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

Figures

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  Fig 1. Hydrogen sulfide (H2S) production is enhanced in cystathionine-γ-lyase (CGL)-overexpressing (CGLTg) gastrocnemius muscle. A, Quantitative real-time polymerase chain reaction (PCR) gene expression analysis of H2S-generating enzymes (CGL [CTH], 3-mercaptopyruvate sulfur transferase [MPST], and cystathionine-β-synthase [CBS]) in gastrocnemius muscle at baseline, normalized to 18S expression (n = 7-10 mice/group). B, CGL protein abundance in gastrocnemius muscle of CGL wild-type (CGLWT) and CGLTg mice at baseline by Western blot (Pierce reversible protein stain [Thermo Fisher Scientific] served as the loading control; n = 4-5 mice/group). C, Representative images of baseline gastrocnemius muscle sections (original magnification ×20) immunostained with CGL and vascular endothelial-cadherin (VE-cad) antibodies. Baseline CGL-mediated H2S production capacity in CGLWT and CGLTg mice gastrocnemius muscle (D) and plasmatic H2S production (E) detected by lead acetate assay (n = 5-6 mice/group). Data presented as mean ± standard error of the mean ∗∗P ≤ .01 and ∗∗∗∗P ≤ .0001, Student's t test.

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  Fig 2. Cystathionine-γ-lyase (CGL)-overexpressing (CGLTg) mice showed improved protection from hindlimb ischemia (HLI). A, Superficial perfusion of mouse hindlimb measured by laser Doppler perfusion imaging (LDPI). Results illustrated by representative LDPI images (Left) and ratio of LDPI perfusion quantification in ischemic and nonischemic limbs (Right; n = 8/group). B, Time (Left) and distance (Right) run on incremental speed test at day 14 after HLI (n = 8/group). C, Gastrocnemius muscle tissue perfusion 21 days after HLI shown in representative images and quantification (Right) of transverse sections stained with vascular endothelial-cadherin (VE-cad; n = 10-20/group). Scale bar = 50 μm. D, Representative transverse sections (Left) and quantification (Right) of laminin staining of gastrocnemius muscle at 21 days after HLI (n = 4-8/group). Scale bar = 50 μm. Data presented as mean ± standard error of the mean ∗P ≤ .05 and ∗∗P ≤ .01, Student's t test.

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  Fig 3. Vascular sprouting was accelerated in cystathionine-γ-lyase (CGL)-overexpressing (CGLTg) mice. A, Average length of microvessel sprouting from aortic ring explants from wild-type CGL (CGLWT) and CGLTg mice incubated in full endothelial cell growth medium-2 (EGM-2; n = 7/group). Scale bar = 100 μm. B, Average length of microvessel sprouting from aortic ring explants from CGLWT and CGLTg mice incubated in vascular endothelial growth factor A (VEGFA)-only EBM-2 (n = 7/group). Scale bar = 200 μm. C, Radial extension of developing mouse retina at postnatal day (D) 5, with representative images (Left) and quantification of radial extension (Right) of whole retina mounts stained with isolectin B4 (n = 4-5/group). Scale bar = 100 μm. Data presented as mean ± standard error of the mean ∗P ≤ .05, Student's t test.

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  Fig 4. Beneficial effects seen in cystathionine-γ-lyase (CGL)-overexpressing (CGLTg) mice was associated with changes in oxidized/reduced nicotinamide adenine dinucleotide (NAD+/NADH) pathways. A, Niacinamide level in CGL wild-type (CGLWT) and CGLTg gastrocnemius muscle (n = 4). P = .0569, Student's t test. B, Quantification of NAD+ and NADH concentration in gastrocnemius muscle (n = 5). Data presented as mean ± standard deviation. ∗∗P ≤ .01 (Student's t test), comparing NADH abundance between groups. C, Quantification of NAD+ and NADH concentration in liver (n = 5). Data presented as mean ± standard deviation. ∗P ≤ .05 (Student's t test), comparing NADH abundance between groups. D, Quantitative real-time polymerase chain reaction (PCR) gene expression analysis of enzymes associated with the NAD+/NADH pathway in gastrocnemius muscle. NAD-generating enzymes: Nmnat1, Nmnat2, Nmnat3, and Nadsyn1. NAD salvage pathway: Slc12a8, Nmrk1, Nmrk2, and Nampt. Preiss-Handler pathway: Naprt. NAD-consuming enzyme: SIRT1. All genes were normalized to 18S expression (n = 7-10/group). E, Endothelial cell (EC) migration across scratch with representative image (Left; original magnification, ×10) and…

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