Beta-Arrestin1 Prevents Preeclampsia by Downregulation of Mechanosensitive AT1-B2 Receptor Heteromers.

Preeclampsia is the most frequent pregnancy-related complication worldwide with no cure. While a number of molecular features have emerged, the underlying causal mechanisms behind the disorder remain obscure. Here, we find that increased complex formation between angiotensin II AT1 and bradykinin B2, two G protein-coupled receptors with opposing effects on blood vessel constriction, triggers symptoms of preeclampsia in pregnant mice. Aberrant heteromerization of AT1-B2 led to exaggerated calcium signaling and high vascular smooth muscle mechanosensitivity, which could explain the onset of preeclampsia symptoms at late-stage pregnancy as mechanical forces increase with fetal mass. AT1-B2 receptor aggregation was inhibited by beta-arrestin-mediated downregulation. Importantly, symptoms of preeclampsia were prevented by transgenic ARRB1 expression or a small-molecule drug. Because AT1-B2 heteromerization was found to occur in human placental biopsies from pregnancies complicated by preeclampsia, specifically targeting AT1-B2 heteromerization and its downstream consequences represents a promising therapeutic approach.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

Distinctive Activation Mechanism for Angiotensin Receptor Revealed by a Synthetic Nanobody.

The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.

Multidimensional Tracking of GPCR Signaling via Peroxidase-Catalyzed Proximity Labeling.

G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.

Structure of the Angiotensin receptor revealed by serial femtosecond crystallography.

Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.

The central renin-angiotensin system: A genetic pathway, functional decoding, and selective target engagement characterization in humans.

Accumulating evidence suggests that the brain renin angiotensin system (RAS) plays a pivotal role in the regulation of cognition and behavior as well as in the neuropathology of neurological and mental disorders. The angiotensin II type 1 receptor (AT1R) mediates most functional and neuropathology-relevant actions associated with the central RAS. However, an overarching comprehension to guide translation and utilize the therapeutic potential of the central RAS in humans is currently lacking. We conducted a comprehensive characterization of the RAS using an innovative combination of transcriptomic gene expression mapping, image-based behavioral decoding, and pre-registered randomized controlled discovery-replication pharmacological resting-state functional magnetic resonance imaging (fMRI) trials (N = 132) with a selective AT1R antagonist. The AT1R exhibited a particular dense expression in a subcortical network encompassing the thalamus, striatum, and amygdalo-hippocampal formation. Behavioral decoding of the AT1R gene expression brain map showed an association with memory, stress, reward, and motivational processes. Transient pharmacological blockade of the AT1R further decreased neural activity in subcortical systems characterized by a high AT1R expression, while increasing functional connectivity in the cortico-basal ganglia-thalamo-cortical circuitry. Effects of AT1R blockade on the network level were specifically associated with the transcriptomic signatures of the dopaminergic, opioid, acetylcholine, and corticotropin-releasing hormone signaling systems. The robustness of the results was supported in an independent pharmacological fMRI trial. These findings present a biologically informed comprehensive characterization of the central AT1R pathways and their functional relevance on the neural and behavioral level in humans.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

A molecular mechanism for angiotensin II receptor blocker-mediated slit membrane protection: Angiotensin II increases nephrin endocytosis via AT1-receptor-dependent ERK 1/2 activation.

Albuminuria is characterized by a disruption of the glomerular filtration barrier, which is composed of the fenestrated endothelium, the glomerular basement membrane, and the slit diaphragm. Nephrin is a major component of the slit diaphragm. Apart from hemodynamic effects, Ang II enhances albuminuria by ß-Arrestin2-mediated nephrin endocytosis. Blocking the AT1 receptor with candesartan and irbesartan reduces the Ang II-mediated nephrin-ß-Arrestin2 interaction. The inhibition of MAPK ERK 1/2 blocks Ang II-enhanced nephrin-ß-Arrestin2 binding. ERK 1/2 signaling, which follows AT1 receptor activation, is mediated by G-protein signaling, EGFR transactivation, and ß-Arrestin2 recruitment. A mutant AT1 receptor defective in EGFR transactivation and ß-Arrestin2 recruitment reduces the Ang II-mediated increase in nephrin ß-Arrestin2 binding. The mutation of ß-Arrestin2K11,K12, critical for AT1 receptor binding, completely abrogates the interaction with nephrin, independent of Ang II stimulation. ß-Arrestin2K11R,K12R does not influence nephrin cell surface expression. The data presented here deepen our molecular understanding of a blood-pressure-independent molecular mechanism of AT-1 receptor blockers (ARBs) in reducing albuminuria.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Angiotensin II enhances bacterial clearance via myeloid signaling in a murine sepsis model.

Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.

The Angiotensin AT2 Receptor: From a Binding Site to a Novel Therapeutic Target.

Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.

The Angiotensin Antagonist Losartan Modulates Social Reward Motivation and Punishment Sensitivity via Modulating Midbrain-Striato-Frontal Circuits.

Social deficits and dysregulations in dopaminergic midbrain-striato-frontal circuits represent transdiagnostic symptoms across psychiatric disorders. Animal models suggest that interactions between the dopamine (DA) and renin-angiotensin system (RAS) may modulate learning and reward-related processes. The present study therefore examined the behavioral and neural effects of the Angiotensin II type 1 receptor (AT1R) antagonist losartan on social reward and punishment processing in humans. A preregistered randomized double-blind placebo-controlled between-subject pharmacological design was combined with a social incentive delay (SID) functional MRI (fMRI) paradigm during which subjects could avoid social punishment or gain social reward. Healthy volunteers received a single-dose of losartan (50 mg, n = 43, female = 17) or placebo (n = 44, female = 20). We evaluated reaction times (RTs) and emotional ratings as behavioral and activation and functional connectivity as neural outcomes. Relative to placebo, losartan modulated the reaction time and arousal differences between social punishment and social reward. On the neural level the losartan-enhanced motivational salience of social rewards was accompanied by stronger ventral striatum-prefrontal connectivity during reward anticipation. Losartan increased the reward-neutral difference in the ventral tegmental area (VTA) and attenuated VTA associated connectivity with the bilateral insula in response to punishment during the outcome phase. Thus, losartan modulated approach-avoidance motivation and emotional salience during social punishment versus social reward via modulating distinct core nodes of the midbrain-striato-frontal circuits. The findings document a modulatory role of the renin-angiotensin system in these circuits and associated social processes, suggesting a promising treatment target to alleviate social dysregulations.SIGNIFICANCE STATEMENT Social deficits and anhedonia characterize several mental disorders and have been linked to the midbrain-striato-frontal circuits of the brain. Based on initial findings from animal models we here combine the pharmacological blockade of the Angiotensin II type 1 receptor (AT1R) via losartan with functional MRI (fMRI) to demonstrate that AT1R blockade enhances the motivational salience of social rewards and attenuates the negative impact of social punishment via modulating the communication in the midbrain-striato-frontal circuits in humans. The findings demonstrate for the first time an important role of the AT1R in social reward processing in humans and render the AT1R as promising novel treatment target for social and motivational deficits in mental disorders.

Reduced cardiac antioxidant defenses mediate increased susceptibility to workload-induced myocardial injury in males with genetic cardiomyopathy.

Ongoing cardiomyocyte injury is a major mechanism in the progression of heart failure, particularly in dystrophic hearts. Due to the poor regenerative capacity of the adult heart, cardiomyocyte death results in the permanent loss of functional myocardium. Understanding the factors contributing to myocyte injury is essential for the development of effective heart failure therapies. As a model of persistent cardiac injury, we examined mice lacking ß-sarcoglycan (ß-SG), a key component of the dystrophin glycoprotein complex (DGC). The loss of the sarcoglycan complex markedly compromises sarcolemmal integrity in this ß-SG-/- model. Our studies aim to characterize the mechanisms underlying dramatic sex differences in susceptibility to cardiac injury in ß-SG-/- mice. Male ß-SG-/- hearts display significantly greater myocardial injury and death following isoproterenol-induced cardiac stress than female ß-SG-/- hearts. This protection of females was independent of ovarian hormones. Male ß-SG-/- hearts displayed increased susceptibility to exogenous oxidative stress and were significantly protected by angiotensin II type 1 receptor (AT1R) antagonism. Increasing general antioxidative defenses or increasing the levels of S-nitrosylation both provided protection to the hearts of ß-SG-/- male mice. Here we demonstrate that increased susceptibility to oxidative damage leads to an AT1R-mediated amplification of workload-induced myocardial injury in male ß-SG-/- mice. Improving oxidative defenses, specifically by increasing S-nitrosylation, provided protection to the male ß-SG-/- heart from workload-induced injury. These studies describe a unique susceptibility of the male heart to injury and may contribute to the sex differences in other forms of cardiac injury.

Bidirectional relation between dipeptidyl peptidase 4 and angiotensin II type I receptor signaling.

Cardiometabolic diseases are often associated with heightened levels of angiotensin II (Ang II), which accounts for the observed oxidative stress, inflammation, and fibrosis. Accumulating evidence indicates a parallel upregulation of dipeptidyl dipeptidase 4 (DPP4) activity in cardiometabolic diseases, with its inhibition shown to mitigate oxidative stress, inflammation, and fibrosis. These findings highlight an overlap between the pathophysiological mechanisms used by Ang II and DPP4. Recent evidence demonstrates that targeted inhibition of DPP4 prevents the rise in Ang II and its associated molecules in experimental models of cardiometabolic diseases. Similarly, inhibitors of the angiotensin I-converting enzyme (ACE) or Ang II type 1 receptor (AT1R) blockers downregulate DPP4 activity, establishing a bidirectional relationship between DPP4 and Ang II. Here, we discuss the current evidence supporting the cross talk between Ang II and DPP4, along with the potential mechanisms promoting this cross regulation. A comprehensive analysis of this bidirectional relationship across tissues will advance our understanding of how DPP4 and Ang II collectively promote the development and progression of cardiometabolic diseases.

Nanobody-Mediated Dualsteric Engagement of the Angiotensin Receptor Broadens Biased Ligand Pharmacology.

Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or ß-arrestin (TRV027). While the nanobody itself acts as a probe-specific neutral or negative allosteric ligand of N-terminally 6e-tagged AT1R, nanobody conjugation to orthosteric ligands had varying effects on Gq dissociation and ß-arrestin plasma membrane recruitment. The potency of certain AngII analogs was enhanced up to 100-fold, and some conjugates behaved as partial agonists, with up to a 5-fold decrease in maximal efficacy. Nanobody conjugation also biased the signaling of TRV055 and TRV056 toward Gq, suggesting that Gq bias at AT1R can be modulated through molecular mechanisms distinct from those previously elucidated. Both competition radioligand binding experiments and functional assays demonstrated that orthosteric antagonists (angiotensin receptor blockers) act as non-competitive inhibitors of all these nanobody-peptide conjugates. This proof-of-principle study illustrates the array of pharmacological patterns that can be achieved by incorporating neutral or negative allosteric pharmacophores into dualsteric ligands. Nanobodies directed toward linear epitopes could provide a rich source of allosteric reagents for this purpose. SIGNIFICANCE STATEMENT: Here we engineer bitopic (dualsteric) ligands for epitope-tagged angiotensin II type 1 receptor by conjugating angiotensin II or its biased analogs to an epitope-specific nanobody (antibody fragment). Our data demonstrate that nanobody-mediated interactions with the receptor N-terminus endow angiotensin analogs with properties of allosteric modulators and provide a novel mechanism to increase the potency, modulate the maximal effect, or alter the bias of ligands.

miR-125a-5p/miR-125b-5p contributes to pathological activation of angiotensin II-AT1R in mouse distal convoluted tubule cells by the suppression of Atrap.

The renin-angiotensin system plays a crucial role in the regulation of blood pressure. Activation of the angiotensin II (Ang II)-Ang II type 1 receptor (AT1R) signaling pathway contributes to the pathogenesis of hypertension and subsequent organ damage. AT1R-associated protein (ATRAP) has been identified as an endogenous inhibitory protein of the AT1R pathological activation. We have shown that mouse Atrap (Atrap) represses various Ang II-AT1R-mediated pathologies, including hypertension in mice. The expression of human ATRAP (ATRAP)/Atrap can be altered in various pathological states in humans and mice, such as Ang II stimulation and serum starvation. However, the regulatory mechanisms of ATRAP/Atrap are not yet fully elucidated. miRNAs are 21 to 23 nucleotides of small RNAs that post-transcriptionally repress gene expression. Single miRNA can act on hundreds of target mRNAs, and numerous miRNAs have been identified as the Ang II-AT1R signaling-associated disease phenotype modulator, but nothing is known about the regulation of ATRAP/Atrap. In the present study, we identified miR-125a-5p/miR-125b-5p as the evolutionarily conserved miRNAs that potentially act on ATRAP/Atrap mRNA. Further analysis revealed that miR-125a-5p/miR-125b-5p can directly repress both ATRAP and Atrap. In addition, the inhibition of miR-125a-5p/miR-125b-5p resulted in the suppression of the Ang II-AT1R signaling in mouse distal convoluted tubule cells. Taken together, miR-125a-5p/miR-125b-5p activates Ang II-AT1R signaling by the suppression of ATRAP/Atrap. Our results provide new insights into the potential approaches for achieving the organ-protective effects by the repression of the miR-125 family associated with the enhancement of ATRAP/Atrap expression.

Acupoint catgut embedding attenuates oxidative stress and cognitive impairment in chronic cerebral ischemia by inhibiting the Ang II/AT1R/NOX axis.

Chronic cerebral ischemia (CCI) is a common neurological disorder, characterized by progressive cognitive impairment. Acupoint catgut embedding (ACE) represents a modern acupuncture form that has shown neuroprotective effects; nevertheless, its effects on CCI and the mechanisms remain largely unknown. Here, we aimed to explore the therapeutic action of ACE in CCI-induced cognitive impairment and its mechanisms. The cognitive function of CCI rats was determined using Morris water maze test, and histopathological changes in the brain were assessed through hematoxylin-eosin (HE) staining. To further explore the molecular mechanisms, the expression levels of oxidative stress markers and the Ang II/AT1R/NOX axis-associated molecules in the hippocampus were evaluated using enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry. Here, we observed that ACE treatment alleviated cognitive dysfunction and histopathological injury in CCI rats. Intriguingly, candesartan (an AT1R blocker) enhanced the beneficial effects of ACE on ameliorating cognitive impairment in CCI rats. Mechanistically, ACE treatment blocked the Ang II/AT1R/NOX pathway and subsequently suppressed oxidative stress, thus mitigating cognitive impairment in CCI. Our findings first reveal that ACE treatment could suppress cognitive impairment in CCI, which might be partly due to the suppression of Ang II/AT1R/NOX axis.

Placental ischemia-upregulated angiotensin II type 1 receptor in hypothalamic paraventricular nucleus contributes to hypertension in rat.

Preeclampsia (PE) is associated with increased angiotensin II sensitivity and poor neurological outcomes marked by temporal loss of neural control of blood pressure. Yet the role of centrally expressed angiotensin II type 1 receptor (AT1R) within the paraventricular nucleus of the hypothalamus (PVN) in the PE model is not understood. In a PE rat model with reduced placental perfusion pressure (RUPP) induced on gestational day 14 (GD14), the PVN expression and cellular localization of AT1R were assessed using immunofluorescence and western blotting. The sensitivity of RUPP to acute angiotensin II infusion was assessed. AT1R was antagonized by losartan (100 µg/kg/day) for 5 days intracerebroventricularly (ICV). Hemodynamic data and samples were collected on GD19 for further analysis. RUPP upregulated (p < 0.05) mRNA and protein of AT1R within the PVN and lowered (p < 0.05) circulating angiotensin II in rats. RUPP increased neural and microglial activation. Cellular localization assessment revealed that AT1R was primarily expressed in neurons and slightly in microglia and astrocytes. Infusion of 100 ng/kg as bolus increased the mean arterial pressure (MAP in mmHg) in both RUPP and Sham. ICV losartan infusion attenuated RUPP-increased MAP (113.6 ± 6.22 in RUPP vs. 92.16 ± 5.30 in RUPP + Los, p = 0.021) and the expression of nuclear transcription factor NF-κB, tyrosine hydroxylase (TH), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) in the PVN. Our data suggest that centrally expressed AT1R, within the PVN, contributes to placental ischemia-induced hypertension in RUPP rats highlighting its therapeutic potential in PE.

Angiotensin II involvement in the development and persistence of amphetamine-induced sensitization: Striatal dopamine reuptake implications.

Amphetamine (AMPH) exposure induces behavioural and neurochemical sensitization observed in rodents as hyperlocomotion and increased dopamine release in response to a subsequent dose. Brain Angiotensin II modulates dopaminergic neurotransmission through its AT1 receptors (AT1-R), positively regulating striatal dopamine synthesis and release. This work aims to evaluate the AT1-R role in the development and maintenance of AMPH-induced sensitization. Also, the AT1-R involvement in striatal dopamine reuptake was analysed. The sensitization protocol consisted of daily AMPH administration for 5 days and tested 21 days after withdrawal. An AT1-R antagonist, candesartan, was administered before or after AMPH exposure to evaluate the participation of AT1-R in the development and maintenance of sensitization, respectively. Sensitization was evaluated by locomotor activity and c-Fos immunostaining. Changes in dopamine reuptake kinetics were evaluated 1 day after AT1-R blockade withdrawal treatment, with or without the addition of AMPH in vitro. The social interaction test was performed as another behavioural output. Repeated AMPH exposure induced behavioural and neurochemical sensitization, which was prevented and reversed by candesartan. The AT1-R blockade increased the dopamine reuptake kinetics. Neither the AMPH administration nor the AT1-R blockade altered the performance of social interaction. Our results highlight the AT1-R's crucial role in AMPH sensitization. The enhancement of dopamine reuptake kinetics induced by the AT1-R blockade might attenuate the neuroadaptive changes that lead to AMPH sensitization and its self-perpetuation. Therefore, AT1-R is a prominent candidate as a target for pharmacological treatment of pathologies related to dopamine imbalance, including drug addiction and schizophrenia.

Significant Association of Candidate Genes (AGTR1 and TGF-Β1) Polymorphism with Diabetic Nephropathy in Diabetes Mellitus Type 2 Patients.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is one of the complications of diabetes mellitus (DM). This study aimed to investigate the association between genetic polymorphisms, specifically AGTR1 (rs5186) and TGF-ß1 (rs1800470), and the risk of developing Diabetic nephropathy (DN) in type 2 diabetes mellitus patients, compared to those without DN and healthy controls. METHODS: A case-control study was conducted on 165 diabetic patients (59 with diabetic nephropathy (DN) and 54 without DN (DM)), and 52 healthy controls (HC). The genotyping was done using amplification refractory mutation system method (ARMS-PCR). Age, gender, and duration of diabetes were matched across groups. Clinical parameters including FBS, RBS, HbA1C, creatinine, urea, SBP, DBP, total cholesterol, triglycerides, LDL, and BMI were assessed. RESULTS: Diabetic patients with nephropathy exhibited significantly higher levels of clinical parameters compared to those without nephropathy and healthy controls. The risk allele of AGTR1 , C (p <0.0001), and risk allele containing genotypes AC (p <0.0001) and CC (p - 0.0010) were significantly higher in DN patients compared to DM and HC groups. Similarly, the TGF-ß1 risk allele C (p - 0.0001), and corresponding genotypes TC (p - 0.0038) and CC (p - 0.0027) were significantly associated with increased risk of diabetic nephropathy compared to DM and HC groups. CONCLUSION: The data showed significant association of AGTR1 (rs5186) and TGF-ß1 (rs1800470) polymorphism with an increased risk of diabetic nephropathy in type 2 diabetes mellitus patients. More investigation will be required to disseminate the results, while increasing the samples size and using whole genome sequencing.