Conclusion
H2S 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.
Methods
We evaluated the pro-angiogenesis effects of H2S 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 H2S. 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 H2S improved angiogenesis in diabetic mice. miR-126-3p was regulated by both diabetes and H2S. Exogenous H2S 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.