Hydrogen Sulfide Improves Angiogenesis by Regulating the Transcription of pri-miR-126 in Diabetic Endothelial Cells

Introduction: Diabetes mellitus results in high rates of cardiovascular disease, such as microcirculation disorder of the lower limbs, with angiogenesis impairment being the main factor. The endothelium functions as a barrier between blood and the vessel wall. Vascular endothelial cell dysfunction caused by hyperglycemia is the main factor leading to angiogenesis impairment. Hydrogen sulfide (H(2)S) and miR-126-3p are known for their pro-angiogenesis effects; however, little is known about how H(2)S regulates miR-126-3p to promote angiogenesis under high-glucose conditions. Objectives: The main objective of this research was to explore how H(2)S regulates the miR-126-3p levels under high-glucose conditions. Methods: We evaluated the pro-angiogenesis effects of H(2)S in the diabetic hindlimb of an ischemia mice model and in vivo Matrigel plugs. Two microRNA datasets were used to screen microRNAs regulated by both diabetes and H(2)S. The mRNA and protein levels were detected through real-time PCR and Western blot, respectively. Immunofluorescent staining was also used to assess the capillary density and to evaluate the protein levels in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were used in in vitro experiments. A scratch wound-healing assay was applied to detect the migration ability of endothelial cells. Methylated DNA immunoprecipitation combined with real-time PCR was chosen to identify the DNA methylation level in the HUVECs. Results: Exogenous H(2)S improved angiogenesis in diabetic mice. miR-126-3p was regulated by both diabetes and H(2)S. Exogenous H(2)S up-regulated the miR-126-3p level and recovered the migration rate of endothelial cells via down-regulating the DNMT1 protein level, which was increased by high glucose. Furthermore, DNMT1 upregulation in the HUVECs increased the methylation levels of the gene sequences upstream of miR-126-3p and then inhibited the transcription of primary-miR-126, thus decreasing the miR-126-3p level. CSE overexpression in the HUVECs rescued the miR-126-3p level, by decreasing the methylation level to improve migration. Conclusion: H(2)S increases the miR-126-3p level through down-regulating the methylation level, by decreasing the DNMT1 protein level induced by high glucose, thus improving the angiogenesis originally impaired by high glucose.