The Interaction of Fibrin with Endothelial Cell Receptor N-Cadherin Promotes Fibrin-Dependent Angiogenesis

We recently discovered that fibrin interacts with the endothelial cell receptor N-cadherin and identified specific amino acid residues in N-cadherin that are critical for this interaction. However, the functional significance of this interaction has remained unclear.Given the structural and functional similarities between N-cadherin and vascular endothelial cadherin, we hypothesized that the interaction of fibrin with N-cadherin may contribute to fibrin-dependent angiogenesis. The primary objective of this study was to test this hypothesis.To test our hypothesis, we first knocked out N-cadherin in immortalized human microvascular endothelial cells (HMEC-1) using CRISPR technology, generating N-cadherin-knockout cells lacking this receptor. Our experiments using a fibrin gel angiogenesis assay revealed that fibrin promoted the formation of capillary-like structures by wild-type HMEC-1 cells, as expected. In contrast, fibrin had no effect on the N-cadherin-knockout cells. These findings highlight the critical role of N-cadherin in fibrin-dependent angiogenesis and suggest that the interaction of fibrin with N-cadherin may contribute to this process. To validate this suggestion, we mutated the amino acid residues in N-cadherin of HMEC-1 that are critical for fibrin binding, generating HMEC-1 cells expressing mutant N-cadherin. Experiments using these mutant cells, using the same angiogenesis assay, produced results nearly identical to those observed with the N-cadherin-knockout cells. Specifically, fibrin did not stimulate angiogenesis in the mutant HMEC-1 cells expressing mutant N-cadherin lacking fibrin-binding ability, thereby providing direct confirmation of our hypothesis.Our study establishes the functional role of the interaction between fibrin and endothelial N-cadherin, demonstrating that this interaction promotes fibrin-dependent angiogenesis.