AVE 0991, a nonpeptide analog of angiotensin-(1-7) [Ang-(1-7)], is an orally active Mas agonist with inhibitory effects on [125I]-Ang-(1-7) binding to bovine aortic endothelial cell membranes, and inhibits astrocyte-mediated neuroinflammation in Alzheimer's disease by enhancing autophagy.

AVE 0991 is a nonpeptide compound that elicits effects on the endothelium similar to Angiotensin-(1-7) [Ang-(1-7)]. AVE 0991 and unlabeled Ang-(1-7) compete for high-affinity binding of [125I]-Ang-(1-7) to bovine aortic endothelial cell membranes, with IC50s of 21±35 and 220±280 nM, respectively. The peak concentrations of NO and O2- release induced by AVE 0991 sodium salt and Ang-(1-7) (both at 10 μM) show no significant difference (NO: 295±20 and 270±25 nM; O2-: 18±2 and 20±4 nM). However, the amount of bioactive NO released is approximately 5 times higher for AVE 0991 compared to Ang-(1-7)[1].

In wild-type (WT) mice, the administration of AVE 0991 at a dose of 0.58 nmol/g leads to a significant reduction in water diuresis compared to vehicle-treated mice (0.06±0.03 mL versus 0.27±0.05; n=9 for each group; P＜0.01). This antidiuretic effect of AVE 0991 is accompanied by an increase in urine osmolality (1669±231.0 mOsm/KgH2O versus 681.1±165.8 mOsm/KgH2O in vehicle-treated mice; P＜0.01).The genetic deletion of Mas, a receptor associated with the effects of AVE 0991, eliminates the antidiuretic impact of AVE 0991 during water loading (0.37±0.10 mL [n=9] versus 0.27±0.03 mL [n=11] in AVE 0991-treated mice). Similar to observations in C57BL/6 mice, the administration of AVE 0991 (0.58 nmol/g) in water-loaded Swiss mice also results in a significant reduction in urinary volume compared to vehicle-treated animals (0.13±0.05 mL [n=16] versus 0.51±0.04 mL [n=40]; P＜0.01).Furthermore, a one-week treatment with AVE-0991 induces a notable decrease in perfusion pressure (56.55±0.86 vs. 68.73±0.69 mmHg in vehicle-treated rats) and an increase in systolic tension (11.40±0.05 vs. 9.84±0.15 g in vehicle-treated rats). Additionally, there is an elevation in the rate of tension rise (+dT/dt; 184.30±0.50 vs. 155.20±1.97 g/s in vehicle-treated rats) and the rate of tension fall (dT/dt; 179.60±1.39 vs. 150.80±2.42 g/s in vehicle-treated rats). A slight increase in heart rate (HR) is also observed (220.40±0.71 vs. 214.20±0.74 beats/min in vehicle-treated rats) [4].

Briefly, 100 μg of membranes from primary cultured bovine aortic endothelial cells (BAECs, passage 1) are incubated in a total volume of 200 μL for 45 minutes at 25°C in HEPES-buffered saline (10 mM HEPES, 0.1 M NaCl, 5 mM MgCl2) containing 0.2% BSA and protease inhibitor cocktail Complete. Saturable binding of [125I]-Ang-(1-7) is calculated by subtracting nonspecific binding (40% to 50%), determined in the presence of 10 μM unlabeled Ang-(1-7) from total binding. Competition experiments with increasing concentrations of AVE 0991 and unlabeled Ang-(1-7) are performed in the presence of 10 nM [125I]-Ang-(1-7). Assays are terminated by vacuum filtration (≤15 mm Hg) over filters (0.65 μm, Opak 96-well plates) presoaked with 1% BSA. The filters are washed 3 times with each 100 μL of PBS (50 mM, NaHPO4 and 0.15 M NaCl, pH 7.2). Radioactivity on dried filters is quantified with a gamma counter [1].

COS cells and CHO cells are stably transfected with rat Mas cDNA driven by a cytomegalovirus promoter and selected by neomycin. 125I-Ang-(1-7) (0.5×10^-9 mol/L) is incubated in 24-well plates for 60 minutes at 4°C in 0.3 mL of serum-free medium (DMEM) supplemented with 0.2% BSA, 0.005% bacitracin, 0.1 mol/L PMSF, and 0.5 mol/L orthophenanthroline with Mas-transfected COS cells in the presence or absence of AVE 0991 (AVE, 10-10 to -5 mol/L). After 2 ishes with ice-cold serum-free DMEM, cells are disrupted with 0.1% Triton X-100. Bound radioactivity is measured in a gamma counter. Binding of rhodamine-Ang-(1-7) in Mas-transfected CHO cells is performed under similar conditions using 2×10^-9 mol/L rhodamine-labeled-Ang-(1-7) in the presence or absence of AVE (10^-6 mol/L), CV11974 (10^-6 mol/L), or PD123319 (10^-6 mol/L). NSB is determined in the presence of 10-6 mol/L Ang-(1-7) [1].

Swiss male mice, Mas-KO (Mas-/-) male mice on the pure genetic background C57BL/6, and WT C57BL/6 control mice (Mas+/+) are used. Water diuresis is induced by intraperitoneal water injection (0.05 mL/g of body weight [BW]) in conscious mice. Drugs are administered in the same injection with water load at prefixed volumes (0.01 mL/g BW). In the first set of experiments, WT mice (C57BL/6, control group) or Mas-KO mice are treated with: (1) 0.58 nmol/g AVE 0991 (n=9, control; n=11, Mas-KO mice); or (2) vehicle for AVE 0991 (10 μM KOH, 0.01 mL/g; n=9, control; n=9, Mas-KO). In the second set, Swiss mice are treated with: (1) vehicle (n=36); (2) 0.58 nmol/g AVE 0991 (n=16); (3) 46 pmol/g Ang-(1-7) antagonist A-779 (n=4); (4) 2 nmol/g losartan or valsartan (n=5); (5) 2 nmol/g AT2 receptor antagonists PD123319 or PD123177 (n=9); (6) AVE 0991 combined with A-779; (7) AVE 0991 combined with losartan or valsartan (n=4 for each); (8) or AVE 0991combined with PD123319 (n=5) or PD123177 (n=4). The urinary volume is measured for 60 minutes after water loading, and urine samples are obtained to determine the osmolality [2]. Male Wistar rats weighing 250-300 g are used. Rats are treated either with AVE-0991 (1 mg/kg, n=9) or vehicle (0.9% NaCl, n=11) administered orally by gavage. At the end of the 7 day period of AVE-0991 treatment, the animals are decapitated 10-15 min after intraperitoneal injection of 400 IU of heparin. After the thorax is opened, the heart is carefully dissected, removed from the thoracic cavity, and placed in a plate containing ice-cold Krebs-Ringer solution (KRS) to attenuate any potential cardiac damage during dissection of aorta artery [3].