Hydrogen sulfide promotes proliferation and regeneration of human cerebral microvascular endothelial cells via the sonic hedgehog signaling pathway

Abstract We investigated the effects of hydrogen sulfide (H 2 S) and sonic hedgehog (SHH) on the proliferation, autophagy, and apoptosis of human microvascular endothelial cells (HCMEC/D3). We also explored the regulatory relationship between cystathionine-β-synthase (CBS) and the SHH pathway. Human microglia cells (HMC3) were stimulated under hypoxia to secrete H 2 S and SHH proteins, which were then co-cultured with HCMEC/D3 cells. The relationship between H 2 S and SHH was investigated by inhibiting the CBS or SHH pathways. Vascular endothelial growth factor (VEGF) and H 2 S levels were detected using ELISA. The mRNA and Protein levels of VEGF, Beclin-1, light chain 3 (LC3), Cysteine aspartic acid protease-3(caspase-3), CBS, SHH, extracellular regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (P-ERK1/2) were determined by RT-PCR and western blot. The results indicated that H 2 S secretion by HMC3 increased during hypoxia, with both CBS and SHH proteins being up-regulated. The inhibition of CBS resulted in decreased levels of H 2 S and SHH in HMC3. When the SHH pathway is inhibited, H 2 S secretion levels remain unaffected. H 2 S and SHH proteins increased VEGF, P-ERK1/2, Beclin-1, and LC3 expression while reducing caspase-3 expression in HCMEC/D3 cells. H 2 S secretion by HMC3 promotes the proliferation and regeneration of HCMEC/D3 by regulating SHH protein and alleviating hypoxic injury.