Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation

Abstract

The aim of the study was to analyze whether cadherin- and Rho-family GTPases-mediated dynamic rearrangement of cell-cell adhesion play an important role during human arterial smooth muscle cell (haSMC) migration. Expression patterns of N-cadherin and beta-catenin were analyzed in a domestic pig restenosis model after 14, 28, and 90 days as well as in quiescent and migratory haSMCs in vitro. N-cadherin expression was upregulated by transient sense; downregulation was induced by antisense transfection. For functional inhibition, antibody GC-4 was used. Cell migration was quantified using Boyden chamber assays. Regulation of RhoA GTPase was tested by assessment of RhoA activity. In vivo analysis of N-cadherin expression in a porcine restenosis model revealed downregulation in the neointima after 14 days. After 28 days, N-cadherin expression was slightly restored, while after 90 days, no difference between medial and neointimal expression was detectable. beta-Catenin levels remained unchanged during the whole period. According to the in vivo situation, N-cadherin was significantly downregulated in migratory haSMCs compared to quiescent cells in vitro. After N-cadherin overexpression, haSMC migration was reduced by 87% (P<0.001). By contrast, inhibition of N-cadherin in quiescent haSMCs by GC-4 increased the migratory potential by 87% (P<0.01). In haSMCs overexpressing N-cadherin, a significant upregulation of RhoA activity was demonstrated, while RhoA activity was blocked by GC-4. These results indicate that the regulation of haSMC attachment by N-cadherins is essential for haSMC migration. Modification of N-cadherin expression and activity induces RhoA signaling with relevance for the reorganization of the actin cytoskeleton.