Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker on oxidative stress and metabolism of elements in kidney of STZ-induced diabetic rats.

In diabetes, increased oxidative stress and impaired trace element metabolism play an important role in the pathogenesis of diabetic nephropathy. The objective of this research was to examine the outcomes of blocking the renin-angiotensin system, using either the angiotensin-converting enzyme inhibitor (ACEI), perindopril, or the angiotensin II type 1 (AT1) receptor blocker, irbesartan, on oxidative stress and trace element levels such as Zn, Mg, Cu, and Fe in the kidneys of diabetic rats that had been induced with streptozotocin. Thirty-two Wistar albino male rats were equally divided into four groups. The first group was used as a control. The second group of rats developed diabetes after receiving a single intraperitoneal dose of STZ. The third and fourth groups of rats had STZ-induced diabetes and received daily dosages of irbesartan (15 mg/kg b.w/day) and perindopril (6 mg/kg b.w/day) treatment, respectively. Biochemical analysis of the kidneys showed a distinct increase in oxidative stress, indicated by heightened levels of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activities, as well as reduced glutathione (GSH) levels in the kidneys of diabetic rats. In the kidneys of diabetic rats, the mean levels of Fe and Cu were found to be significantly higher than those of the control group. Additionally, the mean levels of Zn and Mg were significantly lower in the diabetic rats compared to the control rats. Both perindopril and irbesartan decreased significantly MDA content and increased SOD activities and GSH levels in the kidneys of rats with diabetes. The Zn and Mg concentrations in the kidneys of diabetic rats treated with perindopril and irbesartan were markedly higher than in untreated STZ-diabetic rats, while the Cu and Fe concentrations were significantly lower. The urinary excretion of rats treated with perindopril and irbesartan showed a pronounced increase in Cu levels, along with a significant reduction in Zn and Mg levels. Although diabetic rats demonstrated degenerative morphological alterations in their kidneys, both therapies also improved diabetes-induced histopathological modifications in the kidneys. Finally, the present results suggest that manipulating the levels of Zn, Mg, Cu, and Fe - either through ACE inhibition or by blocking AT1 receptors - could be advantageous in reducing lipid peroxidation and increasing antioxidant concentration in the kidneys of diabetic rats.

Vicinal diaryl pyrazole with tetrazole/urea scaffolds as selective angiotensin converting enzyme-1/cyclooxygenase-2 inhibitors: Design, synthesis, anti-hypertensive, anti-fibrotic, and anti-inflammatory.

As a hybrid weapon, two novel series of pyrazoles, 16a-f and 17a-f, targeting both COX-2 and ACE-1-N-domain, were created and their anti-inflammatory, anti-hypertensive, and anti-fibrotic properties were evaluated. In vitro, 17b and 17f showed COX-2 selectivity (SI = 534.22 and 491.90, respectively) compared to celecoxib (SI = 326.66) and NF-κB (IC50 1.87 and 2.03 µM, respectively). 17b (IC50 0.078 µM) and 17 f (IC50 0.094 µM) inhibited ACE-1 comparable to perindopril (PER) (IC50 0.048 µM). In vivo, 17b decreased systolic blood pressure by 18.6%, 17b and 17f increased serum NO levels by 345.8%, and 183.2%, respectively, increased eNOS expression by 0.97 and 0.52 folds, respectively and reduced NF-κB-p65 and P38-MAPK expression by -0.62, -0.22, -0.53, and -0.24 folds, respectively compared to  l-NAME (-0.34, -0.45 folds decline in NF-κB-p65 and P38-MAPK, respectively). 17b reduced ANG-II expression which significantly reversed the cardiac histological changes induced by L-NAME.

Angiotensinergic neurotransmission in the bed nucleus of the stria terminalis is involved in cardiovascular responses to acute restraint stress in rats.

The brain angiotensin II acting via AT1 receptors is a prominent mechanism involved in physiological and behavioral responses during aversive situations. The AT2 receptor has also been implicated in stress responses, but its role was less explored. Despite these pieces of evidence, the brain sites related to control of the changes during aversive threats by the brain renin-angiotensin system (RAS) are poorly understood. The bed nucleus of the stria terminalis (BNST) is a limbic structure related to the cardiovascular responses by stress, and components of the RAS system were identified in this forebrain region. Therefore, we investigated the role of angiotensinergic neurotransmission present within the BNST acting via local AT1 and AT2 receptors in cardiovascular responses evoked by an acute session of restraint stress in rats. For this, rats were subjected to bilateral microinjection of either the angiotensin-converting enzyme inhibitor captopril, the selective AT1 receptor antagonist losartan, or the selective AT2 receptor antagonist PD123319 before they underwent the restraint stress session. We observed that BNST treatment with captopril reduced the decrease in tail skin temperature evoked by restraint stress, without affecting the pressor and tachycardic responses. Local AT2 receptor antagonism within the BNST reduced both the tachycardia and the drop in tail skin temperature during restraint. Bilateral microinjection of losartan into the BNST did not affect the restraint-evoked cardiovascular changes. Taken together, these data indicate an involvement of BNST angiotensinergic neurotransmission acting via local AT2 receptors in cardiovascular responses during stressful situations.

Sepsis modulates aortic AT1 and P2Y6 receptors to produce vascular hyporeactivity in mice.

PURPOSE: Hyporeactivity to vasopressors leading to multiple organ failure is a serious clinical implication in sepsis. Though the regulatory role of purinoceptors in inflammation is reported, their involvement in sepsis-induced vasoplegia is still unknown. Thus we investigated the effect of sepsis on vascular AT1 and P2Y6 receptors. MATERIALS AND METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Vascular reactivity was assessed by organ bath study and aortic mRNA expression of AT1 and P2Y6 was quantified by qRT-PCR. RESULTS: Both angiotensin-II and UDP produced higher contractions in the absence of endothelium as well as following inhibition of nitric oxide synthase. Angiotensin-II mediated aortic contraction was antagonized by losartan (AT1 antagonist), but not by PD123319 (AT2 antagonist) whereas UDP-induced aortic contraction was significantly inhibited by MRS2578 (P2Y6 antagonist). In addition, MRS2578 significantly inhibited the contractile response of Ang-II. Compared to SO mice, angiotensin-II and UDP-induced maximum contraction were found to be significantly attenuated in sepsis. Accordingly, aortic mRNA expression of AT1a receptors was significantly down-regulated while that of P2Y6 receptors was significantly increased in sepsis. 1400 W (a selective iNOS inhibitor) significantly reversed angiotensin-II-induced vascular hyporeactivity in sepsis without affecting UDP-induced hypo-reactivity. CONCLUSION: Sepsis-induced vascular hyporeactivity to angiotensin-II is mediated by enhanced expression of iNOS. Moreover, AT1R-P2Y6 cross talk/heterodimerization could be a novel target for regulating vascular dysfunction in sepsis.

Effects of intrarenal angiotensin 1-7 infusion on renal haemodynamic and excretory function in anaesthetised two-kidney one-clip and deoxycorticosterone acetate-salt hypertensive rats.

NEW FINDINGS: What is the central question of this study? Are renal functional responses to intrarenal angiotensin 1-7 (Ang (1-7)) infusion dependent on the level of the endogenous renin-angiotensin system (RAS) in the two-kidney one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt animal models of hypertension? What is the main finding and its importance? The renal actions of Ang (1-7) are dependent on the relative endogenous levels of each arm of the classical angiotensin II-angiotensin II type 1 receptor (AT1 R) axis and those of the Ang (1-7)-Mas receptor axis. These findings support the hypothesis that a balance exists between the intrarenal classical and novel arms of the RAS, and in particular the relative abundance of AT1 R to Mas receptor, which may to a large extent determine the renal excretory response to Ang (1-7) infusion. ABSTRACT: This study investigated the action of angiotensin 1-7 (Ang (1-7)) on renal haemodynamic and excretory function in the two-kidney one-clip (2K1C) and deoxycorticosterone acetate (DOCA)-salt rat models of hypertension, in which the endogenous renin-angiotensin system (RAS) activity was likely to be raised or lowered, respectively. Rats were anaesthetised and prepared for the measurement of mean arterial pressure and kidney function during renal interstitial infusion of Ang (1-7) or saline. Kidney tissue concentrations of angiotensin II (Ang II) and Ang (1-7) were determined. Intrarenal infusion of Ang (1-7) into the clipped kidney of 2K1C rats increased urine flow (UV), absolute (UNa V) and fractional sodium (FENa ) excretions by 110%, 214% and 147%, respectively. Renal Ang II concentrations of the clipped kidney were increased with no major changes in Ang (1-7) concentration. By contrast, Ang (1-7) infusion decreased UV, UNa V, and FENa by 27%, 24% and 21%, respectively in the non-clipped kidney in which tissue Ang (1-7) concentrations were increased, but renal Ang II concentrations were unchanged compared to sham animals. Ang (1-7) infusion in DOCA-salt rats had minimal effects on glomerular filtration rate but significantly decreased UV, UNa V and FENa by ∼30%. Renal Ang (1-7) concentrations were higher and Ang II concentrations were lower in DOCA-salt rats compared to sham rats. These findings demonstrate that the intrarenal infusion of exogenous Ang (1-7) elicits different renal excretory responses the magnitude of which is dependent on the balance between the endogenous renal Ang II-AT1 receptor axis and Ang (1-7)-Mas receptor axis.

The AT1/AT2 Receptor Equilibrium Is a Cornerstone of the Regulation of the Renin Angiotensin System beyond the Cardiovascular System.

The AT1 receptor has mainly been associated with the pathological effects of the renin-angiotensin system (RAS) (e.g., hypertension, heart and kidney diseases), and constitutes a major therapeutic target. In contrast, the AT2 receptor is presented as the protective arm of this RAS, and its targeting via specific agonists is mainly used to counteract the effects of the AT1 receptor. The discovery of a local RAS has highlighted the importance of the balance between AT1/AT2 receptors at the tissue level. Disruption of this balance is suggested to be detrimental. The fine tuning of this balance is not limited to the regulation of the level of expression of these two receptors. Other mechanisms still largely unexplored, such as S-nitrosation of the AT1 receptor, homo- and heterodimerization, and the use of AT1 receptor-biased agonists, may significantly contribute to and/or interfere with the settings of this AT1/AT2 equilibrium. This review will detail, through several examples (the brain, wound healing, and the cellular cycle), the importance of the functional balance between AT1 and AT2 receptors, and how new molecular pharmacological approaches may act on its regulation to open up new therapeutic perspectives.

Oxidized LDL but not angiotensin II induces cardiomyocyte hypertrophic responses through the interaction between LOX-1 and AT1 receptors.

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

Regulation of total LC3 levels by angiotensin II in vascular smooth muscle cells.

Hypertension is associated with high circulating angiotensin II (Ang II). We have reported that autophagy regulates Ang II-induced vascular smooth muscle cell (VSMC) hypertrophy, but the mechanism mediating this effect is still unknown. Therefore, we studied how Ang II regulates LC3 levels in VSMCs and whether Bag3, a co-chaperone known to regulate LC3 total levels, may be involved in the effects elicited by Ang II. A7r5 cell line or rat aortic smooth muscle cell (RASMC) primary culture were stimulated with Ang II 100 nM for 24 h and LC3 I, LC3 II and Bag3 protein levels were determined by Western blot. MAP1LC3B mRNA levels were assessed by RT-qPCR. Ang II increased MAP1LC3B mRNA levels and protein levels of LC3 I, LC3 II and total LC3 (LC3 I + LC3 II). Cycloheximide, but not actinomycin D, abolished LC3 II and total LC3 increase elicited by Ang II in RASMCs. In A7r5 cells, cycloheximide prevented the Ang II-mediated increase of LC3 I and total LC3, but not LC3 II. Moreover, Ang II increased Bag3 levels, but this increase was not observed upon co-administration with either losartan 1 µM (AT1R antagonist) or Y-27632 10 µM (ROCK inhibitor). These results suggest that Ang II may regulate total LC3 content through transcriptional and translational mechanisms. Moreover, Bag3 is increased in response to Ang II by a AT1R/ROCK signalling pathway. These data provide preliminary evidence suggesting that Ang II may stimulate autophagy in VSMCs by increasing total LC3 content and LC3 processing.

Angiotensin II and AT1a Receptors in the Proximal Tubules of the Kidney: New Roles in Blood Pressure Control and Hypertension.

Contrary to public perception, hypertension remains one of the most important public health problems in the United States, affecting 46% of adults with increased risk for heart attack, stroke, and kidney diseases. The mechanisms underlying poorly controlled hypertension remain incompletely understood. Recent development in the Cre/LoxP approach to study gain or loss of function of a particular gene has significantly helped advance our new insights into the role of proximal tubule angiotensin II (Ang II) and its AT1 (AT1a) receptors in basal blood pressure control and the development of Ang II-induced hypertension. This novel approach has provided us and others with an important tool to generate novel mouse models with proximal tubule-specific loss (deletion) or gain of the function (overexpression). The objective of this invited review article is to review and discuss recent findings using novel genetically modifying proximal tubule-specific mouse models. These new studies have consistently demonstrated that deletion of AT1 (AT1a) receptors or its direct downstream target Na+/H+ exchanger 3 (NHE3) selectively in the proximal tubules of the kidney lowers basal blood pressure, increases the pressure-natriuresis response, and induces natriuretic responses, whereas overexpression of an intracellular Ang II fusion protein or AT1 (AT1a) receptors selectively in the proximal tubules increases proximal tubule Na+ reabsorption, impairs the pressure-natriuresis response, and elevates blood pressure. Furthermore, the development of Ang II-induced hypertension by systemic Ang II infusion or by proximal tubule-specific overexpression of an intracellular Ang II fusion protein was attenuated in mutant mice with proximal tubule-specific deletion of AT1 (AT1a) receptors or NHE3. Thus, these recent studies provide evidence for and new insights into the important roles of intratubular Ang II via AT1 (AT1a) receptors and NHE3 in the proximal tubules in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

Angiotensin AT1 receptor antagonism by losartan stimulates adipocyte browning via induction of apelin.

Adipocyte browning appears to be a potential therapeutic strategy to combat obesity and related metabolic disorders. Recent studies have shown that apelin, an adipokine, stimulates adipocyte browning and has negative cross-talk with angiotensin II receptor type 1 (AT1 receptor) signaling. Here, we report that losartan, a selective AT1 receptor antagonist, induces browning, as evidenced by an increase in browning marker expression, mitochondrial biogenesis, and oxygen consumption in murine adipocytes. In parallel, losartan up-regulated apelin expression, concomitant with increased phosphorylation of protein kinase B and AMP-activated protein kinase. However, the siRNA-mediated knockdown of apelin expression attenuated losartan-induced browning. Angiotensin II cotreatment also inhibited losartan-induced browning, suggesting that AT1 receptor antagonism-induced activation of apelin signaling may be responsible for adipocyte browning induced by losartan. The in vivo browning effects of losartan were confirmed using both C57BL/6J and ob/ob mice. Furthermore, in vivo apelin knockdown by adeno-associated virus carrying-apelin shRNA significantly inhibited losartan-induced adipocyte browning. In summary, these data suggested that AT1 receptor antagonism by losartan promotes the browning of white adipocytes via the induction of apelin expression. Therefore, apelin modulation may be an effective strategy for the treatment of obesity and its related metabolic disorders.

Telmisartan induces a specific gut microbiota signature which may mediate its antiobesity effect.

Telmisartan prevents diet-induced obesity (DIO) in rodents. Given that the precise underlying mechanism is not known, we examined whether a gut-related mechanism might be involved. Sprague-Dawley rats received cafeteria diet (CD) for 3 months to develop DIO and were administered either telmisartan (8 mg/kgbw) or vehicle. In addition, pair-fed (PF) rats received CD adjusted to TEL and control rats (CON) only received chow. Stool samples were analysed by 16 S rRNA gene amplicon sequencing. CD-fed rats became obese while TEL, PF and CON rats remained lean. Alpha diversity analyses indicated that bacterial diversity was similar before the study but changed over time. Beta diversity revealed a time-, CD- and telmisartan-dependent effect. The Firmicutes/Bacteroidetes ratio and the abundance of Blautia, Allobaculum and Parasutterella were higher in DIO and PF than in CON, but not in TEL. Enterotype (ET)-like clustering analyses, Kleinberg's hub network scoring and random forest analyses also indicated that telmisartan induced a specific signature of gut microbiota. In response to stool transfer from telmisartan-pre-treated donor to high-fat fed acceptor mice, body weight gain was slightly attenuated. We attribute the anti-obesity action of telmisartan treatment to diet-independent alterations in gut microbiota as the microbiota from telmisartan-treated, CD-fed rats clearly differed from those of DIO and PF rats. ET-like clustering network, random forest classification and the higher stability in bacterial co-occurrence network analyses indicate that there is more than one indicator species for telmisartan's specific signature, which is further strengthened by the fact that we cannot identify a single indicator species.

Activation of the angiotensin II receptor promotes autophagy in renal proximal tubular cells and affords protection from ischemia/reperfusion injury.

Roles of the renin-angiotensin system in autophagy and ischemia/reperfusion (I/R) injury in the kidney have not been fully characterized. Here we examined the hypothesis that modest activation of the angiotensin II (Ang II) receptor upregulates autophagy and increases renal tolerance to I/R injury. Sprague-Dawley rats were assigned to treatment with a vehicle or a non-pressor dose of Ang II (200 ng/kg/min) for 72 h before 30-min renal I/R. LC3-immunohistochemistry showed that Ang II treatment increased autophagosomes in proximal tubular cells by 2.7 fold. In Ang II-pretreated rats, autophagosomes were increased by 2.5 fold compared to those in vehicle-treated rats at 4 h after I/R, when phosphorylation of Akt and S6 was suppressed and ULK1-Ser555 phosphorylation was increased. Serum creatinine and urea nitrogen levels, incidence of oliguria, and histological score of tubular necrosis at 24 h after I/R were attenuated by Ang II-pretreatment. In NRK-52E cells, Ang II induced LC3-II upregulation, which was inhibited by losartan but not by A779. The results indicate that a non-pressor dose of Ang-II promotes autophagy via ULK1-mediated signaling in renal tubular cells and attenuates renal I/R injury. The AT1 receptor, but not the Mas receptor, contributes to Ang-II-induced autophagy and presumably also to the renoprotection.

Role of pregnancy on insulin-induced vasorelaxation: the influence of angiotensin II receptors.

Insulin resistance is a feature of pregnancy and is associated with increased levels of angiotensin II (Ang II) and insulin. Therefore, pregnancy may change insulin-induced vasodilation through changes in Ang II receptors. Insulin-induced vasorelaxation was evaluated in phenylephrine-precontracted aortic rings of pregnant and non-pregnant rats, using a conventional isolated organ preparation. Experiments were performed in thoracic or abdominal aorta rings with or without endothelium in the presence and absence of NG-nitro-L-arginine methyl ester (L-NAME) (10-5 M), losartan (10-7 M), or PD123319 (10-7 M). AT1 and AT2 receptor expressions were detected by immunohistochemistry. Insulin-induced vasodilation was endothelium- and nitric oxide-dependent and decreased in the thoracic aorta but increased in the abdominal segment of pregnant rats. The insulin's vasorelaxant effect was increased by losartan mainly on the thoracic aorta. PD123319 decreased insulin-induced vasorelaxation mainly in the pregnant rat abdominal aorta. AT1 receptor expression was decreased while AT2 receptor expression was increased by pregnancy. In conclusion, pregnancy changes insulin-induced vasorelaxation. Moreover, insulin vasodilation is tonically inhibited by AT1 receptors, while AT2 receptors appear to have an insulin-sensitizing effect. The role of pregnancy and Ang II receptors differ depending on the aorta segment. These results shed light on the role of pregnancy and Ang II receptors on the regulation of insulin-mediated vasodilation.

Targeting the Angiotensin II Type 1 Receptor in Cerebrovascular Diseases: Biased Signaling Raises New Hopes.

The physiological and pathophysiological relevance of the angiotensin II type 1 (AT1) G protein-coupled receptor no longer needs to be proven in the cardiovascular system. The renin-angiotensin system and the AT1 receptor are the targets of several classes of therapeutics (such as angiotensin converting enzyme inhibitors or angiotensin receptor blockers, ARBs) used as first-line treatments in cardiovascular diseases. The importance of AT1 in the regulation of the cerebrovascular system is also acknowledged. However, despite numerous beneficial effects in preclinical experiments, ARBs do not induce satisfactory curative results in clinical stroke studies. A better understanding of AT1 signaling and the development of biased AT1 agonists, able to selectively activate the ß-arrestin transduction pathway rather than the Gq pathway, have led to new therapeutic strategies to target detrimental effects of AT1 activation. In this paper, we review the involvement of AT1 in cerebrovascular diseases as well as recent advances in the understanding of its molecular dynamics and biased or non-biased signaling. We also describe why these alternative signaling pathways induced by ß-arrestin biased AT1 agonists could be considered as new therapeutic avenues for cerebrovascular diseases.

Stearoyl-CoA Desaturase (SCD) Induces Cardiac Dysfunction with Cardiac Lipid Overload and Angiotensin II AT1 Receptor Protein Up-Regulation.

Heart failure is a major cause of death worldwide with insufficient treatment options. In the search for pathomechanisms, we found up-regulation of an enzyme, stearoyl-CoA desaturase 1 (Scd1), in different experimental models of heart failure induced by advanced atherosclerosis, chronic pressure overload, and/or volume overload. Because the pathophysiological role of Scd1/SCD in heart failure is not clear, we investigated the impact of cardiac SCD upregulation through the generation of C57BL/6-Tg(MHCSCD)Sjaa mice with myocardium-specific expression of SCD. Echocardiographic examination showed that 4.9-fold-increased SCD levels triggered cardiac hypertrophy and symptoms of heart failure at an age of eight months. Tg-SCD mice had a significantly reduced left ventricular cardiac ejection fraction of 25.7 ± 2.9% compared to 54.3 ± 4.5% of non-transgenic B6 control mice. Whole-genome gene expression profiling identified up-regulated heart-failure-related genes such as resistin, adiponectin, and fatty acid synthase, and type 1 and 3 collagens. Tg-SCD mice were characterized by cardiac lipid accumulation with 1.6- and 1.7-fold-increased cardiac contents of saturated lipids, palmitate, and stearate, respectively. In contrast, unsaturated lipids were not changed. Together with saturated lipids, apoptosis-enhancing p53 protein contents were elevated. Imaging by autoradiography revealed that the heart-failure-promoting and membrane-spanning angiotensin II AT1 receptor protein of Tg-SCD hearts was significantly up-regulated. In transfected HEK cells, the expression of SCD increased the number of cell-surface angiotensin II AT1 receptor binding sites. In addition, increased AT1 receptor protein levels were detected by fluorescence spectroscopy of fluorescent protein-labeled AT1 receptor-Cerulean. Taken together, we found that SCD promotes cardiac dysfunction with overload of cardiotoxic saturated lipids and up-regulation of the heart-failure-promoting AT1 receptor protein.

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-ß1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

The role of the renin-angiotensin system in skin physiology and pathophysiology.

Since its first description around the year 2000, the local renin-angiotensin system (RAS) in skin has been subject of an increasing number of studies with many additions over the last two to three years. A focus of research has been investigations on the role of cutaneous angiotensin receptors and locally synthesised angiotensin II in wound healing, in dermatoses associated with skin fibrosis and in melanoma. This review will provide an introduction into the RAS with emphasis on information relevant for the cutaneous RAS. It will discuss the role of the RAS in skin physiology, followed by a detailed review of the existing literature addressing the role of local angiotensin II and angiotensin AT1 and AT2 receptors in wound healing and in various skin diseases such as hypertrophic scars/keloids, scleroderma, dystrophic epidermolysis bullosa, Dupuytren's disease, squamous cell carcinoma, melanoma and psoriasis. In a final section, the potential relevance of drugs, which interfere with the RAS, for future therapy of dermatological disorders is discussed. Collectively, research about the RAS in skin can currently be described as an area, which has gained increasing attention by basic researchers, thus resulting in a multitude of preclinical studies pointing to the potential relevance of components of the RAS as drug targets in dermatological diseases. With a few small clinical studies already performed successfully for indications such as hypertrophic scars and keloids, it can be said that the skin RAS is now in the critical phase of translation from preclinical evidence to clinical use.

Preeclampsia as predisposing factor for hypertensive retinopathy: Participation by the RAAS and angiogenic factors.

Preeclampsia (PE) is a hypertensive complication of pregnancy. Its cause is still unknown and it could be a risk factor for future ophthalmic problems. Retinal vascular bed alterations have been described as a consequence of PE, suggesting a retinopathy. Factors related to angiogenesis and vascular permeability, such as vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) or components of the renin angiotensin aldosterone system (RAAS), prorrenin/renin receptor ((P)RR) and angiotensin II type I receptor (AT1R) have been located in the retina, participating in other retinopathies, but it is unknown if they could participate in PE. Our aim was to elucidate whether VEGF, PEDF, (P)RR and AT1R could be modified during PE and during hypertension induced in rats with a history of PE. We used female Wistar rats and subrrenal aortic coarctation to induce PE, and after delivery, we induced a second hit by Nω-nitro-L-arginine methyl ester (L-NAME) administration. We measured blood pressure, proteinuria and pups development. In both models, eye fundal exploration and immunoblot for VEGF, PEDF, (P)RR and AT1R were performed. We found that the development of hypertension occurred faster in previously PE rats than in normal animals. VEGF, PEDF, (P)RR and AT1R were increased in PE, but in L-NAME-induced hypertension only (P)RR and AT1R were altered. Eye fundal data indicated that PE induced a level I retinopathy, but L-NAME induced a faster and more severe retinopathy in previously PE animals compared to previously normal pregnancy rats. These results indicate that PE predisposes to development of a faster and more severe retinopathy after a second hit. They also suggest that VEGF and PEDF seem to participate only in PE retinopathy, but in both models, RAAS components seem to have a more critical participation.