Cystathionine ß-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 ß-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.

MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure.

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9-/- (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and -4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

Hydrogen sulfide ameliorates hyperhomocysteinemia-associated chronic renal failure.

Elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with end-stage renal diseases. Hcy metabolizes in the body to produce hydrogen sulfide (H(2)S), and studies have demonstrated a protective role of H(2)S in end-stage organ failure. However, the role of H(2)S in HHcy-associated renal diseases is unclear. The present study was aimed to determine the role of H(2)S in HHcy-associated renal damage. Cystathionine-beta-synthase heterozygous (CBS+/-) and wild-type (WT, C57BL/6J) mice with two kidney (2-K) were used in this study and supplemented with or without NaHS (30 micromol/l, H(2)S donor) in the drinking water. To expedite the HHcy-associated glomerular damage, uninephrectomized (1-K) CBS(+/-) and 1-K WT mice were also used with or without NaHS supplementation. Plasma Hcy levels were elevated in CBS(+/-) 2-K and 1-K and WT 1-K mice along with increased proteinuria, whereas, plasma levels of H(2)S were attenuated in these groups compared with WT 2-K mice. Interestingly, H(2)S supplementation increased plasma H(2)S level and normalized the urinary protein secretion in the similar groups of animals as above. Increased activity of matrix metalloproteinase (MMP)-2 and -9 and apoptotic cells were observed in the renal cortical tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice; however, H(2)S prevented apoptotic cell death and normalized increased MMP activities. Increased expression of desmin and downregulation of nephrin in the cortical tissue of CBS(+/-) 2-K and 1-K and WT 1-K mice were ameliorated with H(2)S supplementation. Additionally, in the kidney tissues of CBS(+/-) 2-K and 1-K and WT 1-K mice, increased superoxide (O(2)(*-)) production and reduced glutathione (GSH)-to-oxidized glutathione (GSSG) ratio were normalized with exogenous H(2)S supplementation. These results demonstrate that HHcy-associated renal damage is related to decreased endogenous H(2)S generation in the body. Additionally, here we demonstrate with evidence that H(2)S supplementation prevents HHcy-associated renal damage, in part, through its antioxidant properties.