Cystathionine β-synthase and cystathionine γ-lyase double gene transfer ameliorate homocysteine-mediated mesangial inflammation through hydrogen sulfide generation.

Elevated level of homocysteine (Hcy) induces chronic inflammation in vascular bed, including glomerulus, and promotes glomerulosclerosis. In this study we investigated in vitro mechanism of Hcy-mediated monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) induction and determined the regulatory role of hydrogen sulfide (H₂S) to ameliorate inflammation. Mouse glomerular mesangial cells (MCs) were incubated with Hcy (75 μM) and supplemented with vehicle or with H₂S (30 μM, in the form of NaHS). Inflammatory molecules MCP-1 and MIP-2 were measured by ELISA. Cellular capability to generate H₂S was measured by colorimetric chemical method. To enhance endogenous production of H₂S and better clearance of Hcy, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) genes were delivered to the cells. Oxidative NAD(P)H p47(phox) was measured by Western blot analysis and immunostaining. Phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH₂-terminal kinase (JNK1/2) were measured by Western blot analysis. Our results demonstrated that Hcy upregulated inflammatory molecules MCP-1 and MIP-2, whereas endogenous production of H₂S was attenuated. H₂S treatment as well as CBS and CSE doubly cDNA overexpression markedly reduced Hcy-induced upregulation of MCP-1 and MIP-2. Hcy-induced upregulation of oxidative p47(phox) was attenuated by H₂S supplementation and CBS/CSE overexpression as well. In addition to that we also detected Hcy-induced MCP-1 and MIP-2 induction was through phosphorylation of ERK1/2 and JNK1/2. Either H₂S supplementation or CBS and CSE doubly cDNA overexpression attenuated Hcy-induced phosphorylation of these two signaling molecules and diminished MCP-1 and MIP-2 expressions. Similar results were obtained by inhibition of ERK1/2 and JNK1/2 using pharmacological and small interferring RNA (siRNA) blockers. We conclude that H₂S plays a regulatory role in Hcy-induced mesangial inflammation and that ERK1/2 and JNK1/2 are two signaling pathways involved this process.