AT2R Agonist Research Articles

The regenerative potential of angiotensin AT2 receptor in cardiac repair

Angiotensin II, the main effector peptide of the renin-angiotensin system, interferes with cardiac remodeling and repair through its receptors, including AT(1) and AT(2) receptor (R). The functional relevance of the previously neglected AT(2)R is currently intensively studied. Pharmacological therapies with AT(1)R blockers have improved outcomes in patients with ischemic heart injury, probably involving an indirect stimulation of AT(2)R. Previous experimental studies have clearly shown a protective action of AT(2)R in tissue repair and regeneration. We have recently identified the c-kit(+)AT(2)R(+) progenitor cell population in rat heart and bone marrow, which increases after induction of myocardial infarction. Further experimental evidence demonstrates that AT(2)R mediates cardiac homing and repair process of the c-kit(+) progenitor cells. AT(2)R stimulation through AT(1)R blockers or directly by AT(2)R agonist or both in combination may potentially offer the translational options to improve the regenerative potentials of stem/progenitor cells derived from patients with cardiovascular disease.

Altered efficacy of AT1R-targeted treatment after spontaneous cancer cell-AT1R upregulation

BackgroundTargeting of the renin angiotensin system (RAS) reduces tumour growth in experimental models of cancer. We aimed to establish if combined targeting of the 'classical' and 'alternative' arms of the RAS could result in synergistic inhibition of colorectal cancer (CRC) liver metastases.MethodsImmediately following induction of CRC liver metastases through intrasplenic injection of murine CRC cells, treatment with irbesartan (AT1R blocker; 50 mg/kg/day s.c.), captopril (ACE inhibitor; 750 mg/kg/day i.p.), CGP42112A (AT2R agonist; 0.6 μg/kg/hr i.p.), and/or ANG-(1-7) (24 μg/kg/hr i.p.) began and continued for 21 days. Liver to body weight ratio and/or stereology were used as a measure of tumour burden. Immunohistochemistry was used to determine AT1R and VEGF expression as well as proliferation (Ki67), apoptosis (active caspase 3) and angiogenesis (CD34).ResultsCombined RAS therapies failed to improve upon single arm therapies. However, while irbesartan previously inhibited tumour growth in this model, in the current experiments irbesartan failed to affect tumour burden. Subsequent analysis showed a cancer-cell specific upregulation of the angiotensin II type I receptor (AT1R) in irbesartan-insensitive compared to irbesartan-sensitive tumours. The upregulation of AT1R was associated with an increase in proliferation and VEGF expression by cancer cells. While animals bearing irbesartan-sensitive tumours showed a marked decrease in the number of proliferating cells in the liver and VEGF-expressing infiltrating cells in the tumour following AT1R treatment, these were unchanged by treatment in animals bearing irbesartan-insensitive (high AT1R expressing) tumours.ConclusionsAlthough the results do not support increased efficacy of combined treatment, they provide intriguing evidence of the importance of RAS expression in determining patient response and tumour growth potential and suggest that components of the RAS could be used as biomarkers to aid in patient selection.

Novel Angiotensin II Type 2 receptor agonist C21 promotes natriuresis predominantly via proximal tubules in obese rats

Earlier we have demonstrated that acute stimulation of angiotensin II type 2 receptors (AT2) promotes natriuresis by inhibiting NKA-pump in the proximal tubules of obese Zucker rats (OZR), but not in lean Zucker rats. In the present study, we used a novel agonist C21 to delineate the tubular, proximal vs distal, action of AT2R, in natriuresis in OZR. Infusion of C21 (1 and 5 μg/kg/min) caused dose-dependent increase in urine flow (UF), urinary Na-volume (UNaV) and nitric oxide which was blocked by pre-infusion of AT2R antagonist PD123319 (50 μg/kg/min), suggesting the involvement of AT2R. Infusion of AM and BFTZ, blockers of 2- main distal tubule apical Na-transporters, caused significant increase in UF and UNaV, which were further increased after C21 (5 μg/kg/min) treatment. There were no changes in GFR or blood pressure in response to these drugs treatment, suggesting a tubular effect. The tubular effects were further validated by the increase in fractional excretion of (FENa) in response to AM+BFTZ and C21 infusion. The difference in UNaV in response to AM+BFTZ+C21 and AM+BFTZ, suggests that AT2 receptors acted predominantly on proximal tubules, which contributed approximately 2.5-fold more in UNaV as compared to distal tubules. This is the first study which demonstrates that selective activation of AT2 receptor with novel AT2R agonist C21 promotes natriuresis predominantly via proximal tubules in obese rats

Stimulation of AT2 receptor exerts beneficial effects in stroke-prone rats: focus on renal damage

Angiotensin II acts through two major receptors: AT1-R and AT2-R. It is known that the stimulation of AT1-R mediates vasoconstriction, cell proliferation and fibrosis, aldosterone release and inflammatory response but, although the stimulation of AT2-R is thought to promote vasodilation and anti-inflammatory effects, its real in-vivo functions are still unclear. The aim of this study was to investigate the effects of specific and selective AT2-R stimulation on the pathological events occurring in spontaneously hypertensive stroke-prone rats (SHRSPs). SHRSPs who were fed a high-salt diet underwent long-term treatment with vehicle or compound 21 (C21), a nonpeptide selective AT2-R agonist, at doses of 0.75, 5 and 10 mg/kg per day. The vehicle-treated rats developed brain abnormalities detectable by magnetic resonance imaging after 42.5 +/- 7.5 days, and died 43 +/- 9.5 days after the start of the dietary treatment. The highest C21 dose delayed the occurrence of brain damage (P < 0.001 vs. vehicle-treated SHRSPs) and prolonged survival (P < 0.001) without affecting blood pressure. These beneficial effects of C21 were abolished by the administration of PD123319, an AT2-R antagonist. C21 treatment preserved renal structure by preventing inflammatory cell infiltration, collagen accumulation, and the neo-expression of vimentin; it also prevented the increased plasma renin activity and accumulation of urinary acute-phase proteins observed in the vehicle-treated rats. Specific and selective AT2-R stimulation has beneficial effects on the pathological events occurring in SHRSPs. These data indicate a new avenue for the pharmacological treatment of diseases in which modulation of the renin-angiotensin system is required.

Potentiation of the antihypertensive action of losartan by peripheral overexpression of the ANG II type 2 receptor

Our previous studies demonstrated that peripheral overexpression of angiotensin II (ANG II) type 2 receptors (AT(2)R) prevents hypertension-induced cardiac hypertrophy and remodeling without altering high blood pressure. This, coupled with the observations that AT(2)R play a role in the antihypertensive actions of ANG II type 1 receptor (AT(1)R) blockers (ARBs), led us to propose that peripheral overexpression of AT(2)R would improve the antihypertensive action of losartan (Los) in Sprague-Dawley (SD) rats made hypertensive via chronic infusion of ANG II. Here we utilized adenoviral vector-mediated AT(2)R gene transfer to test this hypothesis. A single intracardiac injection of adenoviral vector containing genomic AT(2)R (G-AT(2)R) DNA and enhanced green fluorescent protein (EGFP) gene controlled by cytomegalovirus (CMV) promoters (Ad-G-AT(2)R-EGFP; 5 x 10(9) infectious units) into adult SD rats produced robust AT(2)R overexpression in cardiovascular tissues (kidney, lung, heart, aorta, mesenteric artery, and renal artery) that persisted for 3-5 days postinjection. By 7 days post viral injection, the overexpressed AT(2)R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels). In two separate protocols, we demonstrated that the hypotensive effect of Los (0.125, 0.5, and 1.0 mg/kg iv) was significantly greater in the AT(2)R-overexpressing animals (-40.7 +/- 4.3, -41.8 +/- 4.8, and -48.1 +/- 2.6 mmHg, respectively) compared with control vector (Ad-CMV-EGFP)-treated rats (-12.4 +/- 2.2, -20.2 +/- 3.4, and -27.3 +/- 3.4 mmHg, respectively). These results provide support for a depressor role of AT(2)R and the proposal that combined AT(2)R agonist and ARB treatment may be an improved therapeutic strategy for controlling hypertension.

Opposing actions of angiotensin II type 1 and 2 receptors on plasma cholesterol levels in rats

There have been very few studies to examine how angiotensin II (AII) affects lipid metabolism. We examined the roles of AII type 1 and type 2 receptors (AT1R and AT2R) in cholesterol metabolism in rats fed either normal chow or high-fructose diets. AII (100 ng/kg per min) or vehicle (saline) was continuously infused through an osmotic mini-pump to normal chow-fed or 60% fructose-fed rats for 2 weeks. AII infusion markedly elevated both the systolic and diastolic blood pressure in the two animal groups. AII did not affect the plasma total cholesterol (TC) in the chow-fed rats. In the AII-infused rats treated with olmesartan medoxomil, an AT1R blocker, we were interested to observe significant decreases in plasma TC and non-high-density lipoprotein (HDL)-cholesterol (C) (TC minus HDL-C), and liver cholesterol content were also decreased. Simultaneous infusion of AII and PD123319, an AT2R blocker, markedly increased non-HDL-C and hepatic cholesterol. The infusion of CGP42112A, an AT2R agonist, decreased non-HDL-C by 30% in normal rats. The AII infusion led to significant elevations in TC and non-HDL-C in the fructose-fed rats, and olmesartan treatment completely rectified this AII-induced hypercholesterolemia. These results suggest that the AII receptors exert opposing effects on the plasma cholesterol level; that is, AT1R increases plasma cholesterol while AT2R decreases it. Fructose feeding may selectively augment the action of AT1R and thereby enhance the increase in plasma cholesterol levels in response to AII infusion.

Interaction of angiotensin II with the angiotensin type 2 receptor inhibits the cardiac transient outward potassium current.

The Ca2+ -independent transient outward K+ current (Ito) plays a crucial role in shaping the cardiac action potential. In the present study, we examined whether angiotensin II (AngII) regulated the Ito as well as the putative intracellular cascade responsible for the effects. Ito was recorded in rat ventricular myocytes using the nystatin-perforated patch-clamp configuration. AngII (0.1 microM) inhibited Ito (21.9+/-4.8% at +40 mV), but not the IK1, in a voltage- and time-independent manner. The inhibition decreased at concentrations higher than 1 microM resulting in a bell-shaped dose-response curve (IC50 = 3.1+/-1.5 microM). The blocking effects were abolished in the presence of the type 2 AngII receptor (AT2R) antagonist PD123319, but not in the presence of the selective type 1 AngII receptor (AT1R) antagonist candesartan. Moreover, the selective AT2R agonist CGP42112A completely reproduced the effects of AngII (20.5+/-2.4% of block at +40 mV), indicating that AngII-induced Ito block was mediated via stimulation of AT2R. Furthermore, selective stimulation of AT2R by CGP42112A significantly prolonged the rat atrial action potentials recorded using conventional microelectrode techniques. The AngII-induced inhibition of I(to) was not modified by either Npi-nitro-L-arginine-methyl ester (L-NAME) or eicosatetrayonic acid (ETYA), indicating that neither the nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) system nor the arachidonic acid cascade was implicated in the effects of AngII on Ito. However, the AngII-induced Ito inhibition was completely abolished by the serine/threonine phosphatase type 2A (PP2A) inhibitors, okadaic acid and cantharidin, but not by the inactive analog of okadaic acid, 1-norokadaone. Intracellular application of PP2A decreased Kv4.2 currents recorded in transiently transfected Chinese hamster ovary cells (CHO). These results indicate that AngII activates PP2A through the stimulation of the AT2R, resulting in a decrease of the Ito amplitude.

Angiotensin type 2 receptors are reexpressed by cardiac fibroblasts from failing myopathic hamster hearts and inhibit cell growth and fibrillar collagen metabolism.

Angiotensin (Ang) II type 1 receptor (AT1-R) induces cardiomyocyte hypertrophy and fibroblast proliferation, whereas the physiological role of AT2-R in cardiac remodeling remains poorly defined. Using Bio14.6 cardiomyopathic (CM) hamsters, we found that AT2-R sites were increased by 153% during heart failure compared with F1B controls. AT1-R numbers were increased by 72% in the hypertrophy stage and then decreased to the control level during heart failure. Such differential regulation of AT2-R and AT1-R during heart failure was consistent with changes in the respective mRNA levels. Autoradiography and immunocytochemistry revealed that both AT2-R and AT1-R are localized at higher densities in fibroblasts present in fibrous regions. Surrounding myocardium predominantly expressed AT1-R, but the level of expression was less than that in fibrous regions. Cardiac fibroblasts isolated from CM hearts during heart failure but not from control hamsters expressed AT2-R (30 fmol/mg protein). Using the cardiac fibroblasts expressing AT2-R, we found that Ang II stimulated net collagenous protein production by 48% and pretreatment with an AT2-R antagonist, PD123319, evoked a further elevation (83%). Ang II-induced synthesis of fibronectin and collagen type I were enhanced by 40% and 53%, respectively, by pretreatment with PD123319. Ang II-induced DNA synthesis (assessed by [3H]thymidine uptake) was significantly increased by PD123319, and the AT2-R agonist CGP42112A reduced the serum-stimulated increase in cell numbers by 23%. Treatment with an AT1-R antagonist, TCV116, for 20 weeks inhibited progression of interstitial fibrosis by 28%, whereas with 44-week PD123319 treatment but not 20-week treatment, the extent of the fibrous region was increased significantly, by 29%. These findings demonstrate that AT2-R is re-expressed by cardiac fibroblasts present in fibrous regions in failing CM hearts and that the increased AT2-R exerts an anti-AT1-R action on the progression of interstitial fibrosis during cardiac remodeling by inhibiting both fibrillar collagen metabolism and growth of cardiac fibroblasts.