44-OR

Abstract

Aim The presence of HLA Ab in the allograft is associated with transplant vasculopathy (TV). Ligation of HLA class I elicits signaling events and induces the remodeling of actin cytoskeleton leading to cell survival, migration and proliferation in EC. However, the mechanism by which anti-HLA class II Ab contribute to TV development is not clear. We hypothesize that HLA II ligation induces signaling cascades mediating cytoskeleton reorganization. Methods To express HLA II, human aortic EC were infected with adenoviruses encoding Class II transactivator (Ad-CIITA). Expression of HLA II was determined by flow cytometric analysis using Ab against a monomorphic epitope on HLA II or locus specific Ab against HLA-DR, DQ and DP. Ad-CIITA infected EC were stimulated with anti-HLA II Ab and protein phosphorylation was examined by Western blot. Stress fiber formation was evaluated by fluorescence microscopy in EC stained with Texas Red-phalloidin. The FAK siRNA was utilized to deplete FAK expression. Results Infection with Ad-CIITA for 2d induced expression of HLA II in 80% of EC. Staining with locus specific Ab showed HLA-DR was expressed at the highest level with comparable levels of DP and DQ. Treatment of EC with HLA II Ab significantly increased the phosphorylation of FAK, Src, ERK and p70 S6 kinase (S6K). Ligation of HLA II also significantly augmented formation of stress fibers in EC compared to isotype control IgG [Fig. 1] Knockdown of FAK attenuated phosphorylation of Src, ERK and S6K and also abolished class II-mediated stress fiber formation. Download : Download full-size image Conclusions Ab ligation of HLA II molecules stimulates signal transduction and induces actin cytoskeleton remodeling in EC in a FAK dependent manner.