Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblasts.

OBJECTIVE: Scleroderma (systemic sclerosis; SSc) is an autoimmune disease characterized by vasculopathy and widespread organ fibrosis. Altered fibroblast function, both in vivo and in vitro, is well documented and illustrated by augmented synthesis and deposition of extracellular matrix proteins. We undertook this study to investigate the possibility that epigenetic mechanisms mediate the emergence and persistence of the altered SSc fibroblast phenotype. METHODS: The effects of DNA methyltransferase and histone deacetylase inhibitors on collagen expression and the level of epigenetic mediators in fibroblasts were examined. The effects of transient transfection of SSc fibroblasts with FLI1 gene and normal cells with FLI1 antisense construct on collagen expression were determined. The methylation status of the FLI1 promoter was tested in cultured cells and in SSc and normal skin biopsy specimens. RESULTS: Increased levels of epigenetic mediators in SSc fibroblasts were noted. The addition of epigenetic inhibitors to cell cultures normalized collagen expression in SSc fibroblasts. The augmented collagen synthesis by SSc fibroblasts was linked to epigenetic repression of the collagen suppressor gene FLI1. Heavy methylation of the CpG islands in the FLI1 promoter region was demonstrated in SSc fibroblasts and skin biopsy specimens. CONCLUSION: The results of this study indicate that epigenetic mechanisms may mediate the fibrotic manifestations of SSc. The signal transduction leading to the SSc fibrotic phenotype appears to converge on DNA methylation and histone deacetylation at the FLI1 gene.

Inhibitory effect of quercetin on proliferation of human microvascular endothelial cells in vitro.

AIM: To investigate the role of quercetin (Que) in the proliferation of cultured human skin microvascular endothelial cells (MVEC). METHODS: Cell count and [methyl-3H]thymidine ([3H]TdR) uptake assay were used to measure the effect of Que in the proliferation of cultured MVEC. Cytotoxicity of Que on MVEC was also evaluated by 51Cr release assay. RESULTS: When MVEC were treated with Que, the proliferation was significantly inhibited in a time-course and dose-dependent manner. Que 5 micromol/L did not inhibit the proliferation of MVEC. When the concentration of Que increased to 20, 40, 80, and 160 micromol/L, the cell numbers per well were decreased and the inhibition rate was 12.2 %, 23.5 %, 35.3 %, and 54.1 % respectively with IC50 of 138 micromol/L. The inhibitory rate of [3H]-TdR uptake was 18.7 %, 34.4 %, 48.9 %, and 62.5 % respectively (IC(50)=87.5 micromol/L). (51)Cr release assay showed that Que 160 micromol/L incubated with MVEC from 1 to 16 h had no clear cytotoxicity compared with control group. CONCLUSION: Que greatly inhibited the proliferation of cultured human MVEC in vitro. This effect may not be related to the cytotoxicity of Que on MVEC.

[Effects of quercetin on platelet-endothelial cell adhesion and their expression of adhesion molecules].

AIM: To observe the effect of quercetin (Que) on the adhesion of platelets to cultured endothelial cells and adhesion molecule expression by human umbilical vein endothelial cells (HUVEC) and platelets. METHODS: [3H]-Adenine labeled platelets were incubated with HUVEC to investigate the effect of Que on adhesion of platelets to HUVEC. The number of platelets adhering to the HUVEC monolayer was determined by liquid scintillation spectroscopy. TNF-alpha induced HUVEC expression ICAM-1 and thrombin induced platelets expression of P-selectin were measured by flow cytometry. RESULTS: Que (0.3-2.4 mumol.L-1) was shown to inhibit the increase of P-selectin expression of thrombin activated platelets. Pretreatment of HUVEC with tumor necrosis factor (TNF-alpha) significantly increased platelets adhesion to HUVEC and the expression ICAM-1. Que (0.6-2.4 mumol.L-1) inhibited this effect of TNF-alpha in a concentration-dependent manner. CONCLUSION: Que can inhibit the adhesion of platelets to HUVEC and the expression of adhesion molecules (P-selectin and ICAM-1).

Expression of recombinant human ICOS and in vitro characterization of its bioactivity on B lymphocytes.

Inducible costimulator (ICOS) is a novel costimulatory molecule expressed in activated T cell and has critical regulation effect on special immune response. In this study, the cDNA encoding human ICOS was cloned from activated tonsil cells via RT-PCR, and was expressed in E. coli on pET28 expression vector. The recombinant ICOS protein expressed from E. coli showed a molecular weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further confirmed by Western blot. In presence of IL-10, the purified rhICOS significantly increased in vitro B cell growth stimulated by pokeweed mitogen (PWM), and enhanced the secretion of IgG from B cells.

Inhibitory effects of prostaglandin A1 on apoptosis of rat cardiac microvascular endothelial cells was mediated by NF-kappaB.

AIM: To study the effects of prostaglandin A1 (PGA1) on rat cardiac microvascular endothelial cells. METH-ODS: Isolated rat cardiac microvascular endothelial cells were cultured in hypoxia and reoxygen conditions, respectively. Endothelial cell apoptosis was detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining. The activity of NF-kappaB was detected by electrophoretic mobility shift assay. Bcl-2 and Bax protein expression were examined by Western blot and bcl-2 mRNA expression was examined by Northern blot. RESULTS: PGA1 reduced endothelial cell apoptosis markedly, inhibited activity of NF-kappaB, and increased expression of Bcl-2 protein and bcl-2 mRNA. However, PGA1 did not alter Bax protein expression resulting in an increase in the ratio of Bcl-2 to Bax. CONCLUSION: PGA1 can inhibit rat cardiac microvascular endothelial cell apoptosis by inhibiting activity of NF-kappaB.