PURPOSE: Age and elevated intraocular pressure are major risk factors for primary open-angle glaucoma (POAG) which is caused by a restriction in aqueous humor outflow from the anterior chamber. In this study, we investigated whether age-related changes in integrin subunit expression and activity signifies an early event in initiating fibrotic-like changes in the TM that could restrict outflow. METHODS: Human trabecular meshwork (TM) cells from young (<40 years) and old (>50 years) donor eyes were used. Flow cytometry, RT-qPCR, and immunofluorescence microscopy were used to evaluate levels of integrin and αSMA expression. On-cell westerns were used to determine fibronectin levels. Collagen gel contraction assays were used to determine contractile properties of cells and shRNA was used to knockdown α5β1 integrin levels. RESULTS: Studies revealed a significant decrease in α5 integrin expression in TM cells from older individuals. This loss was accompanied by an increase in activated but not total αvβ3 integrin levels. TM cells from older donors expressed higher levels of αSMA mRNA, assembled αSMA-containing stress fibers, and contracted collagen gels significantly more than young TM cells. TM cells from old donors also assembled higher levels of insoluble fibronectin fibrils and contained higher levels of EDB+ fibronectin in their extracellular matrix. shRNA knockdown of α5 integrin subunits showed that the increase in αvβ3 integrin activity was due to lower levels of α5 integrin expression. CONCLUSIONS: These studies suggest that age-related dysregulation of α5β1 and αvβ3 integrin signaling may represent an important early molecular event in inducing fibrogenic pathways associated with POAG.