Fig 2.

PVR vitreous inhibited PDGF-dependent activation of PDGFRα. (a and b) PDGFRα activation mediated by exogenous PDGF was inhibited by PVR vitreous. ARPE-19α cells were cultured and starved as described for Fig. 1. (a) The indicated amount of PDGF-A was added to either DMEM or RV-PVR, and the resulting solutions were added to cells for 5 min at 37°C. A concentration of 10 ng of VEGF-A/ml corresponds to 0.26 nM. (b) Alternatively, cells were treated with either DMEM or 2.5 ng of PDGF-A/ml in the presence of increasing doses of RV-PVR (DMEM was used as a diluent) for 5 min at 37°C. After treatment, the cells were lysed, and the resulting TCLs were subjected to Western analysis and quantification as described for Fig. 1. The results from three independent experiments for each analysis (a and b) revealed that RV-PVR caused a statistically significant (P < 0.05 using a paired t test) decline in PDGFRα phosphorylation at 1, 2.5, 5, and 10 ng of PDGF-A/ml; likewise, RV-PVR titrations greater than 75% of the total treatment volume inhibited PDGF-A-mediated PDGFRα phosphorylation. (c) Cells preconditioned with PVR vitreous were desensitized to subsequent PDGF treatment. ARPE-19α cells, cultured and starved as described in Fig. 1, were either preincubated for 15 min at 37°C with DMEM or RV-PVR (0.2 ml). After incubation, medium or vitreous was removed, and the cells were washed extensively and then treated with serum-free medium alone (—) or supplemented with 10 ng of PDGF-A/ml for 5 min at 37°C. After treatment, the cells were lysed, and the resulting TCLs were subjected to the same Western analysis and quantification as described for Fig. 1. The bar graph shows the mean fold induction of PDGFRα phosphorylation ± the SD obtained for three independent experiments (*, P < 0.05 using a paired t test). These results suggest that PVR vitreous contains PDGFRα-associated agents that prevent PDGF-dependent activation of PDGFRα. (d) Preclearing PVR vitreous with the Fc-extracellular domain PDGFRα fusion protein (TRAP) significantly reduced its ability to inhibit PDGF-dependent PDGFRα activation. RV-PVR (0.2 ml) was either left untouched or precleared with 2 μM TRAP or a 2 μM concentration of control IgG-Fc fragment [Ig(Fc)]. The resulting samples were used to stimulate cells; serum-free media (DMEM) and 10 ng/ml of PDGF-A (PDGF) were the negative and positive controls, respectively. After 5 min treatment at 37°C, cells were lysed and the resulting TCLs subjected to the same Western analysis and quantification as in (a). The bar graph shows the mean fold induction values ± the SD obtained for three independent experiments (*, P < 0.05 using a paired t test). The ability of TRAP to reduce the inhibitory activity of the vitreous suggests the vitreal inhibitors can associate with the extracellular domain of PDGFRα.