Abstract 133: Coagulation FXI Regulates Endothelial Cell Barrier Function By Inducing VE-cadherin Proteolysis In An ADAM10-dependent Manner

Abstract

Introduction: Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI), known for its role in the coagulation cascade, also plays a regulatory role in inflammation. While a relationship between FXI activity, VE-cadherin expression levels, and endothelial barrier function has been observed, the mechanisms by which this process is mediated are unknown. Hypothesis: Activated FXI (FXIa) regulates EC barrier function by inducing VE-cadherin proteolysis. Methods: Human umbilical vein endothelial cells were stimulated with FXIa (30 and 5 nM) for six hours. VE-cadherin proteolysis and expression levels were analyzed by Western blot and immunofluorescence. ECs permeability was quantified by measuring the flux of Evans blue-albumin across the ECs. Immunoprecipitation and Western blot analysis were used to study FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions. Results: FXIa (5 nM) generated a VE-cadherin C-terminal fragment and decreased VE-cadherin expression on ECs; this process was reversed by the serine protease inhibitor, PPACK. Thrombin, kallikrein, or FXIIa did not induce VE-cadherin proteolysis. FXIa increased ECs permeability, yet did not induce the expression of VCAM-1. In contrast, while VCAM-1 expression was upregulated by tumor necrosis factor alpha or vascular endothelial growth factor, neither of these induced VE-cadherin proteolysis. A pharmacological inhibitor of a disintegrin and metalloproteinase 10 (ADAM10) abrogated the effects of FXIa on ECs, including proteolysis of VE-cadherin. Similarly, the low density lipoprotein antagonist, receptor-associated protein (RAP), inhibited FXIa-induced VE-cadherin proteolysis by preventing FXIa complex formation with PAI-1 and VLDLR on ECs. Conclusion: These results suggest that FXIa disrupts VE-cadherin-mediated EC permeability by increasing ADAM10 activity through complex formation and internalization by PAI-1 and VLDLR. It remains to be seen whether this pathway represents a druggable target to protect EC barrier function in inflammatory diseases by inhibiting FXIa.