RESUMO
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 (CGLTg) 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 CGLWT and CGLTg gastrocnemius muscle. Results: The restoration of blood flow occurred more rapidly in CGLTg mice. Compared with the CGLWT mice, the median ± standard deviation running distance and time were increased for the CGLTg 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 CGLTg 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 CGLTg 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 CGLTg 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 CGLTg 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.
RESUMO
PURPOSE: We have introduced an artificial intelligence framework, 31P-SPAWNN, in order to fully analyze phosphorus-31 ( 31 $$ {}^{31} $$ P) magnetic resonance spectra. The flexibility and speed of the technique rival traditional least-square fitting methods, with the performance of the two approaches, are compared in this work. THEORY AND METHODS: Convolutional neural network architectures have been proposed for the analysis and quantification of 31 $$ {}^{31} $$ P-spectroscopy. The generation of training and test data using a fully parameterized model is presented herein. In vivo unlocalized free induction decay and three-dimensional 31 $$ {}^{31} $$ P-magnetic resonance spectroscopy imaging data were acquired from healthy volunteers before being quantified using either 31P-SPAWNN or traditional least-square fitting techniques. RESULTS: The presented experiment has demonstrated both the reliability and accuracy of 31P-SPAWNN for estimating metabolite concentrations and spectral parameters. Simulated test data showed improved quantification using 31P-SPAWNN compared with LCModel. In vivo data analysis revealed higher accuracy at low signal-to-noise ratio using 31P-SPAWNN, yet with equivalent precision. Processing time using 31P-SPAWNN can be further shortened up to two orders of magnitude. CONCLUSION: The accuracy, reliability, and computational speed of the method open new perspectives for integrating these applications in a clinical setting.