Cystathionine-γ-lyase overexpression modulates oxidized nicotinamide adenine dinucleotide biosynthesis and enhances neovascularization

Objective

Hydrogen sulfide is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-γ-lyase (CGL). CGL-dependent hydrogen sulfide production is required for neovascularization in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have not yet been elucidated.

Methods

Male whole body CGL-overexpressing transgenic (CGL^Tg) mice and wild-type (WT) littermates (C57BL/6J) were subjected to the hindlimb ischemia model (age, 10-12 weeks). Functional recovery was assessed via the treadmill exercise endurance test. Leg perfusion was measured by laser Doppler imaging and vascular endothelial-cadherin immunostaining. To examine the angiogenic potential, aortic ring sprouting assay and postnatal mouse retinal vasculature development studies were performed. Finally, comparative metabolomics analysis, oxidized/reduced nicotinamide adenine dinucleotide (NAD⁺/NADH) analysis, and quantitative real-time polymerase chain reaction were performed on CGL^WT and CGL^Tg gastrocnemius muscle.

Results

The restoration of blood flow occurred more rapidly in CGL^Tg mice. Compared with the CGL^WT mice, the median ± standard deviation running distance and time were increased for the CGL^Tg mice after femoral artery ligation (159 ± 53 m vs 291 ± 74 m [P < .005] and 17 ± 4 minutes vs 27 ± 5 minutes [P < .05], respectively). Consistently, in the CGL^Tg ischemic gastrocnemius muscle, the capillary density was increased fourfold (0.05 ± 0.02 vs 0.20 ± 0.12; P < .005). Ex vivo, the endothelial cell (EC) sprouting length was increased in aorta isolated from CGL^Tg mice, especially when cultured in VEGFA (vascular endothelial growth factor A)-only media (63 ± 2 pixels vs 146 ± 52 pixels; P < .05). Metabolomics analysis demonstrated a higher level of niacinamide, a precursor of NAD⁺/NADH in the muscle of CGL^Tg mice (61.4 × 10⁶ ± 5.9 × 10⁶ vs 72.4 ± 7.7 × 10⁶ area under the curve; P < .05). Similarly, the NAD⁺ salvage pathway gene expression was increased in CGL^Tg gastrocnemius muscle. Finally, CGL overexpression or supplementation with the NAD⁺ precursor nicotinamide mononucleotide improved EC migration in vitro (wound closure: control, 35% ± 9%; CGL, 55% ± 11%; nicotinamide mononucleotide, 42% ± 13%; P < .05).

Conclusions

Our results have demonstrated that CGL overexpression improves the neovascularization of skeletal muscle on hindlimb ischemia. These effects correlated with changes in the NAD pathway, which improved EC migration.