Nidogen-2 Maintains the Contractile Phenotype of Vascular Smooth Muscle Cells and Prevents Neointima Formation via Bridging Jagged1-Notch3 Signaling

Background: How the extracellular matrix (ECM) microenvironment modulates the contractile phenotype of vascular smooth muscle cells (VSMCs) and confers vascular homeostasis remains elusive. Methods: To explore the key ECM proteins in the maintenance of the contractile phenotype of VSMCs, we applied protein-protein interaction (PPI) network analysis to explore novel ECM proteins associated with the VSMC phenotype. By combining in vitro and in vivo genetic mice vascular injury model, we identified nidogen-2, a basement membrane (BM) glycoprotein, as a key ECM protein for maintenance of vascular smooth muscle cell identity. Results: We collected a VSMC phenotype-related gene dataset (VSMCPRG dataset) by using Gene Ontology (GO) annotation combined with a literature search. A computational analysis of protein-protein interactions between ECM protein genes and the genes from the VSMCPRG dataset revealed the candidate gene nidogen-2, a BM glycoprotein involved in regulation of the VSMC phenotype. Indeed, nidogen-2-deficient VSMCs exhibited loss of contractile phenotype in vitro , and compared with wild-type (WT) mice, nidogen-2 -/- mice showed aggravated post-wire injury neointima formation of carotid arteries. Further bioinformatics analysis, co-immunoprecipitation assays and luciferase assays revealed that nidogen-2 specifically interacted with Jagged1, a conventional Notch ligand. Nidogen-2 maintained the VSMC contractile phenotype via Jagged1-Notch3 signaling but not Notch1 or Notch2 signaling. Notably, nidogen-2 enhanced Jagged1 and Notch3 interaction and subsequent Notch3 activation. Reciprocally, Jagged1 and Notch3 interaction, signaling activation, and Jagged1-triggered VSMC differentiation were significantly repressed in nidogen-2-deficient VSMCs. In accordance, the suppressive effect of Jagged1 overexpression on neointima formation was attenuated in nidogen-2 -/- mice compared to wild-type mice. Conclusions: Nidogen-2 maintains the contractile phenotype of VSMCs through Jagged1-Notch3 signaling in vitro and in vivo . Nidogen-2 is required for Jagged1-Notch3 signaling.