ANGPTL7, a Gene Induced by Elevated IOP, Affects Adhesion of Trabecular Meshwork Cells to their ECM (2013)
Undergraduates: Will Hope, Juan Carabana
Faculty Advisor: Terete Borras
Department: Biology
Purpose: ANGPTL7 is a potential candidate gene for management of glaucoma. Its encoded protein is more abundant in glaucomatous tissues, and its mRNA is strongly upregulated by elevated IOP, the major risk factor for the disease. Focal Adhesions anchor the cell to its substrate and are a major site of force sensation and mechanotransduction. Our goal was to investigate whether this highly force-responsive gene affects cell-substrate adhesion of trabecular meshwork cells, which could in turn affect outflow facility.
Methods: Overexpression of HTM cells was achieved by nucleofector-transfecting 2 ¿g of DNA (ANGPTL7 and Empty as a control) into 4X10^5 cells. Cells were plated, fixed and treated with primary antibodies against V5 and Paxillin. To determine adhesion, overexpressing cells were plated, grown for 2h, washed and fixed. Number of adhered cells was obtained by crystal violet staining, absorbance reading and correlation to a standard curve.
Results: Overexpression of ANGPTL7 in HTM cells caused a decrease and distributional change in Paxillin. At 24 h post-transfection, 60% of pEmpty-transfected cells adhered to the dish compared to 34% of those overexpressing ANGPTL7 (p=0.014). HTM cells showed a 10% reduction in adhesion to the ANGTPL7-containing matrix compared to the adhesion to the ECM control (p=0.036). Diminished adhesion resulting in the in the loosening of cells to their ECM could be beneficial by increasing aqueous outflow reducing the effects of glaucoma.