ABSTRACT
Purpose: Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvß3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells. Methods: TM-1 cells overexpressing wild-type ß3 (WTß3) or constitutively active ß3 (CAß3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvß3 and α5ß1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTß3 or CAß3 integrin subunits were used to examine the role of αvß3 integrin in HTM cells. The role of the canonical α5ß1 integrin-mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the ß1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632. Results: Activation of αvß3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5ß1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAß3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched. Conclusions: αvß3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a ß1 integrin/ROCK-independent mechanism. Thus, αvß3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.
