Type IV collagen expression is regulated by Notch3-mediated Notch signaling during angiogenesis

Angiogenesis, the process of new blood vessel formation, involves endothelial cell proliferation and migration, accompanied by the remodeling of the extracellular matrix (ECM). Type IV collagen, a major ECM component, plays a critical role in vascular basement membrane regeneration, influencing cell polarity, migration, and survival. This study examines the regulatory role of Notch signaling, mediated by Notch3, in type IV collagen expression using TIG-1 fibroblasts and a co-culture angiogenesis model with human umbilical vein endothelial cells (HUVECs). Using small interfering RNA (siRNA) to suppress Notch3 expression, we observed a significant reduction in COL4A1 gene expression, which encodes the α1 chain of type IV collagen. Conversely, transient expression of the Notch3 intracellular domain (NICD3) activated Notch signaling, resulting in increased COL4A1 expression. In the co-culture model, pre-treatment of TIG-1 cells with Notch signaling inhibitors, including siNotch3 and DAPT, decreased the number of α1(IV)-positive TIG-1 fibroblasts adjacent to HUVECs. This reduction highlights the essential role of Notch3-mediated signaling in promoting type IV collagen expression during angiogenesis. Our findings suggest that Notch signaling regulates type IV collagen expression levels, supporting basement membrane formation and vascular maturation. These results provide insight into the molecular mechanisms of angiogenesis, potentially contributing to therapeutic strategies targeting vascular-related pathologies.