood lipid, as well as serum AngII, Lep, and NPY levels, increase serum NO levels in obese hypertension rats. Rhynchophylline can effectively inhibit the expression of SOCS3 by activating the JAK2/STAT3 pathway in the hypothalamus. Therefore, it is speculated that the JAK2/STAT3 signaling pathway is the main pathway through which Rhynchophylline plays a role in improving selective leptin resistance and reducing blood pressure and weight.

Renal denervation inhibits the renin–angiotensin–aldosterone system in spontaneously hypertensive rats

ABSTRACT This study was conducted to explore the effect of renal denervation (RDN) on the renin–angiotensin–aldosterone system (RAAS) in spontaneously hypertensive rats (SHRs). Our experimental rats were randomly divided into the RDN group conducted by painting 10% phenol on the bilateral renal nerves (RDNX), the shamoperation group simply painting with saline (Sham), and the normotension control group (WKY) following all the animal blood and tissues of kidney, hypothalamus, and adrenal gland collected and examined 2 weeks after RDN operation. We found that the aldosterone (ALD) levels in serum and tissues all decreased in the RDNX group compared with the Sham group (p < .05). Meantime, the expression of angiotensin II type1 receptor (AT1R) mRNA also exhibited significantly reduced by 2.22-fold in the RDNX group compared to the Sham group identical to the expression of AT1R protein in the renal cortex and outer stripe of the outer medulla (OSOM) subjected to denervation surgery, which manifested the lower ATIR protein expression than the Sham group (p < .05). Besides, the expression of angiotensin II (Ang II) protein in the cortex , OSOM, and inner stripe of the outer medulla were all attenuated by RDN in comparison with the Sham group (p < .05). RDN reduced intrarenal RAAS and circulating RAAS to lower blood pressure and repair renal function.

The role of angiotensin signalling in anthracycline-induced cardiotoxicity

Abstract Anthracyclines (e.g. epirubicin, doxorubicin, daunorubicin) are widely used for the treatment of adult and paediatric cancers. Despite their therapeutic efficacy, anthracyclines are associated with both acute and late-onset cardiac toxicities. Meta-analyses report an overt cardiotoxicity incidence of 6.3%, whilst sub-clinical cardiotoxicity incidence is 17.9% (1). Angiotensin converting enzyme (ACE) inhibitors are used to treat anthracycline-induced cardiotoxicity (AIC) (2) and despite their efficacy being well studied for the treatment of heart failure, hypertension and post-acute coronary syndromes, their mechanism(s) for treating and preventing AIC remain unknown. Using in vitro cardiomyocytes, we evaluated the angiotensin signalling mechanisms stimulated by doxorubicin chemotherapy, applying quantitative PCR, immunofluorescence and real-time cell analysis technologies. In vitro adult human ventricular cardiomyocytes (AC10 cell line) treated with clinically relevant sub-toxic concentrations of doxorubicin, demonstrate a dose and time-dependent increase in angiotensin II type-1 receptor (AT1R) gene expression. Maximal AT1R expression was observed after 24 hours' exposure at 250 nanomolar (nM), with qPCR recording up to 13-fold increases in expression relative to control (figure 1). Consistent with gene expression studies, doxorubicin also induced expression of AT1R at the protein level, with immunofluorescence imaging displaying up-regulation of AT1R in association with doxorubicin concentrations up to 500nM (figure 2). Western blot results also support the induction of AT1R, however no relationship was observed between either doxorubicin concentration or drug exposure time. Cellular growth and morphological changes of cardiomyocytes in response to clinically relevant doses of doxorubicin treatment were evaluated with real-time cell analysis using impedance-based xCELLigence technology. During the early phases of doxorubicin exposure, an increase in cell size was observed, whilst experiments modelling the pharmacokinetics and serial half-lives of doxorubicin demonstrated reversibility of doxorubicin-induced cardiomyocyte injury following drug elimination. These data support the mechanistic hypothesis that a relationship exists between AIC and modulation of the angiotensin signalling pathway in cardiomyocytes. We demonstrate that cardiomyocyte exposure to doxorubicin induces AT1R gene and protein expression, whilst doxorubicin-induced cardiomyocyte injury displays reversibility following drug elimination. Genetic polymorphisms within the ACE gene have been associated with cardiomyopathy and left ventricular hypertrophy. Our research now provides the platform to ascertain whether the ACE genotype contributes to heart failure from AIC, and whether an elevation in pro-hypertrophic angiotensin II levels could exacerbate anthracycline-induced hypertrophy and promote the development of late-onset anthracycline-induced heart failure. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Cancer Research UK PhD research grant

Valsartan Prevented Neointimal Hyperplasia and Inhibited SRSF1 Expression and the TLR4-iNOS-ERK-AT1 Receptor Pathway in the Balloon-injured Rat Aorta.

Valsartan has the potential to attenuate neointimal hyperplasia and to suppress the inflammatory response. This study aimed to evaluate the role of valsartan in neointimal hyperplasia and the toll-like receptor 4 (TLR4)-nitric oxide synthase (NOS) pathway in the balloon-injured rat aorta.Forty-eight Wistar rats were randomly allocated to three groups: sham control (control), balloon-injured group (surgery), and balloon-injured+valsartan-treated group (valsartan). Rats were killed at 14 and 28 days after balloon-injury, and then the aortic tissues were collected for morphometric analysis as well as for measurements of the mRNA or protein expression of angiotensin II, angiotensin II type 1 (AT1) receptor, angiotensin II type 2 (AT2) receptor, TLR4, endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), serine/arginine-rich splicing factor 1(SRSF1) and extracellular signal regulated kinase (ERK). Valsartan at a dose of 20 mg/kg/day markedly decreased neointimal hyperplasia in the aorta of balloon-injured rats, and significantly reduced the mRNA or protein expression of TLR4, AT1 receptor, SRSF1 and phosphorylated-ERK (p-ERK) as well as the aortic levels of iNOS (all p < 0.05). Moreover, valsartan increased the eNOS level and AT2 receptor mRNA and protein expression levels (all p < 0.05). Valsartan prevented neointimal hyperplasia and inhibited SRSF1 expression and the TLR4-iNOS-ERK-AT1 receptor pathway in the balloon-injured rat aorta.

Increased AT1 receptor expression mediates vasoconstriction leading to hypertension in Snx1-/- mice.

Angiotensin II type 1 receptor (AT1R) is a vital therapeutic target for hypertension. Sorting nexin 1 (SNX1) participates in the sorting and trafficking of the renal dopamine D5 receptor, while angiotensin and dopamine are counterregulatory factors in the regulation of blood pressure. The effect of SNX1 on AT1R is not known. We hypothesized that SNX1, through arterial AT1R sorting and trafficking, is involved in blood pressure regulation. CRISPR/Cas9 system-generated SNX1-/- mice showed dramatic elevations in blood pressure compared to their wild-type littermates. The angiotensin II-mediated contractile reactivity of the mesenteric arteries and AT1R expression in the aortas were also increased. Moreover, immunofluorescence and immunoprecipitation analyses revealed that SNX1 and AT1R were colocalized and interacted in the aortas of wild-type mice. In vitro studies revealed that AT1R protein levels and downstream calcium signaling were upregulated in A10 cells treated with SNX1 siRNA. This may have resulted from decreased AT1R protein degradation since the AT1R mRNA levels showed no changes. AT1R protein was less degraded when SNX1 was downregulated, as reflected by a cycloheximide chase assay. Furthermore, proteasomal rather than lysosomal inhibition increased AT1R protein content, and this effect was accompanied by decayed binding of ubiquitin and AT1R after SNX1 knockdown. Confocal microscopy revealed that AT1R colocalized with PSMD6, a proteasomal marker, and the colocalization was reduced after SNX1 knockdown. These findings suggest that SNX1 sorts AT1R for proteasomal degradation and that SNX1 impairment increases arterial AT1R expression, leading to increased vasoconstriction and blood pressure.

Complex interactions between the angiotensin II type 1 receptor, the epidermal growth factor receptor and TRIO-dependent signaling partners

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.

Angiotensin II Type 1 Receptor Antagonist Azilsartan Restores Vascular Reactivity Through a Perivascular Adipose Tissue-Independent Mechanism in Rats with Metabolic Syndrome.

Perivascular adipose tissues (PVAT) are involved in the regulation of vascular tone. In mesenteric arteries, the compensatory vasodilatory effects of PVAT appear when vascular relaxation is impaired and disappear at around 23weeks of age in SHRSP.Z-Leprfa/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome (MetS). The renin-angiotensin system is involved in the development of endothelium and vascular dysfunction. Therefore, we investigated whether azilsartan, a potent angiotensin II type 1 (AT1) receptor antagonist, can protect against the deterioration of the PVAT compensatory vasodilator function that occurs with aging in MetS. Two age groups of SHRSP.ZF rats (13 and 20weeks of age) were administered azilsartan or vehicle through oral gavage once daily for 10weeks. The vasodilation response of the isolated superior-mesenteric arteries upon addition of endothelium-dependent and -independent agonists was determined in the presence or absence of PVAT using organ bath methods. In vivo treatment with azilsartan improved the acetylcholine-induced vasodilation in mesenteric arteries with and without PVAT at both time-points. The mRNA levels of AT1 receptor and AT1 receptor-associated protein were unchanged in PVAT upon azilsartan treatment. Furthermore, in vitro treatment with azilsartan (0.1 and 0.3μM for 30min) did not affect the compensatory effect of PVAT on vasodilation in response to acetylcholine in SHRSP.ZF rat mesenteric arteries. Our results provide evidence supporting the use of azilsartan for the long-term protection against vascular dysfunctions in MetS. Azilsartan did not improve the dysfunction of PVAT-mediated modulation of vascular tone during MetS. The protective effect of azilsartan is mediated by restoring the endothelium- and vascular smooth muscle-mediated mechanisms.

Anti-Apoptotic Effect of G-Protein-Coupled Receptor 40 Activation on Tumor Necrosis Factor-α-Induced Injury of Rat Proximal Tubular Cells

G-protein-coupled receptor 40 (GPR40) has an anti-apoptotic effect in pancreatic β-cells. However, its role in renal tubular cell apoptosis remains unclear. To explore the role of GPR40 in renal tubular apoptosis, a two-week unilateral ureteral obstruction (UUO) mouse model was used. The protein expression of GPR40 was decreased, while the Bax/Bcl-2 protein expression ratio, the expression of tumor necrosis factor (TNF)-α mRNA, and angiotensin II type 1 receptor (AT1R) protein were increased in mice with UUO. In vitro, pretreatment of rat proximal tubular (NRK52E) cells with GW9508, a GPR40 agonist, attenuated the decreased cell viability, increased the Bax/Bcl-2 protein expression ratio, increased protein expression of cleaved caspase-3 and activated the nuclear translocation of nuclear factor-κB (NF-κB) p65 subunit induced by TNF-α treatment. TNF-α treatment significantly increased the expression of AT1R protein and the generation of reactive oxygen species (ROS), whereas GW9508 treatment markedly reversed these effects. Pretreatment with GW1100, a GPR40 antagonist, or silencing of GPR40 in NRK52E cells promoted the increased expression of the cleaved caspase-3 protein by TNF-α treatment. Our results demonstrate that decreased expression of GPR40 is associated with apoptosis via TNF-α and AT1R in the ureteral obstructed kidney. The activation of GPR40 attenuates TNF-α-induced apoptosis by inhibiting AT1R expression and ROS generation through regulation of the NF-κB signaling pathway.

Upregulation of AT1 Receptor Mediates a Pressor Effect Through ROS-SAPK/JNK Signaling in Glutamatergic Neurons of Rostral Ventrolateral Medulla in Rats With Stress-Induced Hypertension.

The present study examined whether angiotensin II (Ang II) mediates the pressor effect through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS)-mitogen-activated protein kinase (MAPK) signaling in the glutamatergic neurons of the rostral ventrolateral medulla (RVLM) in stress-induced hypertensive rats (SIHR). The SIHR model was established using electric foot-shocks combined with noises for 15 days. We observed that Ang II type 1 receptor (AT1R) and the glutamatergic neurons co-localized in the RVLM of SIHR. Furthermore, glutamate levels in the intermediolateral column of the spinal cord were higher in SIHR than in controls. Microinjection of Ang II into the RVLM of SIHR activated stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), extracellular signal-regulated protein kinase (ERK) 1/2, and p38MAPK. Compared with controls, the activation of SAPK/JNK, ERK1/2, p38MAPK, and ROS in the RVLM were higher in SIHR, an effect that was blocked by an NADPH oxidase inhibitor (apocynin) and an AT1R antagonist (candesartan). RVLM microinjection of apocynin or a SAPK/JNK inhibitor (SP600125), but not an ERK1/2 inhibitor (U0126) or a p38MAPK inhibitor (SB203580), decreased AT1R mRNA and mean arterial blood pressure (MABP) in SIHR. The increase of AT1R protein expression and MABP was inhibited by intracerebroventricular infusion (ICV), for 14 days, of SP600125, but not U0126 or SB203580 in SIHR. We conclude that Ang II modulates the pressor effect through AT1R-dependent ROS-SAPK/JNK signaling in glutamatergic neurons in the RVLM of SIHR.

Superoxide via Sp3 mechanism increases renal renin activity, renal AT1 receptor function, and blood pressure in rats.

We tested a hypothesis that superoxide, by inducing Sp3, increases renal renin activity, renal angiotensin II type 1 receptor (AT1R) function, and blood pressure (BP) in rats. Group 1 rats were treated with vehicle, saline. Group 2 rats were treated with superoxide dismutase (SOD) inhibitor diethylthiocarbamate (DETC). Group 3 rats were treated with DETC and an SOD mimetic, tempol. Group 4 rats were treated with tempol only. All four groups of rats were treated for 2 wk then anesthetized, and BP was recorded. Thereafter, diuresis and natriuresis in response to AT1R blocker candesartan were determined. When compared with vehicle rats, BP increased in DETC rats. The increased BP in DETC rats decreased with tempol. Diuresis and natriuresis in response to candesartan increased in controls, and this further increased in DETC rats and decreased with tempol. A second set of four groups of rats underwent the same treatment as above and were anesthetized, and their kidneys were obtained for biochemical studies. The levels of superoxide but not hydrogen peroxide increased, whereas SOD activities decreased further in the renal cortical tissues of DETC rats than vehicle rats. These effects were attenuated with tempol in DETC rats. Moreover, tissue renin activity and abundance of membranous AT1R proteins increased more in DETC rats than vehicle rats, and decreased with tempol in DETC rats. Furthermore, the levels of lysine-acetylated, but not serine-phosphorylated, Sp3 increased more in the nuclei of DETC rats than vehicle rats. The increased levels of Sp3 lysine acetylation decreased in DETC rats with tempol. Taken together, our results suggest that superoxide activates renal Sp3 via lysine acetylation increasing renin activity, AT1R function, and BP in rats.

Renal sympathetic denervation lowers arterial pressure in canines with obesity-induced hypertension by regulating GAD65 and AT1R expression in rostral ventrolateral medulla

ABSTRACTTo explore the roles of glutamate acid decarboxylase 65 (GAD65) and angiotensin II type 1 receptor (AT1R) in the action of renal sympathetic denervation (RSD) on obesity-induced hypertension in canines. Thirty-two beagles were randomly divided into a hypertensive model (n = 22) and control (n = 10) groups. A hypertensive canine model was established by feeding a high-fat diet. Twenty hypertensive beagles were randomized equally to a sham-surgery and RSD-treated group receiving catheter-based radiofrequency RSD. Compared with the control group, the sham-surgery group exhibited significant increases in blood pressure, serum angiotensin II level, rostral ventrolateral medulla (RVLM) glutamate level, and AT1R mRNA and protein expression and decreases in γ-amino acid butyric acid (γ-GABA) level and GAD65 mRNA and protein expression in the RVLM (all P < 0.05). Treatment with RSD significantly attenuated the above abnormal alterations (all P < 0.05). Linear correlation analysis revealed that angiotensin II level was positively correlated with glutamate level (r = 0.804) and inversely correlated with γ-GABA level (r = −0.765). GAD65 protein expression was positively correlated with γ-GABA level (r = 0.782). Catheter-based radiofrequency RSD can decrease blood pressure in obesity-induced hypertensive canines. The antihypertensive mechanism might be linked to upregulation of GAD65 and downregulation of AT1R in the RVLM.

Abstract P353: Attenuation of Cardiac Fibrosis, Hypertrophy and Myopathy by AT2R Agonist NP-6A4

Adverse cardiac remodeling (hypertrophy, fibrosis and myopathy) underlies cardiac dysfunction and heart failure. Male Zucker obese (ZO:fa/fa) rat that suffers from cardiac dysfunction, adverse remodeling, and heart failure with preserved ejection fraction is a useful model to study the effects of cardioprotective drugs. We reported that NP-6A4 (Novopyxis Inc. Cambridge, MA), a peptide agonist of the Angiotensin II Type 2 Receptor (AT2R), protected mouse cardiomyoblast HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient serum deficiency stress better than β-AR-Blockers, ARBs, and AT2R agonist CGP42112A. This effect was inhibited by AT2R-specific antagonist PD123319, confirming that NP-6A4 acts through AT2R. AT2R is a cardiac and vascular reparative molecule that protects the heart and vasculature from structural damage and fibrosis. No current drugs increase AT2R expression. We report that NP-6A4 treatment (1μM) increased AT2R mRNA (up to 4 fold; p≤0.05) in hCAVSMCs and human coronary artery endothelial cells (up to 8 fold; p≤0.05). This effect was inhibited by PD123319 (10 μM). Treatment of 11-week old male ZO rats with heart disease with NP-6A4 (1.8mg/kg/day in saline, delivered once daily subcutaneously; N=7) increased cardiac AT2R expression and mitigated cardiac dysfunction and adverse remodeling. Quantitative RT-PCR and immunohistochemistry analysis showed that 2 weeks of NP-6A4 treatment increased cardiac AT2R mRNA (up to 9 fold) and protein (1.5-3 fold) compared to controls (N=6) receiving saline (p&lt;0.01). NP-6A4 treatment improved cardiac parameters; endocardial circumferential strain (p≤0.05), myocardial performance index (MPI) (p≤0.005), and E/E’ ratio (p≤0.002). NP-6A4 treatment also increased cardiac capillary density (118% compared to saline treatedl; p≤0.002), and reduced interstitial fibrosis (77% compared to saline treated; p≤0.039.) At 19-weeks (after 8 weeks of treatment), cardiac tissues from 5 out of 6 control rats exhibited regions of myopathy. Only 2 out 7 NP-6A4 treated rats exhibited similar levels of myopathy in the heart. We propose that NP-6A4 attenuates adverse remodeling and improves cardiac function by increasing AT2R expression in cardiac cells.

Pharmacodynamic study of eprosartan mesylate-loaded transfersomes Carbopol® gel under Dermaroller® on rats with methyl prednisolone acetate-induced hypertension.

The objective of present study was to prepare eprosartan mesylate (EM)-loaded transfersomes Carbopol® gel and characterized for various parameters, including in vitro skin permeation, in vivo antihypertensive study, skin irritation, and histological study. Furthermore, effect of transfersomes gel on angiotensin II type-1 receptor (AT1R) mRNA and protein expressions on smooth vascular muscles of aorta was determined by real-time polymerase chain reaction (RT-PCR) and western blot analysis. The physical evaluation parameters were detected to be in correspondence with reference marketed gel formulation. The transdermal flux, permeability coefficient, and Tlag of EM from transfersomes gel were found to be 26.76 ± 1.66μg/cm2/h, 8.93 ± 0.55 ×10-3 cm/h, and 2.17 ± 0.29h, respectively, across rat skin pretreated with microneedle (Dermaroller®). Pharmacodynamic study showed prolonged and better management of hypertension after the application of transfersomes gel in experimentally induced hypertensive Wistar rats as compared with oral control formulation. The in vivo angiotensin II type-1 blocking efficacy of prepared transfersomes gel and control formulation was also supported with RT-PCR and western blot analysis of AT1R mRNA and protein expressions on smooth vascular muscles of aorta. Skin irritation and skin histological assessment showed that the prepared transfersomes Carbopol® gel was safe to be used for transdermal route. It is concluded that the incorporation of transfersomes into gel formulation offered enhanced skin contact, ease of application, and found to be a suitable drug reservoir for the transdermal delivery of EM for the management of hypertension in Wistar rats.