IntroductionHyperhomocysteinemia (HHcy) impairs nitric oxide endothelium-dependent vasodilation, consequently leading to atherosclerosis, a risk factor for cardiovascular disease. Novel treatments for HHcy are necessary. AimWe tested the hypothesis that alamandine, a vasoactive peptide of the renin-angiotensin system (RAS), could reverse HHcy-induced vascular dysfunction through the MrgD receptor and that this is mediated by the protein kinase A (PKA) pathway. Furthermore, we sought to determine a putative binding model of alamandine to the MrgD receptor through docking and molecular dynamics simulations. MethodThe abdominal aorta was excised from New Zealand white rabbits (n=15) and incubated with 3 mmol/L Hcy (to mimic HHcy) to induce vascular dysfunction in vitro. Vascular function was assessed by vasodilatory responses to cumulative doses of acetylcholine. ResultVasodilation was significantly impaired in HHcy-incubated aortic rings while alamandine reversed this effect (control, 74.25.0%; Hcy, 30.3 +/- 9.8%; alamandine+Hcy, 59.7 +/- 4.8%, P<.0001). KT5720 (PKA inhibitor) significantly inhibited the ability of alamandine to attenuate the impaired vasodilation caused by HHcy (KT5720+Hcy+alamandine, 27.1 +/- 24.1, P<.01). Following immunohistochemistry analysis, the MrgD receptor was highly expressed within the media and endothelial layer of aortic rings in HHcy compared to control (media: 0.23 +/- 0.003 vs control 0.16 +/- 0.01, P<.05 and endothelium: 0.68 +/- 0.07 vs control 0.13 +/- 0.02, P<.01, in PA/I (A.U) units). Computational studies also propose certain interactions of alamandine within the MrgD transmembrane domain. ConclusionThis study shows that alamandine is effective in reversing HHcy-induced vascular dysfunction, possibly through the PKA signaling pathway via MrgD. Our results indicate a therapeutic potential of alamandine in reversing the detrimental effects of HHcy.