AT2 Receptor Knockout Research Articles

Absence of Angiotensin ll type 2 (AT2) Receptors Increases Airway Inflammation and Responsiveness in an Allergic Mouse Model of Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: Ang II receptor type‐1 and type‐2 (AT1 and AT2). This lab has previously found that AT2 receptor agonist plays a significant role in reducing airway inflammation and reactivity (FASEB J April 2018 32: 829.6). For the current study, we characterized the role of AT2 receptors in airway hyperreactivity and inflammation using AT2 receptor knockout (AT2KO; Hein L, Barsh et. al. Nature. 1995;377:744–747) and corresponding wild‐type (WT) mice that were divided into two experimental groups each: control (CON) and allergen sensitized‐challenged (SEN). Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in AT2KO SEN mice compared to WT SEN (324.7±66% in AT2KO SEN vs 166±56.3%, p<0.05). Total cell count analysis showed increased number of cells in AT2KO SEN (3.3±0.2 × 106 vs. 1.4±5 × 106 in WT SEN, p<0.001). Differential BAL cell analysis showed increased eosinophils (73±8% in AT2KO SEN vs. 50.11±2.84% in WT SEN, p<0.05). Macrophages were lower in AT2KO SEN than WT SEN (15±4% vs. 40±3%; p<0.001). These data indicate that absence of AT2 receptors increases airway inflammation and responsiveness in our model of asthma.Support or Funding InformationInstitutional Research Grant Long Island UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardioprotective effect of thyroid hormone is mediated by AT2 receptor and involves nitric oxide production via Akt activation in mice.

Studies have demonstrated that thyroid hormone (T3) can precondition the heart against ischaemic injury and improve post-ischaemic recovery. This study investigated whether the AT2 receptor (AT2R) is involved in cardioprotection and the potential molecular mechanism responsible for this effect. Hyperthyroidism was induced in male wild-type (WT) and AT2R knockout (KO) mice by administering daily intraperitoneal injections of T3 (7μg/100g body weight) for 14days. The mouse hearts were harvested and perfused with a Krebs-Henseleit solution at a constant flow in a Langendorff set-up. After 30min of stabilization, the hearts were subjected to global ischaemia for 20min and reperfused for 45min. Baseline cardiac function was assessed by measuring four parameters: LVDP (mmHg), heart rate (bpm), +dP/dt and -dP/dt (mmHg/s). After reperfusion, the total protein from cardiac ventricles was obtained, and the Akt signalling pathway and NO production were evaluated. Post-ischaemic functional recovery was significantly greater (p<0.05) in the T3-treated WT mice compared to the control, demonstrating the cardioprotective effect of T3. This effect was abolished in T3-treated KO mice, demonstrating the physiological relevance of AT2R to the cardioprotective phenotype induced by T3. Akt activation, iNOS expression and NO production increased in cardiac tissue after T3 treatment in the WT animals, but no difference was observed after treatment in the KO mice. This study indicates that AT2R acts as a cardioprotector in the case of hyperthyroidism. Strategies targeting AT2R agonists might improve cardiac function through NO production and suggest potential therapeutic targets for heart diseases.

Chronic angiotensin AT2R activation prevents high-fat diet-induced adiposity and obesity in female mice independent of estrogen

ObjectiveObesity is a known risk factor for various metabolic disorders and cardiovascular diseases. Recently we demonstrated that female angiotensin AT2 receptor (AT2R) knockout mice on high-fat diet (HFD) had higher body weight and adiposity with a parallel reduction in estrogen (17,β-estradiol/E2). The present study investigated whether the anti-adiposity effects of the AT2R are estrogen-dependent in female mice. MethodsFemale C57BL/6 ovary-intact (Ovi) mice were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.). Ovariectomized (Ovx) mice, supplemented with E2 (5μg/day, pellets implanted subcutaneously), were treated with an AT2R agonist (C21, 0.3mg/kg, daily i.p.) or vehicle. After 4-days of pre-treatment with C21, Ovi and Ovx mice were placed on either normal diet (ND) or HFD while the C21 treatment continued for the next 10days. For a long-term study, Ovi mice were placed on HFD and treated with C21 for 12weeks. ResultsOvi mice fed the HFD had increased parametrial white adipose tissue (pWAT) weight, plasma free fatty acid and triglycerides compared to Ovi mice on ND. Ovariectomy alone caused similar changes in these parameters which were further increased by HFD-feeding. C21 treatment attenuated these HFD-induced changes in Ovi as well as Ovx mice. HFD also, increased the liver/body-weight ratio and decreased the liver expression of the β-oxidation enzyme, carnitine palmitoyltransferase 1 (CPT1-A). C21 treatment attenuated these changes as well. The long-term C21 treatment of Ovi mice lowered the HFD-induced body weight gain, increase in pWAT weight, parametrial adipocyte size and hyperinsulinemia induced by HFD. Finally, HFD drastically reduced urinary estrogen and the beneficial metabolic changes in response to C21-treatment occurred without significantly increasing urinary estrogen. ConclusionWe suggest that the pharmacological activation of AT2R by the agonist C21 reduces adiposity and body weight gain independent of estrogen in female mice. Improvement in fatty acid metabolism is a potential mechanism by which the AT2R exerts anti-adiposity effects.

Abstract 465: AT 2 Receptor Signaling Plays An Important Role In Conversion Of From White To Beige Fat

Background: We have previously reported that treatment with angiotensin II type-2 (AT 2 ) receptor agonist (compound 21; C21) ameliorated insulin resistance with the improvement of adipocyte dysfunction in obese type 2 diabetic mouse model, KKAy. Until quite recently, adipose tissue has been divided into two groups that have directory-opposed capacities; white adipose tissue is best known for its role in energy store, while brown adipose tissue functions for energy expenditure (thermogenesis). In addition, new clusters of adipocyte with thermogenic capability have been discovered in white adipose tissue, which are distinct origins from brown adipose tissue. These adipocytes have been named ‘beige’. However, the role of AT 2 receptor signaling in white-to-beige fat conversion is not known. Methods: KKAy mice were subjected to i.p. injection of C21 (10ug/kg/day) for 2 weeks. After the treatment, the expression of thermogenic genes (Ucp1, Cidea, Pgc1α) in adipose tissue were determined with real-time RT-PCR. Next, we examined the difference in conversion from white to beige fat between wild-type (WT) mice and AT 2 receptor knockout (AT 2 KO) mice. Eight-week-old mice were fed a high-fat and high-cholesterol diet (HFCD) (10% coconut oil + 1.25% cholesterol) for 2 weeks. To examine the effect of exercise on fat conversion, 9-week-old mice were forced swim test (2 minх3 times/day) for 7days. After the swim test, mice were sacrificed for examinations. Results: In KKAy mice, treatment of C21 increased the expression of thermogenic genes in subcutaneous fat, but not in visceral fat. Adipose tissue weight was not changed by treatment of C21. In AT 2 KO mice fed HFCD for 2 weeks, visceral fat was greater than that in WT mice, but subcutaneous fat was not. The expression of thermogenic genes in subcutaneous fat was lower in AT 2 KO mice than in WT mice. In both mice, swim exercise increased the expression of thermogenic genes; however, the up-regulation of these genes in AT 2 KO mice did not reach the level of that in WT mice. Conclusion: These results suggest that AT 2 receptor signaling has an important role in white-to-beige fat conversion, and therefore AT 2 receptor signaling might be a potential therapeutic target for lipid metabolic disorders.

Possible Role of Angiotensin-Converting Enzyme 2 and Activation of Angiotensin II Type 2 Receptor by Angiotensin-(1–7) in Improvement of Vascular Remodeling by Angiotensin II Type 1 Receptor Blockade

Cross talk between the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis and the ACE2/Ang-(1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Furthermore, possible stimulation of the Ang II type 2 (AT2) receptor by Ang-(1-7) has been highlighted as a new pathway. Therefore, we examined the possibility of whether the ACE2/Ang-(1-7)/Mas axis and Ang-(1-7)/AT2 receptor axis are involved in the inhibitory effects of AT1 receptor blockers on vascular remodeling. Wild-type, Mas-knockout, and AT2 receptor knockout mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. Some mice were treated with azilsartan, an AT1 receptor blocker, or Ang-(1-7). Neointimal formation 2 weeks after cuff placement was more marked in Mas-knockout mice compared with wild-type mice. Treatment with azilsartan or Ang-(1-7) attenuated neointimal area, vascular smooth muscle cell proliferation, increases in the mRNA levels of monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-1β, and superoxide anion production in the injured artery; however, these inhibitory effects of azilsartan and Ang-(1-7) were less marked in Mas-knockout mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA and increased AT2 receptor mRNA but did not affect AT1 receptor mRNA or the decrease in Mas mRNA. The inhibitory effect of Ang-(1-7) on neointimal formation was less marked in AT2 receptor knockout mice compared with wild-type mice. These results suggest that blockade of the AT1 receptor by azilsartan could enhance the activities of the ACE2/Ang-(1-7)/Mas axis and ACE2/Ang-(1-7)/AT2 receptor axis, thereby inhibiting neointimal formation.

Abstract 162: Possible Roles of Crosstalk Between Angiotensin-converting Enzyme 2/Angiotensin (1-7)/Mas Axis and Angiotensin (1-7)/angiotensin II Type 2 Receptor Axis in Vascular Remodeling

Objectives: Crosstalk between the angiotensin (Ang)-converting enzyme (ACE)/Ang II/ Ang II type 1 (AT 1 ) receptor axis and ACE2/Ang (1-7)/Mas axis plays a role in the pathogenesis of cardiovascular remodeling. Moreover, possible stimulation of Ang II type 2 (AT 2 ) receptor by Ang-(1-7) has been highlighted as new axis; however, pathophysiological significance of this possibility has to be elucidated in more detail. Therefore, we examined the possibility whether ACE2/Ang (1-7)/Mas axis and Ang (1-7)/AT 2 receptor axis is involved the inhibitory effects of AT 1 receptor blocker (ARB) on vascular remodeling. Methods: Ten-week-old male C57BL/6J (wild type: WT), Mas knockout (MasKO), and AT 2 receptor knockout (AT 2 KO) mice were used in this study. Vascular injury was induced by polyethylene-cuff placement around the mouse femoral artery. After cuff placement, some mice were treated with azilsartan (0.3 mg/kg/day), an ARB, or Ang (1-7) (0.5 mg/kg per day). Two weeks after operation, femoral arteries were removed, fixed and stained by Elastica van Gieson staining for evaluating neointimal formation. Results: Neointima formation induced by cuff placement was further increased in MasKO mice compared with WT mice. Treatment with azilsartan or Ang-(1-7) decreased neointima area and vascular smooth muscle cell proliferation, and attenuated the increases in the mRNA level of MCP-1, TNF-α and IL-1β in the injured artery in WT mice without influencing blood pressure. These inhibitory effects of azilsartan or Ang-(1-7) were less in Mas KO mice. One week after cuff placement, the mRNAs of ACE2 and Mas in the injured artery were markedly decreased in WT mice. Administration of azilsartan or Ang-(1-7) attenuated the decrease in ACE2 mRNA, but not affect the decrease in Mas mRNA. Neointima formation in AT 2 KO mice was more exaggerated compared with that in WT mice. Interestingly, we observed that inhibitory effect of Ang-(1-7) on neointima formation was less in AT 2 KO mice compared with WT mice. Conclusion: These results suggest that blockade of AT 1 receptor by azilsartan could enhance the activities of the ACE2/Ang (1-7)/Mas axis and ACE2/Ang-(1-7)/AT 2 receptor axis, thereby inhibiting the neointima formation.

Commercially Available Angiotensin II At2 Receptor Antibodies Are Nonspecific

Commercially available angiotensin II AT2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available AT2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and AT2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or AT2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of AT2 receptor binding or gene expression. We conclude that none of these commercially available AT2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study AT2 receptor expression.

Compound 21 Induces Vasorelaxation via an Endothelium- and Angiotensin II Type 2 Receptor-Independent Mechanism

Angiotensin II type 2 (AT(2)) receptor stimulation has been linked to vasodilation. Yet, AT(2) receptor-independent hypertension and hypotension (or no effect on blood pressure) have been observed in vivo after application of the AT(2) receptor agonist compound 21 (C21). We, therefore, studied its effects in vitro, using preparations known to display AT(2) receptor-mediated responses. Hearts of Wistar rats, spontaneously hypertensive rats (SHRs), C57Bl/6 mice, and AT(2) receptor knockout mice were perfused according to Langendorff. Mesenteric and iliac arteries of these animals, as well as coronary microarteries from human donor hearts, were mounted in Mulvany myographs. In the coronary vascular bed of Wistar rats, C57Bl/6 mice, and AT(2) receptor knockout mice, C21 induced constriction followed by dilation. SHR hearts displayed enhanced constriction and no dilation. Irbesartan (angiotensin II type 1 receptor blocker) abolished the constriction and enhanced or (in SHRs) reintroduced dilation, and PD123319 (AT(2) receptor blocker) did not block the latter. C21 relaxed preconstricted vessels of all species, and this did not depend on angiotensin II receptors, the endothelium, or the NO-guanylyl cyclase-cGMP pathway. C21 constricted SHR iliac arteries but none of the other vessels, and irbesartan prevented this. C21 shifted the concentration-response curves to U46619 (thromboxane A(2) analog) and phenylephrine (α-adrenoceptor agonist) but not ionomycine (calcium ionophore) to the right. In conclusion, C21 did not cause AT(2) receptor-mediated vasodilation. Yet, it did induce vasodilation by blocking calcium transport into the cell and constriction via angiotensin II type 1 receptor stimulation. The latter effect is enhanced in SHRs. These data may explain the varying effects of C21 on blood pressure in vivo.

Podocytes of AT2 Receptor Knockout Mice Are Protected from Angiotensin II-Mediated RAGE Induction

Background/Aims: The interaction of ‘advanced glycation end products’ (AGEs) and their receptor ‘RAGE’ plays an important role in diabetic nephropathy. We have previously found that in cultured differentiated podocytes, angiotensin II (ANG II) induces RAGE expression via an AT2 receptor-mediated pathway. Methods: To further confirm our results in an in vivo study, AT2 receptor knockout mice (AT2^–/–) and wild-type mice were infused with ANG II by osmotic minipumps for 14 days. Results: As shown by immunohistochemistry, ANG II treatment of wild-type animals (C57BL6) allowed a significantly increased RAGE expression in renal podocytes in comparison to sham-operated C57BL6 mice. In contrast, RAGE expression in podocytes of ANG II-treated knockout mice (AT2^–/–) was only moderately higher than in control animals, but significantly lower than in ANG II-treated wild-type mice. For the AGE species NΕ-carboxymethyllysine, a similar immunohistochemical staining pattern was found. There was no significant change in glomerular AT1a receptor expression. However, no difference in RAGE mRNA expression could be found between ANG II-infused wild-type and AT2^–/– animals by real-time PCR using whole kidney mRNA, presumably due to the low abundance of podocyte mRNA in these preparations. No effects were seen on glomerular apoptosis. Conclusion: These data support the fact that ANG II-mediated RAGE induction in podocytes occurs via AT2 receptors. The present findings may suggest that not all ANG II-mediated changes in diabetic nephropathy can be treated with AT1 receptor blockers.

Renal vasoconstrictor and pressor responses to angiotensin IV in mice are AT1a-receptor mediated

Angiotensin (Ang) IV was reported to induce renal vasoconstriction or vasodilation in rats via AT1 or AT4 receptors, respectively, whereby the latter one has been identified to be the insulin-regulated aminopeptidase (IRAP). We investigated the effects of Ang IV on mean arterial pressure (MAP) and renal cortical blood flow (CBF) in AT1a, AT1b, AT2 receptor and IRAP knockout (-/-) mice and their corresponding wild-type littermates. Ang II, known as a renal vasoconstrictor in mice, was used as a reference. MAP was recorded via a femoral catheter and CBF was measured using a light amplification by stimulated emission of radiation (LASER) Doppler probe; cortical vascular resistance (CVR) was calculated as MAP divided by CBF. Baseline MAP, CBF and CVR in AT1a (-/-) mice were significantly lower than wild-type mice. AT2 (-/-) mice had a significantly higher baseline MAP, but similar CBF. In wild-type mice, Ang IV and Ang II induced dose-dependent pressor and renal vasoconstrictor responses, which were antagonized by the AT1 receptor blocker candesartan. These responses were almost completely absent in AT1a (-/-) mice, but were enhanced in AT2 (-/-) mice; responses in AT1b (-/-) and IRAP (-/-) mice were comparable to those in corresponding wild-type mice. Ang IV mediates pressure and renal vasoconstrictor effects in mice via AT1a receptors, whereas IRAP/AT4 is not involved.

Angiotensin II type 2 receptor deficiency aggravates renal injury and reduces survival in chronic kidney disease in mice

Angiotensin II (Ang II) activates at least two receptors, AT1 and AT2, with the majority of its effects-such as vasoconstriction, inflammation, and matrix deposition-mediated by the AT1 receptor. It is thought that the AT2 receptor counteracts these processes; however, recent studies have found proinflammatory and hypertrophic effects of this receptor subtype. To identify the physiological roles of the AT2 receptor in chronic kidney disease, we performed renal ablation in AT2 receptor knockout and wild-type mice. Renal injury caused a greater impairment of renal function, glomerular injury, albuminuria, and mortality in the knockout mice than in the wild-type mice. There was increased fibronectin expression and inflammation in the knockout mice, as shown by augmented monocyte/macrophage infiltration and higher chemokine monocyte chemotactic protein-1 (MCP-1) and RANTES expression in the remnant kidney. The higher mortality and renal morbidity of the knockout mice was not due to differences in systemic blood pressure, glomerular volume, AT1 receptor, renin, or endothelial nitric oxide synthase expression. Whether activation of the AT2 receptor will have therapeutic benefit in chronic kidney disease will require further study.

Renal cortical cyclooxygenase 2 expression is differentially regulated by angiotensin II AT 1 and AT 2 receptors

Macula densa cyclooxygenase 2 (COX-2)-derived prostaglandins serve as important modulators of the renin-angiotensin system, and cross-talk exists between these two systems. Cortical COX-2 induction by angiotensin-converting enzyme (ACE) inhibitors or AT(1) receptor blockers (ARBs) suggests that angiotensin II may inhibit cortical COX-2 by stimulating the AT(1) receptor pathway. In the present studies we determined that chronic infusion of either hypertensive or nonhypertensive concentrations of angiotensin II attenuated cortical COX-2. Angiotensin II infusion reversed cortical COX-2 elevation induced by ACE inhibitors. However, we found that angiotensin II infusion further stimulated cortical COX-2 elevation induced by ARBs, suggesting a potential role for an AT(2) receptor-mediated pathway when the AT(1) receptor was inhibited. Both WT and AT(2) receptor knockout mice were treated for 7 days with either ACE inhibitors or ARBs. Cortical COX-2 increased to similar levels in response to ACE inhibition in both knockout and WT mice. In WT mice ARBs increased cortical COX-2 more than ACE inhibitors, and this stimulation was attenuated by the AT(2) receptor antagonist PD123319. In the knockout mice ARBs led to significantly less cortical COX-2 elevation, which was not attenuated by PD123319. PCR confirmed AT(1a) and AT(2) receptor expression in the cultured macula densa cell line MMDD1. Angiotensin II inhibited MMDD1 COX-2, and CGP42112A, an AT(2) receptor agonist, stimulated MMDD1 COX-2. In summary, these results demonstrate that macula densa COX-2 expression is oppositely regulated by AT(1) and AT(2) receptors and suggest that AT(2) receptor-mediated cortical COX-2 elevation may mediate physiologic effects that modulate AT(1)-mediated responses.

Anti-fibrogenic function of angiotensin II type 2 receptor in CCl 4-induced liver fibrosis

The renin-angiotensin system (RAS) contributes to fibrogenesis in a variety of organs. We recently showed that a lack of angiotensin (Ang) II type 1 (AT1) receptor activity reduces liver fibrosis. In this study, we investigated whether the Ang II type 2 (AT2) receptor is implicated in the development of liver fibrosis. A comparison was made between AT2-receptor knockout (AT2KO) and wild type (WT) mice after 4 weeks of treatment with carbon tetrachloride (CCl 4). Fibrosis was assessed by Azan–Mallory staining and hepatic hydroxyproline (HP) content. The expression of fibrogenic mRNA was measured by real-time quantitative reverse-transcription polymerase chain reaction (PCR). Liver fibrosis evaluated by regular histological analyses and immunohistochemical α-SMA staining was observed in both groups of mice. The extent of fibrosis was greatest in the AT2KO mice. Fibrosis was associated with increases in hepatic HP content and mRNA expression for TGF-β1 and α-SMA, as well as an increase in hepatic TBARS. These findings suggest that CCl 4 induces oxidative stress which leads to activation of hepatic stellate cells (HSCs). These changes were considerably more pronounced in the AT2KO mice than the WT mice. Taken together, we conclude that AT2 signal has anti-fibrogenic and/or cytoprotective effects on oxidative stress-induced liver fibrosis. We therefore suggest that RAS-associated liver fibrogenesis may be determined by the balance between AT1 and AT2 signals.

Insights into angiotensin II receptor function through AT2 receptor knockout mice.

Angiotensin II signals via at least two receptors termed AT1 and AT2. The function of the AT1 receptor is well defined, while that of the AT2 receptor is still shrouded in uncertainty. AT2 gene-deficient (-/-) mice have been helpful in unravelling the function of the AT2 receptor. We have studied AT2-/- and AT2+/+ mice with classical physiological techniques developed for the rat. We found that although AT2-/- mice have normal glomerular filtration rate, the pressure-natriuresis relationship in these mice, compared with AT2+/+ mice, is shifted rightward. Moreover, medullary blood flow fails to increase with increased perfusion pressure while the AT1 receptor expression in the kidneys is increased. We used telemetry and found that AT2-/- mice have about 10 mmHg higher blood pressures than AT2+/+ mice and that their circadian rhythm is disturbed. Moreover, their baroreflexes, as measured by spectral analyses, differs from AT2+/+ controls. The cardiac function of AT2-/- mice is remarkably preserved and the differences are subtle. However, if the mice are given l-NAME hypertension, they exhibit an end-systolic pressure-volume relationship that reveals decreased contractility and probable increased vascular stiffness. Furthermore, the hearts of AT2-/- mice hypertrophy more in response to l-NAME than those of AT2+/+ mice and perivascular fibrosis is increased. DOCA-salt treatment also shows a more rightward pressure-natriuresis relationship in AT2-/- compared with AT2+/+ mice. The renal iNOS expression is increased with DOCA-salt treatment. Our findings support the notion that the AT2 receptor signals antiproliferative and antifibrotic effects and that its presence results in lower blood pressures and lesser responses to secondary forms of hypertension. Technical advances that have allowed us to adapt methods for the rat to the much smaller mouse have facilitated our studies.

Differential regulation of in vivo angiogenesis by angiotensin II receptors.

Angiotensin II (ANG II), a key regulator of blood pressure and body fluid homeostasis, exerts mitogenic effects on endothelial cells. We therefore hypothesized that ANG II could be a mediator between homeostatic changes within the vascular perfusion bed and growth factor-driven angiogenesis. In the present study, we applied the alginate implant angiogenesis model in mice with normal ANG II levels, elevated ANG II levels by transgenic overexpression of angiotensinogen (AOGEN), or in AT2 receptor-deficient mice. We demonstrate that a decrease in the amount of circulating ANG II by the angiotensin-converting enzyme (ACE) inhibitor enalapril or the AT1 receptor antagonist losartan induced a stimulation of in vivo angiogenesis implying an inhibitory function of ANG II through the AT1 receptor. However, the strong increase of angiogenesis in AOGEN-transgenic mice compared with mice with normal ANG II levels suggests additional stimulatory activity. We showed that the ANG II-induced stimulation of angiogenesis is linked to the AT2 receptor as an impaired induction of angiogenesis was obtained in AT2 receptor knockout mice. These findings provide the first evidence that the AT2 receptor mediates a stimulation of in vivo angiogenesis and indicate that ANG II is a humoral regulator of peripheral angiogenesis involving two receptor subtypes with opposing actions.

Angiotensin II and tubular development.

Accumulating evidence suggests that angiotensin II (ANG II) plays an important role in the complex affair of renal organogenesis. The renin-angiotensin system (RAS) is up-regulated during renal development and in the perinatal period. On the other hand, inhibition of the RAS, for example by angiotensin-converting enzyme (ACE) inhibitors, may produce specific renal abnormalities including abnormal renal vessels, failure to develop a renal pelvis and tubular atrophy associated with expansion of the interstitium. AT2 receptors are expressed abundantly during fetal development and are down-regulated markedly after birth, whereas the abundance of AT1 receptors increases as maturation proceeds. Mice with targeted deletions of genes for angiotensinogen or ACE revealed severe renal abnormalities. In contrast, AT1A, AT1B and AT2 receptor knockout animals exhibited milder abnormalities of the kidney. These findings suggest that AT1 and AT2 receptors are both involved in the development of the nephron, and that ANG II provides signals through both receptors. ANG II exerts in vitro growth-stimulatory effects on tubular cells. Moreover, ANG II induces synthesis of collagen type IV in tubular cells, a necessary prerequisite for successful basement membrane formation. These effects are mediated through AT1 receptors. Thus, it is feasible that blockade of the RAS during kidney organogenesis leads to a decrease in the growth factor ANG II that may be pivotal for tubular growth and differentiation. On the other hand, ANG II's growth-stimulatory effects through AT1 receptors may be counterbalanced by AT2 receptor-mediated apoptosis and growth inhibition. Therefore, alterations in AT2 receptor signalling may alter the delicate balance between growth stimulation and inhibition, leading to alterations in tubular formation.

Angiotensin II receptor antagonism and ace inhibition ameliorate hyperinsulinemia and obesity in a murine model of polygenic obesity

Background: Amlodipine decreases myocardial oxygen consumption (MVO2) via R+ enantiomer mediated endothelial nitric oxide (NO) release. We investigated the role of angiotensin (AT) 2 and 4 receptors in mediating this effect. Methods: We measured MVO2 in vitro using the Clark type oxygen electrode in isolated LV myocardial segments obtained from (i) wild-type and AT2 receptor knock-out (KD) mice (n=5), and (ii) explanted failing human hearts obtained at the time of heart transplantation (n=2). We studied the effect of increasing doses of R+ enantiomer (10-7M -105 M) on MVO2 with and without (i) NO synthase inhibitor, nitro-L-arginine methyl ester (LNAME, 10-3 M), (ii) AT2 receptor blocker, PD 123319, 10-6-10-5M, and (iii) specific AT4 receptor blocker, divalyl angiotensin 4,10-5M. Results: Wild-type mice: R+ caused a dose-dependent decrease in MVO2 (-24±8% at highest dose, p<O,05). This effect was inhibited by L-NAME (-17±7%) and 10-5M PD 123319 (2±7%). AT2 KO mice: R+ caused a dose-dependent decrease in MVO2 (25±3% at highest dose, p<0.05) and this was not blocked by 10-6M PD123319, selective AT2 receptor antagonist suggesting an alternate receptor/pathway. At higher concentration of 10-5M, PD123319 blocked the effect of R+ (-5±2%, p<0.01). At this dose, PD compound appears to be a nonselective AT receptor antagonist possibly acting on AT4 receptor, L-NAME caused a 33% reduction in the effect of R+. Human myocardium: R+ decreased MVO2 with a -20±1% decrease at highest dose. This effect was attenuated by both L-NAME and 10-5M PD123319. Also, specific AT4 receptor blocker caused a 50% attenuation of R+ effect. Conclusion: The R+ enantiomer of amlodipine decreases MVO2 by AT4 receptor mediated NO release in AT2 receptor knock-out mice. In failing human myocardium, this effect also appears to be mediated by AT4 receptors.

Left ventricular function in mice lacking the AT2 receptor.

The role of the AT2 receptor in the heart is incompletely understood. We investigated left ventricular performance in AT2 receptor knockout mice, with and without deoxycorticosterone acetate (DOCA)-salt treatment. Given the putative opposing functions of the AT1 and AT2 receptor, we also analysed AT1 receptor expression in the left ventricle. We used a miniaturized conductance-manometer system to measure pressure-volume loops for analysing left ventricular performance under baseline conditions and after increasing peripheral vascular resistance. We determined left ventricular AT1-receptor expression by RNase-protection assays. In AT2 receptor knockout mice, end-systolic and end-diastolic volumes were lower than in wild-type mice, so that pressure-volume loops were shifted leftward. Left ventricular systolic and diastolic kinetics were not different between the groups. AT2 receptor knockout mice and wild-type mice both stabilized their reduced stroke volume after laparatomy as peripheral resistance was increased. DOCA-salt treatment increased elastance in AT2 receptor knockout mice, compared to controls. Furthermore, AT2 receptor knockout mice had a steeper increase in dP/dtmax. Left ventricular AT1 receptor gene expression was increased in AT2 receptor knockout mice and was not down-regulated in response to DOCA-salt treatment. Finally, the hearts of AT2 receptor knockout mice were smaller than controls, but increased in size in response to DOCA-salt treatment. AT2 receptor knockout mice displayed no major changes in left ventricular function at baseline or in response to DOCA-salt treatment, compared to wild-type mice. The AT2 receptor may be important to AT1 receptor expression in response to DOCA-salt challenge and may have some influence on cardiac growth responses.

Role of the At 2 Receptor in the Cardioprotective Effect of At 1 Antagonist in Mice with Chronic Heart Failure Induced by Coronary Ligation

P63 Angiotensin II (Ang II) acts mainly on two receptor subtypes: type 1 (AT 1 ) and type 2 (AT 2 ). Most of the known biological actions of Ang II are mediated via AT 1 receptors; however, the role of the AT 2 receptor is less well defined. We hypothesized that blockade of AT 1 receptors increases circulating Ang II levels, which in turn activates the AT 2 receptor and induces cardioprotection. In mice which lack AT 2 receptors, the effect of an AT 1 antagonist (AT 1 -ant) would be diminished or absent. AT 2 receptor knockout mice (-/-) and their wild-type littermates (+/+) were subjected to myocardial infarction (MI) by ligating the left anterior descending coronary artery. One month after MI, each strain of mice received either vehicle or AT 1 -ant (valsartan, 50 mg/kg/day in drinking water) for 3 months. Systolic blood pressure (SBP) was measured weekly and echocardiography performed once a month. Basal SBP and cardiac function did not differ between +/+ and -/-. Three months after MI, SBP and cardiac function changed similarly in both strains receiving vehicle. Valsartan significantly increased EF and decreased LVDd and mass and these effects were diminished in -/- (table). Our results suggest that under basal conditions, the AT 2 receptor may not play an important role in regulation of blood pressure and cardiac function; however, it does appear to be an important component in the cardioprotective effect of AT 1 -ant.

Inflammation influences vascular remodeling through AT2 receptor expression and signaling.

The AT(2) receptor, which exerts growth inhibitory effects in cell culture, is present scantily in the adult vasculature but is reexpressed after vascular injury. To examine the in vivo role of this receptor in vascular diseases, we developed a mouse model of vascular remodeling and compared the responses in wild-type (Agtr2(+)) and AT(2) receptor knockout (Agtr2(-)) mice. Polyethylene cuff placement on the femoral artery led to the vascular expression of cytokines, the transcriptional factor interferon regulatory factor-1 (IRF-1), and both the AT(1) and AT(2) receptors. Although the expressions of IRF-1 and AT(1) receptor were induced to comparable levels in both the Agtr2(+) and Agtr2(-) mice, the neointimal lesion size and the smooth muscle cell proliferation were twice greater in the Agtr2(-) than in the Agtr2(+) mouse. Correlated with this difference, AT(2) receptor expression was induced predominantly in the smooth muscle cells of Agtr2(+) mouse. These results demonstrate that the AT(2) receptor plays an important role in nonocclusive inflammatory injury by mediating the effects of inflammation on vascular smooth muscle growth inhibition.