Abstract:Aim To investigate the effects of peptide specific to CCR5 on monocyte chemotaxis. Methods The effects of peptide on the binding of mAb 2D7 to monocyte were surveyed by flow cytometry; Competitive binding of peptide and RANTES on monocyte was measured through Quantikine Human RANTES kit; Chemotaxis assay of monocyte toward peptide was performed in vitro;Effects of peptide on atherosclerosis formation was detected in vivo in mice. Results Peptide could inhibit the binding of 2D7 and RANTES to monocyte (IC50 was about 3.98 μmol/L). In vitro,peptide showed no chemotactic effect to monocyte(P=0.074) and could inhibit chemotactic activity of monocyte toward RANTES,the cell number of migration in the peptide+RANTES groups (23±10) was lower than that of the RANTES groups(62±13). Peptide could impair the influx of monocyte to the aortic root in mice(p<0.05). Conclusion Peptide could target to monocyte and suppress chemotactic activity of monocyte toward RANTES and reduce atherosclerosis.

Abstract:Aim To investigate whether homocysteine (HCY) can induce cultured human umbilical vein endothelial cells (hUVEC) to express regulated upon activation, normal T expressed and secreted (RANTES) protein. Methods After exposure of the cultured hUVEC to HCY at increasing concentrations for 8 h, the RANTES protein expression was determined by immunocytochemistry and Western blot analysis. Results Cultured hUVEC could express RANTES protein. Immunocytochemistry showed the mean absorbance values of RANTES protein expression in hUVEC exposed to HCY at different concentrations (0.1, 0.5 and 1 mmol/L HCY) for 8 h were 0.0434±0.0063, 0.0788±0.0053 and 0.1061±0.0215, respectively, which were significantly higher than that of the control group (0.0200±0.0032). Analysis of variance proved a significant difference between groups (F=319.03, p<0.01). Western blot analysis displayed that exposure of hUVEC to HCY at gradient concentrations (0.1, 0.5 and 1 mmol/L) for 8 h resulted in a 2.29 fold, a 2.63 fold and a 2.78 fold increase in the expression of RANTES protein in the cells, compared with the control group. Conclusions The cultured hUVEC could express RANTES protein, and HCY was able to induce hUVEC to express RANTES protein at a higher level.

Abstract:Aim To understand whether lipid peroxidation injury to endothelial cells ( ECs ) induces the production of monocyte chemotactic factor RANTES. Methods After a four hour exposure of the cultured human umbilical vein ECs to 5 μmol/L diamide, the conditioned medium (CM) was collected. To obtain a conditioned medium containing RANTES (MW 8 kDa ), the above CM was put into the double layer dialysis molecular porous membrane tubings (Spectrum Lab. Inc. ) with the molecular weight cut off at 10 kDa ( inner layer ) and 3.5 kDa ( outer layer ) respectively. Appropriate amount of 0.01 mol/L PBS was injected into the interspace between the two membranes. The whole set of the membrane tubings was suspended in a pyramid flask containing PBS, and then the samples were dialysed against PBS. Finally, a conditioned medium with MW of 3.5～10 kDa was taken out from the interspace between the two membranes. The chemotactic activity of this conditioned medium for monocytes was tested by micropore filter method using a modified Boyden chamber. For antibody inhibition studies, the conditioned medium was incubated with anti-RANTES antibody and assayed again. Results The monocyte migration distance induced by diamide-CM group (89.75±11.33 μm) is markedly longer than that of the nondiamide-CM group(77.30±11.53 μm)and that of the random migration group(76.18±10.50 μm ). Analysis of variance showed that there was a significant difference between groups(F=47.20,P<0.01). After the addition of anti-RANTES antibody, monocyte chemotactic activity was markedly inhibited (F=21.31,P<0.01 ). Conclusion On the basis of functional assay it suggests that lipid peroxidation injury might induce ECs to produce increased RANTES and may play an role in the recruitment of monocytes into the intima in atherogenesis.