ABSTRACT
Objective To investigate mechanisms underlying the regulation of the permeability of vascular endothelial cells by the Treponema pallidum membrane protein Tpp47.Methods Human umbilical vascular endothelial cell (HUVEC) monolayers were established as a model,and were directly cultured with the presence of recombinant Tpp47 protein (rTpp47-treated group),or boiled and inactivated rTpp47 (negative control group).Some HUVEC monolayers,which were pretreated with the RhoA/ROCK signal pathway inhibitor Y-27632 for 30 minutes and then cultured with the presence of rTpp47,served as the pretreatment group.After 1-and 4-hour additional culture,enzymelinked immunosorbent assay (ELISA) was performed to estimate the permeability of these cell monolayers to horseradish peroxidase (HRP).After 12 hours of culture,rhodamine-phalloidin was used to stain cytoskeletal proteins,and confocal laser scanning microscopy was performed to observe the arrangement of the cytoskeletal protein F-actin.Western-blot analysis was conducted to measure the expressions of RhoA in HUVECs treated with rTpp47 or inactivated rTpp47.Results The supernatant level of HRP (expressed as the absorbance value at 450 nm) was significantly higher in the rTpp47-treated group than in the negative control group (0.81 ± 0.10 vs.0.39 ± 0.09,P ＜ 0.05),but no significant difference was observed between the pretreatment group (0.51 ± 0.10) and rTpp47-treated group or negative control group (both P ＞ 0.05) after 1-hour culture.Similarly,the rTpp47-treated group showed significantly increased levels of HRP compared with the pretreatment group and negative control group (2.31-± 0.14 vs.1.21 ± 0.12 and 0.73 ± 0.12,both P ＜ 0.05),while there was no significant difference between the pretreatment group and negative control group after 4-hour culture.The expression of RhoA in HUVECs treated with rTpp47 was significantly higher than that in those treated with inactivated rTpp47.Confocal laser scanning microscopy showed that rTpp47 treatment led to the rearrangement of F-actin in HUVECs followed by the formation of stress fibers in cytoplasm,while Y-27632 could partly inhibit the rearrangement of F-actin.Conclusion The recombinant Treponema pallidum membrane protein Tpp47 can regulate the permeability of vascular endothelial cells through the RhoA/ROCK signal pathway.
ABSTRACT
Objective To evaluate the effect of Treponema pallidum membrane protein Tpp47 on vascular endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs) were classified into multiple groups to be cultured with various concentrations (50,100,200,400 and 800 μg/L) of the recombinant protein Tpp47 or lipopolysaccharide (LPS) for different durations (3,6,12,24 and 48 hours).Then,enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin in the culture supernatant of,fluorescence-based real-time quantitative PCR to quantify the mRNA expressions of ICAM-1 and E-selectin in,HUVECs.The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation activity of HUVECs treated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours.To estimate the effect on adhesion ability,some HUVECs were pretreated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours followed by coculture with THP-1 human monocytic leukaemia cells for 6 hours,then,the adhesion of HUVECs to THP-1 cells was visualized by fluorescence microscopy.The cells receiving no treatment served as the blank control.Results A significant increase was observed in the supernatant level (expressed as the absorbance value at 450 nm) of ICAM-1 for HUVECs treated with Tpp47 of 400 μg/L for 24 hours (1.28 ± 0.03 vs.0.90 ± 0.01,t =18.28,P ＜ 0.05) and that of E-selectin for HUVECs treated with Tpp47 of 400 μg/L for 12 hours (0.51 ± 0.01 vs.0.13 ± 0.03,t =18.19,P＜ 0.05) compared with untreated HUVECs.The adhesion rate to THP-1 cells was significantly higher in HUVECs pretreated with Tpp47 for 24 hours than in untreated HUVECs (56.1％ ± 1.9％ vs.16.3％ ± 2.1％,x2 =12.65,P ＜ 0.05).The cell proliferation rate was 19.5％ ± 1.7％ in HUVECs treated with Tpp47,significantly higher than that in untreated HUVECs (10.0％ ± 3.1％,x2 =3.92,P＜ 0.05),but lower than that in those treated with LPS (41.2％ ± 3.7％,x2 =10.42,P ＜ 0.05).Conclusions The recombinant membrane protein Tpp47 could enhance HUVECs to proliferate and adhere to monocytic THP-1 cells,suggesting a certain role of Tpp47 in the pathogenesis of syphilis.