Cystathionine gamma lyase overexpression enhances neovascularization through NAD-dependent mechanisms

Abstract

ObjectiveHydrogen sulfide (H2S) is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-gamma-lyase (CGL). CGL-dependant H2S production is required for neovasculariation in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have yet to be elucidated. Methods10 weeks old male whole-body CGL overexpressing mice (CGLTg) and wild type littermates (C57BL/6J) were subjected to the hindlimb ischemia model. Functional recovery was assessed through treadmill exercise endurance testing, while ischemic leg perfusion recovery was measured by laser Doppler perfusion imaging and tissue immunohistochemistry. To examine angiogenic potential, aortic ring sprouting assay and post-natal mouse retinal vasculature development studies were performed. Lastly, comparative metabolomics, NAD+/NADH analysis, and quantitative real-time PCR were performed on WT and CGLTg gastrocnemius muscles. ResultsThe restoration of blood flow upon femoral ligation occurred more rapidly in CGLTg mice. CGLTg mice were able to run further and for longer compared to WT mice. In ischemic gastrocnemius, capillary density was increased in mice overexpressing CGL. Endothelial cell sprouting was increased in aorta isolated from CGLTg mice, especially when cultured in VEGF-only media. Metabolomics analysis demonstrated an increased presence of niacinamide, a precursor of nicotinamide adenine dinucleotide (NAD+/ NADH) in the muscle of CGLTg mice. Finally, CGL overexpression and NMN supplementation improved endothelial cell migration in vitro. ConclusionsTaken together, our results demonstrate that CGL overexpression improves the neovascularization of skeletal muscle upon hindlimb ischemia. These effects are mediated by changes in the NAD pathway, which improves endothelial cell migration.