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LaKisha K. Buie, Nuria Comes, Terete Borras; Angiopoietin-like 7 (ANGPTL7) Modulates DEX Induction and Fibronectin (FN1) Fibrils Formation in the Human Trabecular Meshwork (HTM). Invest. Ophthalmol. Vis. Sci. 2011;52(14):4622.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we had identified ANGPTL7 as a potential candidate for regulating IOP. Since ANGPTL7 is secreted and extracellular matrix (ECM) remodeling is critical for aqueous humor resistance, we investigated the effects of ANGPTL7 on relevant TM ECM genes and proteins.
Primary HTM cell lines were generated from nonglaucomatous human donors. A full coding ANGPTL7 cDNA was isolated from DEX-treated HTM cells. An ANGPTL7 expression vector was constructed using the pcDNA 3.1.V5-His-TOPO plasmid (pNC1). HTM primary cells were nucleofector-transfected with pNC1 or ANGPTL7.siRNA and harvested for RNA at 72h. For the DEX-induction interference study, cells were pre-silenced with ANGPTL7- or scrambled-siRNA and treated with DEX or vehicle at 24h. At 48h post-DEX, cells were harvested for expression analysis by TaqManPCR normalized to 18S. Immunocytochemistry of FN1 was performed on nonpermeabilized pNC1- or control-transfected HTM cells. Human anterior segments were perfused with 100nM of ANGPTL7- or scrambled-siRNA in the presence and absence of DEX for 72h. Secreted proteins were analyzed by WB and ELISA.
Overexpression of ANGPTL7 in HTM cells significantly decreased expression of FN1 (-3.2X), collagens type I, IV, and V (-4.8X, -1.2X -2.0X), myocilin (-1.5X) and versican (-2.1X) while it increased that of MMP1 (1.9X). Silencing ANGPTL7 reversed the effect. Overexpression also caused decrease of secreted FN1 and the disruption of FN1 fibrils. Further, in DEX-treated cells, reducing ANGPTL7 DEX upregulation (4.6X) by its own siRNA affected the expression of other steroid responding proteins. DEX-upregulation of FN1 and myocilin were significantly reduced (3.4X and 2.3X respectively). In DEX-treated organ cultures, ANGPTL7.siRNA perfused eye showed a 7.9X reduction compared to the scrambled.perfused contralateral eye. This reduction caused a significant decrease on the DEX induction of FN1 and myocilin (1.36X and 2.5X respectively).
ANGPTL7 influences the expression of TM genes involved in ECM organization and affects FN1 fibrils assembly, suggesting a role for this gene in maintaining ECM structure and flexibility. In addition, ANGPTL7 appears to acts as a mediator of glucocorticoid induction in the TM cells and tissue. Together with previous findings, these new results strengthen the ANGPTL7’s candidacy for regulating IOP and glaucoma.
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