Effect Of Aspergillus Fumigatus Infection On TF, VWF And Apoptosis Of HUVEC

Objective An in vitro model of human umbilical vein endothelial cell (HUVEC) infected by aspergillus fumigatus hypha was established to observe the levels of tissue factor (TF) and von Willebrand factor (vWF) expressed by HUVEC following aspergillus fumigatus infecting alone or treatment with cytochalasin D, N-cadherin monoclonal antibody, dexamethasone, respectively, so as to explore the mechanism of angioinvasion in invasive aspergillosis.Methods After HUVEC were grown to confluency in 24-well plates, pre-configured aspergillus fumigatus hypha suspension (1×1O5CFU/ml) was added into each well, then the model was established. The experiment was divided into six groups, and total reaction volume was 200μ1 per well, that was①negative control group:no stimulus to HUVEC;②TNF-a group:stimulation with TNF-α(100 ng/ml);③aspergillus fumigatus hypha group:stimulation with aspergillus fumigatus hypha (105 CFU/ml);④cytochalasin D group:grown to confluency, HUVEC were pretreated with cytochalasin D solution (70 nmol/L) 10 min before experiment, then aspergillus fumigatus hypha suspension (105 CFU/ml) was added;⑤N-cadherin antibody group:grown to confluency, HUVECs were pretreated with N-cadherin monoclonal antibody (1:100) 1 h before experiment, then aspergillus fumigatus hypha suspension (105 CFU/ml) was added;⑥dexamethasone group:dexamethasone (10-4mol/L) and aspergillus fumigatus hypha (105 CFU/ml) were added at the same time. After coculture with aspergillus fumigatus hypha, the liquid of cell freezing and thawing was acquired to detect the level of TF at 0 h,2 h,6 h, 12 h and 18 h, and cell culture supernatant was extracted to detect that of vWF at 2 h,6 h, 12 h and 18 h. Enzyme linked immunosorbent assay (ELISA) was used.Results1. TF changes:Except negative control group, compared to the Oh, the expression of TF increased at 2 h,6 h,12 h, and 18 h (P< 0.05), however, there was no change in negative control group. The level of TF at 0 h had no statistical significant difference between groups. Compared to negative control group, the level of TF in each experiment group increased at 2 h,6 h,12 h, and 18 h (P< 0.05). The level of TF in the TNF-a group was higher than the aspergillus fumigatus hypha group at 2 h,6 h and 12 h (P< 0.01). The level of TF had no statistical significant difference between the cytochalasin D group and the aspergillus fumigatus hypha group at each time point. The level of TF was lower in the N-cadherin antibody group than in the aspergillus fumigatus hypha group at 2 h,6 h and 12 h (P< 0.05). The level of TF had no statistical significant difference between the dexamethasone group and the aspergillus fumigatus hypha group at each time point.2. vWF changes:Compared to that of vWF at 2 h, the level was higher at 18 h in negative control group and TNF-a group, and higher at 6 h,12 h and 18 h in the other groups. (P< 0.05). Compared to negative control group, the level of vWF in each experiment group increased at 2 h,6 h,12 h, and 18 h except that in N-cadherin antibody group at 2 h (P< 0.05). The level of vWF in TNF-a group was higher than that in the aspergillus fumigatus hypha group at 2 h, but lower at 18 h. (P< 0.05). The level of vWF had no statistical significant difference between the cytochalasin D group and the aspergillus fumigatus hypha group at each time point. The level of vWF was lower in the N-cadherin antibody group than that in the aspergillus fumigatus hypha group at 2 h and 6 h (P< 0.05). The level of vWF had no statistical significant difference between the dexamethasone group and the aspergillus fumigatus hypha group at each time point.Conclusion The results of this study have suggested that:①To establish an in vitro model of HUVEC infected by aspergillus fumigatus is feasible;②HUVEC infected by aspergillus fumigatus hypha results in cell damage and overexpression of TF and vWF;③Cytochalasin D has no significant effect on the HUVEC infected by aspergillus fumigatus hypha to express TF and vWF.④N-cadherin monoclonal antibody can suppress the action of aspergillus fumigatus hypha on HUVEC to express TF and vWF.⑤Dexamethasone has no significant effect on HUVEC infected by aspergillus fumigatus hypha to express TF and vWF.Part 2 Aspergillus fumigatus hypha induces HUVEC apoptosisObjective Using the in vitro model of HUVECs infected by aspergillus fumigatus to observe the apoptosis rate of HUVEC.Methods An in vitro model of HUVEC infected by aspergillus fumigatus was established (reference to the part 1). The experiment was divided into three groups:①negative control group:no stimulus to HUVEC;②aspergillus fumigatus hypha group I: stimulated by aspergillus fumigatus hypha (105 CFU/ml);③aspergillus fumigatus hypha groupⅡ:stimulated by aspergillus fumigatus hypha (106 CFU/ml). Using the flow cytometry to detect the apoptosis of HUVEC at 2h,4h, and 8h.Results Except negative control group, the apoptosis rate of HUVEC increased gradually in each group at 2h,4h and 8h (P< 0.05). Compared to negative control group at the corresponding time points, the apoptosis rate of HUVEC in aspergillus fumigatus hypha group increased at 2,4, and 8 h (P< 0.01), moreover, it was higher in aspergillus fumigatus hypha group II than I (P< 0.05).Conclusion Aspergillus fumigatus hypha can induce HUVEC apoptosis. Within a certain period of time, apoptosis rate increases with the more time HUVECs contacting with hypha. At the same time point, the higher concentration of aspergillus fumigatus hypha results in the higher apoptosis rate of HUVEC.