Staphylococcus epidermidis has become a serious nosocomial pathogen frequently causing infections associated with implanted foreign materials. Biofilm formation is considered a major factor determining S. epidermidis pathogenicity in such device-associated infections. Here, evidence is presented that extracellular DNA is important for the initial phase of biofilm development by S. epidermidis on polystyrene or glass surfaces under static or hydrodynamic conditions. Comparative PCR amplification from S. epidermidis chromosomal and extracellular DNA indicated that the extracellular DNA is similar to chromosomal DNA. Experiments involving the S. epidermidis wild-type and an isogenic atlE mutant indicated that most of the extracellular DNA in S. epidermidis cultures and biofilms is generated through activity of the autolysin AtlE. The presented results suggest that extracellular DNA is generated in S. epidermidis populations through AtlE-mediated lysis of a subpopulation of the bacteria, and that the extracellular DNA promotes biofilm formation of the remaining population.

Objective: Hydrogen sulfide (H 2 S) has been reported to be a gasotransmitter which regulates cardiovascular homeostasis. The present study aims to examine the hypothesis that hydrogen sulfide is able to promote angiogenesis. Methods: Angiogenesis was assessed using in vitro parameters (i.e. endothelial cell proliferation, adhesion, transwell migration assay, scratched wound healing and formation of tube-like structure) and in vivo by assessing neovascularization in mice. Phosphorylation of Akt was measured using Western blot analysis. Results: Exogenously administered NaHS (H 2 S donor) concentration-dependently (10-20 μmol/l) increased cell growth, migration, scratched wound healing and tube-like structure formation in cultured endothelial cells. These effects of NaHS on endothelial wound healing and tube-like structure formation were prevented by either the phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294002 (5 μmol/l) or transfection of a dominant-negative mutant of Akt. NaHS increased Akt phosphorylation and this effect was also blocked by either LY 294002 or wortmannin (25 nmol/l). NaHS did not significantly alter the levels of vascular endothelial growth factor, mRNA expression of fibroblast growth factor and angiopoietin-1, or nitric oxide metabolites. NaHS treatment (10 and 50 μmol kg − 1 day − 1 ) significantly promoted neovascularization in vivo in mice. Conclusion:The present study reports a novel proangiogenic role of H 2 S which is dependent on activation of Akt.

H(2)S (hydrogen sulfide), regarded as the third gaseous transmitter, is implicated in ulcerative colitis and colorectal cancers. The present study investigates the effects of H(2)S on cell proliferation in human colon cancer HCT 116 cells and SW480 cells. We identified the two key enzymes, CBS and CSE, for H(2)S synthesis in HCT 116 cells. An exogenously administered H(2)S donor NaHS induced cell proliferation in a concentration-dependent manner, with optimal proliferative concentration at 200 micromol/l. NaHS administration increased Akt and ERK phosphorylation. Blockade of Akt and ERK activation attenuated NaHS-induced cell proliferation. Cell-cycle analysis showed that NaHS treatment for 6 h decreased the proportion of cells in G(0)-G(1) phase and increased the proportion of cells in S phase. Protein expressions of Cyclin D1 and PCNA (proliferating cell nuclear antigen) were not altered, but the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) was inhibited significantly by NaHS treatment. NaHS significantly reduced NO metabolite levels. In conclusion, NaHS induced human colon cancer cell proliferation. This effect might be mediated by the increase of Akt and ERK phosphorylation and the decrease of p21(Waf1/Cip1) expression and NO production. The results suggested a role for H(2)S in human colonic cancer development.

It is not known whether H(2)S can promote angiogenesis with improvement of regional blood flow in ischemic organs. Sodium hydrosulfide (NaHS, a H(2)S donor) was administered once a day for 4 w following femoral artery ligation. Collateral vessel growth, capillary density, regional tissue blood flow, the expression of endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2) and Akt were examined during or at the end of the treatment period. NaHS treatment significantly increased collateral vessel growth, capillary density, and regional tissue blood flow in ischemic hind limb muscles compared with the controls. These effects were associated with an increase in VEGF expression in the skeletal muscles and VEGFR2 phosphorylation in the neighboring vascular endothelial cells, suggesting a role of VEGF in mediating the NaHS effects in a cell-cell interaction pattern. Moreover, NaHS treatment also resulted in an increase in Akt phosphorylation in ischemic hind limb muscles. In conclusion, our observations with NaHS strongly suggest that H(2)S is a proangiogenic factor in chronic ischemia. The proangiogenic effect of NaHS may be mediated by interaction between the upregulated VEGF in the skeletal muscle cells and the VEGFR2 as well as its downstream signaling element Akt in the vascular endothelial cells.

Aims: The potential receptor for hydrogen sulfide (H 2 S) remains unknown. Results: H 2 S could directly activate vascular endothelial growth factor receptor 2 (VEGFR2) and that a small interfering RNA (siRNA)-mediated knockdown of VEGFR2 inhibited H 2 S-induced migration of human vascular endothelial cells. H 2 S promoted angiogenesis in Matrigel plug assay in mice and this effect was attenuated by a VEGF receptor inhibitor. Using tandem mass spectrometry (MS), we identified a new disulfide complex located between Cys1045 and Cys1024 within VEGFR2 that was labile to H 2 S-mediated modification. Kinase activity of the mutant VEGFR2 (C1045A) devoid of the Cys1045-Cys1024 disulfide bond was significantly higher than wild-type VEGFR2. Transfection with vectors expressing VEGFR2 (C1045A) caused a significant increase in cell migration, while the migrationpromoting effect of H 2 S disappeared in the cells transfected with VEGFR2 (C1045A). Therefore, the Cys1045-Cys1024 disulfide bond serves as an intrinsic inhibitory motif and functions as a molecular switch for H 2 S. The formation of the Cys1045-Cys1024 disulfide bond disrupted the integrity of the active conformation of VEGFR2. Breaking the Cys1045-Cys1024 disulfide bond recovered the active conformation of VEGFR2. This motif was prone to a nucleophilic attack by H 2 S via an interaction of their frontier molecular orbitals. siRNA-mediated knockdown of cystathionine c-lyase attenuated migration of vascular endothelial cells induced by VEGF or moderate hypoxia.

H(2)S is a novel inhibitor of L-type calcium channels in cardiomyocytes. Moreover, H(2)S-induced inhibition of [Ca(2+)]i appears to be a secondary effect owing to its initial action towards I(Ca,L). The inhibitory effect of H(2)S on I(Ca,L) requires further investigation, particularly in the exploration of new pathways involved in cardiac calcium homeostasis and disease pathology.

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