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Jose M. Gonzalez, Yujie Hu, B'Ann T. Gabelt, Paul L. Kaufman, Donna M. Peters; Identification of the Active Site in the Heparin II Domain of Fibronectin that Increases Outflow Facility in Cultured Monkey Anterior Segments. Invest. Ophthalmol. Vis. Sci. 2009;50(1):235-241. doi: 10.1167/iovs.08-2143.
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purpose. To determine the active site in the Heparin II (HepII) domain of fibronectin that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in transformed human trabecular meshwork (TM-1) cells.
methods. Outflow facility was determined by two-level, constant-pressure perfusion in cultured anterior segments of rhesus and cynomolgus monkey eyes. One segment from each pair was exchanged with either the HepII domain or an integrin/syndecan binding peptide (IDAPS or PPRARI) from the HepII domain. To assay changes in the actin cytoskeleton, TM-1 cells were incubated for 24 hours with or without the HepII domain, PPRARI, or IDAPS. Changes were monitored with phase and immunofluorescence microscopy.
results. HepII domain (100 μg/mL) and PPRARI (500 μg/mL) increased outflow facility by 31% ± 13% (n = 9, P < 0.05) and 24% ± 9% (n = 8, P < 0.05), respectively in cultured anterior segments after an overnight infusion. Perfusion with IDAPS (500 μg/mL) had no effect on outflow facility. In TM-1 cultures, 250 μg/mL of the HepII domain or 4 mg/mL of PPRARI disrupted the assembly of actin filaments. A lower concentration of PPRARI (2 mg/mL) disrupted the actin cytoskeleton when used in combination with a nondisrupting concentration of the HepII domain (30–60 μg/mL). In contrast, IDAPS did not disrupt the actin cytoskeleton under any condition tested.
conclusions. The active site in the HepII domain that regulates outflow facility in cultured anterior segments and disrupts the actin cytoskeleton in TM-1 cells is the syndecan/integrin binding sequence, PPRARI.
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