Knockdown of AAMP Impacts on HECV Cell Functions In Vitro and Affects the Expression of VE-Cadherin

Abstract

Background: Angio-Associated Migratory Cell Protein (AAMP) is a 52 kDa protein expressed in a variety of human cell lines. Previous studies have demonstrated that AAMP is involved in endothelial cell adhesion, migration, and tube formation and plays roles in signalling pathways, such as RhoA-ROCK Methods: In this study, we knocked down the expression of AAMP in HECV cells using hammerhead ribozymes and assessed the influence of AAMP on endothelial cells. In order to explore potential mechanisms, Wnt/β catenin inhibitors (FH535 or IWP2) were also used in a number of the functional assays and the immunofluorescent staining patterns of several key molecules were explored.Results: Knockdown of AAMP expression was observed in the HECV cell line following transfection with the ribozyme transgene. Cellular migration and attachment, assessed using ECIS methods, and tubule formation were significantly inhibited by the knockdown of AAMP. Additionally, cell growth and cell matrix adhesion was also substantially reduced following AAMP knockdown, though this did not reach significance. Cell aggregation levels showed no statistical difference between AAMP knockdown cells and control cells. Interestingly, immunofluorescence staining showed AAMP knockdown cells had a reduced expression of VE-cadherin. Some overlap of function was seen between AAMP knockdown and the FH535 inhibitor.Conclusion: AAMP appears to influence endothelial cell migration and tubule formation and potentially, to a lesser effect, cell matrix adhesion and growth and also affects the expression of VE-cadherin. This data suggests that AAMP may play a role in angiogenesis. Assays performed with the FH535 inhibitor suggest a potential relationship between AAMP and Wnt/β catenin signalling.