Effect of Doxycycline on vasculogenic mimicry in human pterygium fibroblasts

Abstract

AIM: To investigate the effects of doxycycline(DOX)on vasculogenic mimicry(VM)in human pterygium fibroblasts(HPFs)and its molecular mechanisms.METHODS: Primary cultured HPFs were identified by Vimentin and CK through immunocytochemical staining. HPFs were divided into control group and DOX group including low, medium and high concentrations(50, 100, 200mg/L). The activity and migration of HPFs were detected by cell counting kit-8(CCK-8)and wound healing assay. The density of VM was observed by three-dimensional cell culture and periodic acid schiff(PAS)staining and compared the differences of VM formation in each group. Western blot was used to analyze the expression of matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor(VEGF).RESULTS:Immunocytochemical staining results showed that the cells were spindle shaped, meanwhile, they were positive for Vimentin and negative for CK, which were consistent with the characteristics of fibroblasts. Compared with the control group, the cell activity, mobility, VM density and the expression of MMP-9 and VEGF proteins in the DOX group were significantly decreased(P<0.05). Compared among different concentrations of DOX groups, the differences were statistically significant(P<0.05). Correlation analysis indicated that VM density formed by HPFs was significantly positively correlated with the protein expression of MMP-9 and VEGF(r=0.949, 0.960, all P<0.05).CONCLUSION: DOX can inhabit HPFs activity, migration, VM density by reducing the expression of MMP-9 and VEGF, suggesting that MMP-9 and VEGF may be the molecular mechanisms of VM formation in pterygium.

Keywords

• pterygium
• vasculogenic mimicry
• three-dimensional culture
• doxycycline
• matrix metalloproteinase-9
• vascular endothelial growth factor