Renin-angiotensin system and inflammation update.

The most classical view of the renin-angiotensin system (RAS) emphasizes its role as an endocrine regulator of sodium balance and blood pressure. However, it has long become clear that the RAS has pleiotropic actions that contribute to organ damage, including modulation of inflammation. Angiotensin II (Ang II) activates angiotensin type 1 receptors (AT1R) to promote an inflammatory response and organ damage. This represents the pathophysiological basis for the successful use of RAS blockers to prevent and treat kidney and heart disease. However, other RAS components could have a built-in capacity to brake proinflammatory responses. Angiotensin type 2 receptor (AT2R) activation can oppose AT1R actions, such as vasodilatation, but its involvement in modulation of inflammation has not been conclusively proven. Angiotensin-converting enzyme 2 (ACE2) can process Ang II to generate angiotensin-(1-7) (Ang-(1-7)), that activates the Mas receptor to exert predominantly anti-inflammatory responses depending on the context. We now review recent advances in the understanding of the interaction of the RAS with inflammation. Specific topics in which novel information became available recently include intracellular angiotensin receptors; AT1R posttranslational modifications by tissue transglutaminase (TG2) and anti-AT1R autoimmunity; RAS modulation of lymphoid vessels and T lymphocyte responses, especially of Th17 and Treg responses; interactions with toll-like receptors (TLRs), programmed necrosis, and regulation of epigenetic modulators (e.g. microRNAs and bromodomain and extraterminal domain (BET) proteins). We additionally discuss an often overlooked effect of the RAS on inflammation which is the downregulation of anti-inflammatory factors such as klotho, peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), transient receptor potential ankyrin 1 (TRPA1), SNF-related serine/threonine-protein kinase (SNRK), serine/threonine-protein phosphatase 6 catalytic subunit (Ppp6C) and n-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP). Both transcription factors, such as nuclear factor κB (NF-κB), and epigenetic regulators, such as miRNAs are involved in downmodulation of anti-inflammatory responses. A detailed analysis of pathways and targets for downmodulation of anti-inflammatory responses constitutes a novel frontier in RAS research.

Role of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in airway reactivity and inflammation in an allergic mouse model of asthma.

Objective: Angiotensin II (Ang II) exerts its effects through two G-protein coupled receptors: angiotensin II type 1 receptors (AT1) and type 2 receptors (AT2). Both these receptor subtypes are poorly understood in asthma. In this study, we investigated effects of AT1 receptor antagonist losartan, novel AT2 receptor agonist novokinin and AT2 receptor antagonist PD 123319 in a mouse model of asthma. Methods: Mice were divided into control (CON) and allergen sensitized (SEN) groups. SEN was sensitized with ovalbumin (OVA) on days 1 and 6 (30 µg; i.p.), followed by 5% OVA aerosol challenge (days 11-13). Treatments included (a) losartan (SEN + LOS; 20 mg/kg i.p., day 14), (b) novokinin (SEN + NOV; 0.3 mg/kg i.p., day 14), and (c) PD 123319 (SEN + PD; 5 mg/kg i.p., day 14). Experiments for airway responsiveness, bronchoalveolar lavage, and tracheal ring reactivity using isolated organ bath were performed. Results: Airway responsiveness to methacholine (MCh) (48 mg/mL) was significantly higher in SEN (563.71 ± 40% vs. 294.3 ± 123.84 in CON). This response was potentiated in SEN + PD group (757 ± 30%; p < .05 compared to SEN). SEN + LOS (247.61 ± 86.85%) and SEN + NOV (352 ± 11%) had significantly lower response compared to SEN. SEN + LOS (26.22 ± 0.29%) and SEN + NOV (46.20 ± 0.76%) treatment significantly (p < .001) attenuated total cell count and eosinophils compared to SEN group (69.38 ± 1.5%), while SEN + PD (73.04 ± 0.69%) had highest number of eosinophils. Tracheal response to MCh was significantly higher in SEN group compared to controls, and this response was significantly lowered with the losartan and novokinin treatments. Conclusions: These data suggest that AT1 and AT2 receptors have opposite effects in modulating airway hyperresponsiveness and inflammation in asthma.

Prevention of lipopolysaccharide-induced CD11b+ immune cell infiltration in the kidney: role of AT2 receptors.

Immune cell infiltration plays a central role in mediating endotoxemic acute kidney injury (AKI). Recently, we have reported the anti-inflammatory and reno-protective role of angiotensin-II type-2 receptor (AT2R) activation under chronic low-grade inflammatory condition in the obese Zucker rat model. However, the role of AT2R activation in preventing lipopolysaccharide (LPS)-induced early infiltration of immune cells, inflammation and AKI is not known. Mice were treated with AT2R agonist C21 (0.3 mg/kg), with and without AT2R antagonist PD123319 (5 mg/kg) prior to or concurrently with LPS (5 mg/kg) challenge. Prior-treatment with C21, but not concurrent treatment, significantly prevented the LPS-induced renal infiltration of CD11b+ immune cells, increase in the levels of circulating and/or renal chemotactic cytokines, particularly interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) and markers of renal dysfunction (blood urea nitrogen and albuminuria), while preserving anti-inflammatory interleukin-10 (IL-10) production. Moreover, C21 treatment in the absence of LPS increased renal and circulating IL-10 levels. To investigate the role of IL-10 in a cross-talk between epithelial cells and monocytes, we performed in vitro conditioned media (CM) studies in human kidney proximal tubular epithelial (HK-2) cells and macrophages (differentiated human monocytes, THP-1 cells). These studies revealed that the conditioned-media derived from the C21-treated HK-2 cells reduced LPS-induced THP-1 tumor necrosis factor-α (TNF-α) production via IL-10 originating from HK-2 cells. Our findings suggest that prior activation of AT2R is prophylactic in preventing LPS-induced renal immune cell infiltration and dysfunction, possibly via IL-10 pathway.

Circulating angiotensin type II receptor: Possible marker for antibody mediated rejection after renal transplantation?

BACKGROUND: Presence of antibody [Ab] against angiotensin receptor [AT1R] indicates heightened risk for antibody mediated rejection [AMR] after transplantation but is insufficient as a marker. We speculated AT1R might be released systemically because of AMR and might be a useful biomarker. METHODS: AT1R was measured in blood from 73 Normals and 72 renal patients pre- and post-transplantation. Patients were stratified as AMR-free [Gp1], AMR<1yr [Gp2] and AMR>1yr [Gp3]. RESULTS: AT1R was higher [13±26vs.367±537, p<0.01)] and more prevalent [20% vs. 92%, p<0.01] among renal patients than Normals. Pretransplant levels were similar [p=ns] between groups. One-year posttransplant levels approached [p<0.01] normalcy for Gps1+3 but spiked during AMR and remained elevated [155±58, p<0.01] for 50% Gp2 patients. One-year AT1R levels were higher among subsequent graft failures than surviving grafts [171±267vs. 38±50, p<0.01]. CONCLUSIONS: Pretransplant AT1R was abnormally elevated: possibly indicating ongoing tissue injury. Pretransplant AT1R didn't predict risk for AMR. However, AT1R spiked during early AMR and sustained elevations were associated with poorer outcomes.

Autoantibodies Toward the Angiotensin 2 Type 1 Receptor: A Novel Autoantibody in Alzheimer's Disease.

BACKGROUND: Autoantibodies with agonist function are described in cardiovascular disorders. Since vascular risk factors are associated with an increased risk for Alzheimer's disease (AD), we investigated a potential association between antibodies to the angiotensin 2 type 1 receptor (anti-AT1R) and AD. OBJECTIVE: The primary objective of this study was to investigate the association between anti-AT1R and AD. The secondary objective was to investigate the association between clinical or biomarker features of AD and anti-AT1R. METHODS: Samples from patients with mild AD participating in a longitudinal study in Western Norway (n = 92, 65 [71%] females, mean age 74.8 [range 50-89]) and age- and gender-matched healthy controls (n = 102) were included. Cerebrospinal fluid (CSF) AD biomarkers were assessed in a subgroup of patients. Patients were examined annually, including Mini-Mental State Examination. ELISA was used to measure anti-AT1R in serum. Non-parametric tests were used for statistical calculations and a p <  0.05 was considered significant. RESULTS: AD patients had significantly higher levels of anti-AT1R compared with healthy controls (10.2 U/mL versus 8.1 U/mL, p = 0.04). This difference was found only in patients without hypertension and diabetes. Anti-AT1R levels correlated with CSF total tau (p = 0.03) and phosphorylated tau (p = 0.03) levels, and inversely with blood pressure in AD (Spearman R -0.277, p = 0.008). DISCUSSION: AD is associated with increased levels of anti-AT1R, and the antibodies correlated with CSF total, and phosphorylated tau levels. Further research is needed to understand the blood pressure response in AD without hypertension and a potential link between tau and anti-AT1R in AD.

AT2R autoantibodies block angiotensin II and AT1R autoantibody-induced vasoconstriction.

Activating autoantibodies to the angiotensin type 1 receptor (AT1R) are associated with hypertensive disorders. The angiotensin type 2 receptor (AT2R) is known to counter-regulate the actions of AT1R. We investigated whether AT2R autoantibodies produced in immunized rabbits will activate AT2R and suppress the vasopressor responses to angiotensin II and AT1R-activating autoantibodies. Five rabbits immunized with a peptide corresponding to the second extracellular loop of AT2R developed high AT2R antibody titers. Rabbit anti-AT2R sera failed to directly dilate isolated rat cremaster arterioles; however, when co-perfused with angiotensin II or AT1R-activating autoantibodies, the anti-AT2R sera significantly inhibited their contractile effects. Rabbit anti-AT2R sera recognized a predominant sequence near the N-terminus of the AT2R second extracellular loop. A decoy peptide based on this sequence effectively reversed the opposing effect of the anti-AT2R sera on angiotensin II-induced contraction of rat cremaster arterioles. A similar blockade of the anti-AT2R sera effect was observed with the AT2R antagonist PD 123319 and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Rabbit anti-AT2R sera reacted specifically with AT2R. No cross-reactivity with AT1R was observed. Blood pressure did not change in immunized animals. However, the pressor responses to incremental angiotensin II infusions were blunted in immunized animals. Thirteen subjects with primary aldosteronism demonstrated increased AT2R autoantibody levels compared with normal controls. In conclusion, AT2R autoantibodies produced in immunized rabbits have the ability to activate AT2R and counteract the AT1R-mediated vasoconstriction. These autoantibodies provide useful and selective tools for the study of their roles in blood pressure regulation and possible therapeutic intervention.

Role of angiotensin II type 1 receptor-activating antibodies in solid organ transplantation.

Angiotensin type I receptor (AT1R) mediates physiologic and pathophysiologic actions of its ligand, angiotensin II. Overactivity of the AT1R and angiotensin II interaction results in hypertension and vascular remodeling. Antibodies to AT1R have been implicated in several vascular pathologies. In renal transplantation, elevated levels of anti-AT1R antibodies have been associated with antibody mediated rejection (AMR) in the absence of donor HLA specific antibodies. In heart transplantation, increased levels of anti-AT1R antibodies have been associated with cellular and AMR as well as an early onset of microvasculopathy. This review summarizes the current investigations regarding the impact of anti-AT1R antibodies in solid organ transplantation and provides insight into the humoral response in the presence of non-HLA and HLA specific antibodies.

Angiotensin II type 1 receptor antagonist attenuates lacrimal gland, lung, and liver fibrosis in a murine model of chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2(d)) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

Immunohistochemical detection of angiotensin II receptors in mouse cerebellum and adrenal gland using "in vivo cryotechnique".

Angiotensin II (AT) receptors, including AT receptor type 1 (AT1R) and type 2 (AT2R), are expressed in the rodent central nervous system, but their distributions and activation states are still unclear. In this study, we have performed immunohistochemical analyses of AT receptors in mouse cerebellum and adrenal gland using our "in vivo cryotechnique" (IVCT). We used antibodies against amino-terminal domains of AT receptors, which are considered to undergo conformational changes upon the binding of AT. Immunoreactivity of AT1R was detected in mouse cerebellum, and was highest in the outer tissue areas of molecular layers using IVCT. The AT1R immunostaining largely overlapped with glial fibrillary acidic protein (GFAP), a marker of Bergmann glia. Surprisingly, the AT1R immunoreactivity in the cerebellar cortex was remarkably reduced following 5 and 10 min of hypoxia or direct administration of an AT1R antagonist, losartan. By contrast, in the adrenal cortex, such AT1R immunoreactivity detected at the zona glomerulosa did not change even after 15 min of hypoxia. The correlation of localization with GFAP and also hypoxia-induced decrease of its immunoreactivity were similarly observed by immunostaining of AT2R in the cerebellar specimens. These findings demonstrated that IVCT is useful to reveal dynamically changing immunoreactivities usually affected by receptor-ligand binding as well as hypoxia, and also suggested that functional activities of AT receptors are time-dependently modulated under hypoxia in the central nervous system in comparison with the adrenal glands.

Angiotensin type 1 receptor autoantibody from preeclamptic patients induces human fetoplacental vasoconstriction.

Increased vascular resistance in the fetoplacental circulation is a characteristic of preeclampsia. However, the potential molecular mechanisms of this condition remain obscure. The current study aimed to determine the direct effect of the peptide antigen corresponding to the second extracellular loop of the angiotensin II type 1 receptor (AT1R-EC(II) ) activating autoantibody (AT1-AA), a novel risk factor in preeclamptic patients, on fetoplacental villus stem blood vessels. Immunohistochemistry revealed that AT1 receptors were localized in the veins and arteries of human placental villi. Among 58 serum samples from preeclamptic patients, 28 (48.28%) were proved AT1-AA-positive by enzyme-linked immunosorbent assay [P<0.01 vs. 2/51 (3.92%) in the normal pregnancy group]. Total IgGs purified from AT1-AA-positive patients' sera (AT1-AA-IgGs) were added to isolated normal human placental blood vessels. The IgG significantly constricted both the villus veins and arteries in a dose-dependent manner in vitro, which could be blocked by the peptide corresponding to the human AT1R-EC(II) , anti-human IgG or the AT1 receptor antagonist losartan. Additionally, the venous constriction induced by AT1-AA-IgGs remained unchanged even at the end of the experiment (about half an hour), but the vasoconstriction caused by the AT1 receptor agonist angiotensin II underwent desensitization within three minutes. Collectively, our results demonstrated that AT1-AA in preeclamptic sera can directly constrict fetoplacental villus blood vessels without desensitization via the AT1 receptor in vitro, which might contribute to poor fetoplacental perfusion in preeclampsia.

Ontogeny of angiotensin type 2 and type 1 receptor expression in mice.

In the current experiment, we determined angiotensin type 2 receptor (AT2R) and angiotensin type 1 receptor (AT1R) protein expression by western blot analysis in developing normal mice. The results indicate that: (1) in all detected brain regions and in the spinal cord, adult mice exhibited significantly higher AT2R expression and lower AT1R expression in total protein extracts compared to fetuses and neonates; (2) other major organs, including heart, lung, liver and kidney, exhibited the same expression pattern as the brain and spinal cord; (3) reciprocal changes in AT2R and AT1R expression were found in the total protein extracts from the brainstems of mice from one-day prenatal to six weeks of age, and there was a negative correlation between AT2R and AT1R protein expression; (4) in both membrane and cytosolic fractions from the brainstem, adult mice exhibited higher AT2R and lower AT1R expression than did fetuses and neonates; and (5) in the brainstem, there were no significant differences in AT2R and AT1R messenger RNA (mRNA) levels among fetal, neonatal and adult mice. The above results reconfirmed our previous finding in rats that adult animals have higher AT2R and lower AT1R expression compared to fetuses and neonates. These data imply an involvement of AT1R in fetal development and of AT2R in adult function.

Identification of noncytotoxic and IL-10-producing CD8+AT2R+ T cell population in response to ischemic heart injury.

Emerging evidence suggests a cardioprotective role of the angiotensin AT2R, albeit the underlying cellular mechanisms are not well understood. We aimed in this article to elucidate a potential role of cardiac angiotensin AT2R in regulating cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, double-immunofluorescence staining showed that AT2R was detected in a fraction of CD8(+) T cells infiltrating in the peri-infarct myocardium. We developed a method that allowed the isolation of myocardial infiltrating CD8(+)AT2R(+) T cells using modified MACS, and further characterization and purification with flow cytometry. Although the CD8(+)AT2R(-) T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes (CMs), the CD8(+)AT2R(+) T cells were noncytotoxic to these CMs. The CD8(+)AT2R(+) T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression when compared with CD8(+)AT2R(-) T cells. We further showed that IL-10 gene expression was enhanced in CD8(+) T cells on in vitro AT2R stimulation. Importantly, in vivo AT2R activation engendered an increment of CD8(+)AT2R(+) T cells and IL-10 production in the ischemic myocardium. In addition, intramyocardial transplantation of CD8(+)AT2R(+) T cells (versus CD8(+)AT2R(-)) led to reduced ischemic heart injury. Moreover, the CD8(+)AT2R(+) T cell population was also demonstrated in human peripheral blood. Thus, we have defined the cardioprotective CD8(+)AT2R(+) T cell population, which increases during ischemic heart injury and contributes to maintaining CM viability and providing IL-10, hence revealing an AT2R-mediated cellular mechanism in modulating adaptive immune response in the heart.

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

Anti-cancer actions of a recombinant antibody (R6313/G2) against the angiotensin II AT1 receptor.

Although several tumour types express both AT1 and AT2 angiotensin II receptors, and angiotensin II stimulates cell proliferation, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are not effective anti-cancer agents. Development of a biologically active monoclonal antibody (6313/G2) against the AT1 receptor prompted the testing of a recombinant short-chain variable fragment form (R6313/G2) against breast cancer cells in vitro and in vivo. Cell lines MCF-7, MDA-MB-231 and T47D all expressed both receptor subtypes. In vitro, R6313/G2 suppressed cell proliferation in the presence of 100 nM angiotensin II, with IC50s of 30 nM, 153 nM and 2.8 microM for the three cell types respectively; in contrast, the AT1 receptor blocker losartan was effective only in T47D cells, at 25 microM. Studies on MCF-7 and T47D cells showed R6313/G2 also opposed the angiotensin II-induced inhibition of caspase-3/7 activity. In vivo, hollow fibres containing the cell lines were implanted in nu/nu balb-c mice at two sites, s.c. and i.p. Treatments of R6313/G2 at 2.5 nmol/kg and 25 nmol/kg twice per day for 7 days dose dependently reduced cell numbers for all three cell lines, but here MCF-7 cells responded most sensitively and MDA-MB-231 cells least. Although T47D cells were refractory at the s.c. site, growth was inhibited at the i.p. location, and otherwise results were similar at the two sites. In xenografts, MCF-7 cell tumours were dose dependently reduced by R6313/G2, and 13 and 27 nmol/kg R6313/G2 twice/day gave means of 74 and 76% tumour regression after 7 days. The data suggest that the anti-cancer action of R6313/G2 is considerably more effective than AT1 antagonists.

Angiotensin II type 2 receptor provides an endogenous brake during inflammation-induced microvascular fluid leak.

BACKGROUND: The dual actions of angiotensin II (AngII) on microvascular fluid leak remain enigmatic. Our hypothesis was that the AngII type 2 (AT2) receptor decreases microvascular fluid leak during inflammation. The purposes of this study were to determine the activity of the AT2 receptor during stimulation by endogenous AngII, during stimulation by exogenous AngII, and during inflammation. STUDY DESIGN: Hydraulic permeability (L(p)) of rat mesenteric venules was measured using a microcannulation technique. L(p) was measured during perfusion with the AT1 receptor antagonist, ZD7155, and also with exogenous AngII during AngII type 1 receptor (AT1) blockade. Inflammation was induced with platelet activating factor (PAF), and L(p) was measured during perfusion of AngII with AT1 blockade and also with an AT2 receptor agonist, CGP42112. RESULTS: AT2 receptor activation by endogenous AngII slightly decreased L(p) over that of the control (p=0.02). Exogenous AngII increased L(p) fivefold (L(p)=4.83+/-1.32; p < 0.001). Addition of AT1 receptor blockade decreased L(p) by 74% (to 1.24+/-0.03; p < 0.01). PAF activation increased L(p) fourfold (L(p)=4.49+/-0.74; p < 0.0001). After PAF activation, exogenous AngII then decreased L(p) by 39% (to 2.74+/-0.12; p < 0.01). Exogenous AngII during AT1 receptor blockade after PAF activation decreased L(p) by 61% (from 4.49+/-0.74 to 1.77+/-0.22; p < 0.0001), and selective AT2 receptor stimulation after PAF activation decreased L(p) by 69% (from 4.49+/-0.74 to 1.40+/-0.04; p < 0.001). CONCLUSIONS: This study further supports a dual role for AngII. AngII increases microvascular fluid leak during basal conditions but appears to decrease it during inflammation. Alterations in AT2 receptor activity may be responsible for these different effects.

High blood pressure reduction reverses angiotensin II type 2 receptor-mediated vasoconstriction into vasodilation in spontaneously hypertensive rats.

BACKGROUND: We have previously shown that angiotensin II type 2 receptor (AT(2)R) stimulation causes endothelium-dependent vasodilation that does not desensitize after chronic angiotensin II type 1 receptor (AT1R) blockade, suggesting a role for AT2R in antihypertensive treatment. METHODS AND RESULTS: We recorded mean arterial pressure (MAP) and investigated AT2R by Western blot analysis, immunohistochemistry, and function in isolated mesenteric resistance arteries (205 microm in diameter) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) receiving the following for 4 weeks in drinking water: placebo, AT1R blockade (candesartan; 2 mg/kg per day), ACE inhibitor (perindopril; 3 mg/kg per day), nonselective vasodilator (hydralazine; 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day). In precontracted isolated arteries, AT2R stimulation (angiotensin II in the presence of candesartan) caused vasodilation in WKY rats (MAP=118 mm Hg) and vasoconstriction in SHR (MAP=183 mm Hg). In SHR treated with candesartan (MAP=146 mm Hg) or hydralazine (16 mg/kg per day; MAP=145 mm Hg), AT2R-induced contraction was reduced by 50%. In SHR treated with perindopril (MAP=125 mm Hg), AT2R stimulation induced vasodilation. In SHR treated with hydralazine (24 mg/kg per day; MAP=105 mm Hg) and in SHR treated with hydralazine (16 mg/kg per day) plus candesartan (MAP=102 mm Hg), an AT2R-mediated vasodilation was restored. Immunochemistry and Western blot analysis showed that AT2R expression, lower in SHR than in WKY rats, was restored to normal levels by treatments reducing arterial pressure in SHR. CONCLUSIONS: Our results suggest that in resistance arteries of SHR, (1) AT2R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT(2)R expression and vasodilator functions.