Increased endogenous H₂S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H₂S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H₂S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H₂S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H₂S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H₂S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H₂S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H₂S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.