Interaction of TGF-β and Notch Signaling Pathway by Smad4 to Induce Extracellular Matrix Accumulation of Vascular Smooth Muscle Cells

Abstract

Background: Ductus arteriosus (DA) close depends on the mature development and extracellular matrix (ECM) accumulation of vascular smooth muscle cells (VSMCs). This study aims to explore the function and interaction of transforming growth factor β (TGF-β) and Notch signaling pathway on VSMCs. Methods: An in vitro experiment was conducted on mouse VSMCs and supplementations of TGF-β, Notch signaling pathway ligand (Jagged-1), and inhibitor SAHM1. Transfections were constructed on VSMCs with two siRNAs of Smad4 and overexpressed Smad4 plasmid. ELISA measured the reactive oxygen species (ROS) level. Cell apoptosis was evaluated by TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry. Immunofluorescence (IF) analysis was used to detect the expression and localization of Smad4. The expression levels of ECM-related proteins and Smads were measured by western blotting and real-time quantitative PCR (RT-qPCR) analyses. Results: The higher concentration of TGF-β1 induced cell apoptosis (p < 0.001), increased ROS level (p < 0.001), and promoted ECM accumulation by regulating nuclear Smad4. Silencing Smad4 significantly impeded ECM accumulation induced by TGF-β1. The Notch pathway ligand Jagged-1 promoted ECM formation and activated Smad2–4 proteins, while Notch pathway inhibitor SAHM1 had the opposite effect. Further results confirmed that the Notch pathway induced Smad4 transcription (p < 0.001), thereby enhancing the function of the TGF-β1/Smads pathway. Moreover, Smad4 overexpression was found to induce ECM accumulation. Conclusions: Our present study explored the interaction of the Notch and TGF-β signaling pathways on ECM accumulation of VSMCs. The Notch pathway induced the transcription of Smad4, leading to enhanced ECM accumulation in VSMCs. This effect is achieved through co-regulation with TGF-β1.