Abstract
Introduction: H2S is a gaseous signaling molecule primarily produced in the vasculature by Cystathionine γ-lyase (CGL/CSE) enzyme, that regulates many biological functions including vasodilation, anti-inflammation, cytoprotection and angiogenesis. CSE genetic deficiency and subsequent reduction in H2S levels attenuates ischemic vascular remodeling. However, effects of CSE/H2S in relation to vasculogenesis have not been investigated. Objective: To investigate the effect of endothelial specific CSE (EC-CSE) transgenic overexpression in restoring blood flow and vasculogenesis in the ischemic hind limb model. Methods: Unilateral femoral artery ligation was performed in wild type (WT), CSE-/- and EC-CSE mice, treated with PBS or H2S donor, DATS (n=8-10, in each group). Tissue angiogenic and proliferation indices were determined by the ratios of CD31/DAPI and Ki67/DAPI staining respectively. Expression of CSE and SDF-1 in the ischemic and non-ischemic limbs was determined by immunohistochemistry; CSE and HIF-1α were measured by qRT-PCR; and ELISA to measure VEGF levels. Plasma and tissue sulfide levels were measured using the MBB/HPLC method. Flow cytometry was used to analyze bone marrow-derived, blood and skeletal muscle EPCs positive for CD34/FLK-1 staining. Results: Ischemic hind limb perfusion was significantly impaired in CSE-/- compared to WT mice, while blood flow was restored in DATS treated cohort groups and EC-CSE transgenic mice by day 14. Angiogenic and proliferation indices were decreased in CSE-/- mice, and restored by DATS treatment and in EC-CSE transgenic mice. These findings corresponded with significantly increased circulating EPCs at days 3, 5 and 7 upon DATS treatment and EC-CSE transgenic overexpression. Lastly, we observed that CSE/H2S induces EPC recruitment via SDF-1/VEGF mediated pathways that augments vasculogenesis and restoration of ischemic hind limb blood flow. Conclusion: Endothelial specific overexpression of CSE (EC-CSE) and exogenous H2S delivery reversed impaired blood flow in CSE-/- mice through EPC mediated revascularization during hind limb ischemia. Modulation of CSE mediated H2S induced revascularization might serve as a novel therapeutic approach for ischemic vascular remodeling.
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