ANGPTL7, a Gene Highly Induced by Elevated IOP, Affects Adhesion of Trabecular Meshwork Cells to their Extracellular Matrix

Abstract

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: : Primary HTM cell lines were generated from nonglaucomatous human donors. A full coding ANGPTL7 cDNA isolated from HTM cells was cloned into the pcDNA 3.1.V5-His-TOPO plasmid and fused to its V5 tag by overriding the stop codon (pWH1). Overexpression was achieved by nucleofector-transfecting 2 µg of DNA into 4X10⁵ cells using an empty vector as a control (pEmpty). Cells were plated in gelatin-coated chamber slides, fixed at 48 h post-transfection and treated with primary antibodies against V5 and the focal adhesion marker Paxillin. To determine adhesion, equal number of cells (1x10⁵) were transfected with pWH1 and pEmpty, plated and grown for 24 h to allow ANGPTL7 overexpression. Number of adhered cells was obtained by crystal violet staining, absorbance reading and correlation to a standard curve. To determine the effect of an ANGPTL7-containing extracellular matrix (ECM) in HTM cell adhesion, HEK-293 cells were transfected with pWH1 or pEmpty and allowed to lay out their matrices. After 4 d, the 293 cells were gently detached (3 mM EDTA) and the ECM dishes seeded with different concentrations of HTM cells. At 2.5 h, adhered HTM cells were calculated as above.

Results: : Overexpression of ANGPTL7 in HTM cells caused a decrease and distributional change in Paxillin in V5 positive cells. At 24 h post-transfection, 60% of pEmpty-transfected cells adhered to the dish compared to 34% of those overexpressing ANGPTL7 (pWH1-transfected) (p=0.014). HTM cells showed a 10% reduction in adhesion to the ANGTPL7-containing matrix compared to the adhesion to the ECM control (pEmpyty) (p=0.036) . Attachment experiments of ANGPTL7 to specific ECM proteins are in progress.

Conclusions: : Increased expression of ANGPTL7 in both cells and isolated matrices seems to induce a diminished adherence in the HTM cells. A loosening of the HTM cells to their ECM during an elevated presence of ANGPTL7 would suggest a beneficial effect of this gene in aqueous humor outflow, which could potentially be used in the treatment of glaucoma patients with elevated IOP.

Keywords: extracellular matrix • cell adhesions/cell junctions • trabecular meshwork