Mas and ET B receptors physically interact in endothelial cells (ECs) and are involved in the protective actions of angiotensin 1-7 (Ang (1-7)). We assessed whether the MAS/ET B R interaction plays a role in EC signalling and whether strategies to enhance MAS/ET B R association influence vascular responses. Human ECs were stimulated with Ang (1-7) (10 -7 M) in the presence/absence of A779 (Mas receptor antagonist, 10 -5 M) and BQ788 (ET B R antagonist, 10 -5 M). Protein expression and signalling activation were assessed by immunoblotting. NO production was evaluated by DAF-FM fluorescence and ROS production by chemiluminescence (superoxide anion) or amplex red (hydrogen peroxide (H 2 O 2 )). mRNA expression was assessed by qPCR. Endothelial function was assessed in mouse intact arteries by myography. Ang (1-7), through Mas and ET B R induced phosphorylation of eNOS (35%); followed by an increase in NO production (2.0 fold) (p<0.05 vs ctl). High throughput screening of protein:protein interaction compounds in an in-house library identified 23 potential enhancers of the MAS/ET B R interaction. Fluorescence polarization assays were used to further select the most potent enhancers and define their working concentration for testing in ECs (Enh1-4: 10 -5 M). Enh4 increased superoxide anion (55.6±26.3% vs ctl, p<0.05) and H 2 O 2 production (54.7±11.1% vs ctl, p<0.05), while Enh3 increased H 2 O 2 generation (48.1±15.4% vs ctl, p<0.05) in ECs. Moreover, Enh4 increased Nrf2 (3.0 fold), Sod1 (2.0 fold) and Nqo1 (3.1 fold) mRNA expression (p<0.05 vs ctl). Enh3 and Enh4 increased NO production (Enh3: 21.2±7.4%; Enh4: 23.6±8.2% vs veh, p<0.05) in ECs. Acetylcholine (Ach) curves were performed to assess endothelium-dependent relaxation in the absence and presence of enhancers. Enh4 increased ACh-induced relaxation (Emax%: 96.7±4.6 vs ctl: 70.4±3.3, p<0.05), while other enhancers did not improve endothelial function. Taken together, increasing MAS/ET B R interaction with specific enhancers augments protective signalling in ECs and promotes endothelial-dependent vasorelaxation, particularly with Enh4. In conclusion, enhancing interactions between MasR and ET B R may be a new vasoprotective strategy to improve vascular function in cardiovascular disease.

The intake of various types and amounts of dietary fats influences metabolic and cardiovascular health. Hence, this study evaluated the impact of routinely consumed Pakistani dietary fats on their cardiometabolic impact. For this, we made four groups of mice, each comprising 5 animals: (1) C-ND: Control mice on a normal diet, (2) HFD-DG: High-fat diet mice on a normal diet plus 10% (w/w) desi ghee, (3) HFD-O: Mice on normal diet plus 10% (w/w) plant oil (4) HFD-BG: Mice on normal diet plus 10% (w/w) banaspati ghee. Mice were fed for 16 weeks, and blood, liver, and heart samples were collected for biochemical, histological, and electron microscopic analysis. The physical factors indicated that mice fed on HFD gained more body weight than the C-ND group. Blood parameters do not show significant differences, but overall, the glucose and cholesterol concentrations were raised in the mice fed with a fat-rich diet, with the highest concentrations in the HFD-BG group. The mice fed with HFD-BG and HFD-O had more lipid droplets in the liver, compared to HFD-DG and C-ND.

Introduction The ET‐1 system plays an important role in the pathophysiology of pulmonary hypertension (PH). We previously demonstrated that treatment of hypoxic mice (an experimental model of PH) with Ang(1‐7) lead to amelioration of right ventricle hypertrophy and systolic pressure, vascular remodelling and hypercontractility to ET‐1. The protective effects of Ang(1‐7) were associated with an increase in ETBR expression. Peptide array analysis identified that the Ang(1‐7) MAS receptor physically interacts with the ETBR. In this study, we evaluated the vascular effects of such interaction and whether enhancers of MAS/ETBR would modulate endothelial and vascular function. Methods Human aortic endothelial cells (HAEC) were used and stimulated with Ang(1‐7) (0.1 μM) in the presence/absence of A779 (MAS antagonist, 1μM), BQ788 (ETBR antagonist, 1μM) and MAS/ETBR peptide disruptor (10μM). Protein expression and signalling activation was assessed by immunoblotting, NO production by DAF‐FM fluorescence and vascular function by wire myography. Results In HAECs, ETBR expression was increased after stimulation with Ang(1‐7) (Control=100% vs. 2h=147%±6.8, 4h=146.1%±5.5 and 24h=176%±21.9) in a MAS receptor dependent manner (+A779 ‐ 2h= 88.2%±1.5, 4h= 84.3%±5.1 and 24h =86.7%±3.6) (p<0.05). Ang(1‐7) also increased AKT (40%) and eNOS (35%) phosphorylation; followed by an increase in NO production (Control=100% vs. 10min= 242.5%±25.4) (p<0.05). The effects on endothelial cell signalling and No production were inhibited by MAS and ETBR antagonists, as well as, a MAS/ETBR interaction peptide disruptor. In a high through put screening of a 20K protein:protein interaction modulator library, we discovered 23 potential enhancers of the MAS/ETBR interaction, where we tested 4 in HAECs (Enh1‐4: 10μM). From all enhancers, only enhancers 3 and 4 increased eNOS activation (Vehicle=100% vs. En3 10min= 202%±18.2, Enh4 10min= 194.7%±19.6) and NO production (Vehicle=100% vs. En3 10min= 121.2%±7.4, Enh4 10min= 123.6%±8.2). Mesenteric resistance arteries were pre‐incubated with Enh3‐4 for 30 minutes prior to acetylcholine (Ach) curves, to assess endothelium‐dependent relaxation. Only Enh4 increased ACh‐induced relaxation (Emax: 96.7±4.6 vs Emax control: 70.4±3.3), p<0.05. Conclusion Our data demonstrates that the MAS/ETBR interaction has protective effects in the endothelium. Enhancing MAS/ETBR crosstalk leads to NO production and improvement of endothelial function, demonstrating potential in the treatment of vascular dysfunction and associated diseases.

Objective: We demonstrated that the MAS and ETB receptors physically interact and may be involved in the protective actions of Ang(1–7) in endothelial cells (ECs). We assessed the biological role of MAS/ETBR interaction in the vasculature, focusing on the NO pathway. Design and method: Human ECs were stimulated with Ang(1–7) (10-7 M) in the presence/absence of A779 (MAS antagonist, 10-5 M), BQ788 (ETBR antagonist, 10-5 M) and MAS/ETBR peptide disruptor (10-5 M). ETBR expression and AKT/eNOS activation were evaluated by immunoblotting and NO production by DAF-FM fluorescence. Results: Ang(1–7) increased ETBR expression (50%), an effect blocked by A779 (p < 0.05 vs control). Ang(1–7) also induced AKT (40%) and eNOS (35%) activation; followed by an increase in NO production (2 fold) (p < 0.05 vs control). These effects were inhibited by A779, BQ788 and the MAS/ETBR disruptor. In a high throughput screening of a 20K protein:protein interaction modulator library, we discovered 23 potential enhancers of the MAS/ETBR interaction, where we tested 4 in ECs (Enh1–4: 10–5 M). All four MAS/ETBR enhancers increased eNOS activation with variable responses (Enh1: 44.2%; Enh2: 65.4%; Enh3: 80.7%; Enh4: 94.9% vs control, p < 0.05), but only treatment with Enh1 and Enh2 increased NO production (Enh1: 109.4%; Enh2: 46.5% vs control, p < 0.05) in ECs. Mesenteric resistance arteries were pre-incubated with Enh1–4 for 30 minutes prior to acetylcholine (Ach) curves, to assess endothelium-dependent relaxation. Enh1 decreased ACh-induced relaxation (Emax: 52.8 ± 2.9 vs Emax control: 70.4 ± 3.3), while Enh4 increased ACh-induced relaxation (Emax: 96.7 ± 4.6 vs Emax control: 70.4 ± 3.3), p < 0.05. Conclusions: Promoting the MAS/ETBR interaction with specific enhancers leads to eNOS activation and vasorelaxation, where Enh1–4 have variable effects. Current studies are focusing on identifying the most sensitive enhancer that will provide vasoprotection, and potentially new strategies in the treatment of vascular disease.