Ranibizumab is a potential prophylaxis for proliferative vitreoretinopathy, a nonangiogenic blinding disease

Abstract

Proliferative vitreoretinopathy (PVR) exemplifies a disease that is difficult to predict, lacks effective treatment options, and substantially reduces the quality of life of an individual. Surgery to correct a rhegmatogenous retinal detachment fails primarily because of PVR. Likely mediators of PVR are growth factors in vitreous, which stimulate cells within and behind the retina as an inevitable consequence of a breached retina. Three classes of growth factors [vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family)] are relevant to PVR pathogenesis because they act on PDGF receptor α, which is required for experimental PVR and is associated with this disease in humans. We discovered that ranibizumab (a clinically approved agent that neutralizes VEGF-A) reduced the bioactivity of vitreous from patients and experimental animals with PVR, and protected rabbits from developing disease. The apparent mechanism of ranibizumab action involved derepressing PDGFs, which, at the concentrations present in PVR vitreous, inhibited non-PDGF-mediated activation of PDGF receptor α. These preclinical findings suggest that available approaches to neutralize VEGF-A are prophylactic for PVR, and that anti-VEGF-based therapies may be effective for managing more than angiogenesis- and edema-driven pathological conditions.

Figure 1

Vitreous-driven signaling events and cellular responses associated with PVR were potentiated by vitreal VEGF-A. A: The functional relationship between three classes of growth factors present in PVR vitreous. VEGF-A antagonizes the action of PDGFs, which block non-PDGFs (growth factors outside of the PDGF family) that activate PDGFRα indirectly and thereby drive experimental PVR.B: Neutralizing vitreal VEGF-A prevented PVR vitreous-driven signaling events. Primary RCFs were serum starved overnight and either lysed immediately without treatment (—) or continuously treated for 48 hours with 400 μL RV-PVR supplemented with 10 μg/mL nonimmune IgG, 25 μg/mL neutralizing anti-VEGF antibody, ranibizumab (α-VEGF), 20 ng/mL PDGF-A, or a combination of 10 μg/mL α-VEGF and 2 μmol/L PDGF TRAP. After treatment, cells were lysed and the resulting TCLs were subjected to Western blot analysis using the indicated antibodies and quantified (see Materials and Methods). Ratios representing normalized band intensities are shown under each immunoblot. Blots shown are representative of three independent experiments. C: Neutralizing vitreal VEGF-A suppressed PVR vitreous-driven cell contraction. RCFs were preconditioned for 48 hours with serum-free medium alone (—) or 400 μL RV-PVR supplemented with 10 μg/mL nonimmune IgG, 25 μg/mL α-VEGF, 2 μmol/L α-VEGF + PDGF TRAP, or 20 ng/mL PDGF-A; in addition, cells were preconditioned with α-VEGF, PDGF TRAP, or PDGF-A alone as controls. After preconditioning, cells were transferred to collagen gels containing the same treatment and subjected to the collagen gel contraction assay. Gel area was measured after 24 hours. Data are presented as percentage contraction of collagen gels measured after 24 hours, and are represented as mean percentage contraction ± SDs obtained for three independent experiments. D: Neutralizing vitreal VEGF-A prevented PVR vitreous-driven cell survival. Near-confluent RCFs were placed in starvation medium (DMEM without serum) for 72 hours as an inducement of apoptosis, during which time they were conditioned with the same treatments as described in B. At 72 hours, surviving cells were quantified as those cells whose nuclei failed to stain positive for apoptosis (by TUNEL assay, see Materials and Methods). The graph presents data from three independent experiments showing the mean percentages of cells (± SD) surviving starvation. ^∗P < 0.05 using a paired t-test. In each experiment, 12 randomly chosen fields were counted. Original magnification, ×100.

Figure 2

Neutralizing vitreal VEGF-A safely and effectively prevented experimental PVR (A). One week after an intravitreal gas injection, rabbits received three separate 0.1-mL injections of PVR-inducing RCFs, PRP, and either 0.05 mg of α-VEGF ranibizumab (RBZ) or an equimolar amount of isotype control IgG. The concentration (calculated based on vitreous volume) of RBZ injected was 10-fold less than the amount typically used in human eyes. For each rabbit, only one eye was injected. Rabbits were examined and scored for development of PVR over a 4-week period; the results from the last time point (day 28) are shown, and the results from all other time points scored are shown in Supplemental Figure S3.Horizontal bars represent the mean PVR stage of each group (n = 11 for each group). Statistically significant differences at each time point were determined by Mann-Whitney analysis (P < 0.001). B: Treatment of rabbits with α-VEGF did not interfere with retinal function. Single-flash ERGs were obtained from rabbits on day 26 after injection; readouts were obtained for both injected and noninjected eyes of the same rabbit after dark adaptation. The ERGs shown span 100 milliseconds and are representative of three individual rabbits per group. The amplitude from the baseline to the a-wave trough (a) reflects the general physiological health of the outer retina (particularly the photoreceptors), while the amplitude from the a-wave trough to the b-wave peak (b) reflects the health of the inner retinal layers. RBZ-treated rabbit eyes elicited the same electrophysiological response as their noninjected counterpart eyes. Light-adapted, single-flash and light-adapted flicker ERGs also showed no significant difference between RBZ-injected and noninjected eyes (data not shown). C: Treatment of rabbits with RBZ did not cause major morphological changes in the retina. Eyes enucleated from a representative RBZ-treated rabbit (PVR stage 0) and a noninjected eye (PVR stage 0) were fixed in 10% formalin, embedded in methacrylate, divided into sections, and the resulting sections were stained with H&E. Representative IgG-treated rabbits had retinal detachments; thus, their morphological characteristics were not included in this analysis. A representative region of the neural retina is shown in each panel. Retinal layers are indicated: GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; PRL, photoreceptor layer. These data indicate that α-VEGF/RBZ treatment did not adversely affect the retina.

Figure 3

PDGF-mediated dimerization attenuated indirect activation of PDGFRα. A: Reduction of PDGFRα levels by shRNA did not attenuate indirect PDGFRα signaling. Lentiviruses were used to stably express shRNAs directed against green fluorescent protein (GFP; shGFP) or PDGFRα (shPDGFRα) in MEFs; shPDGFRα MEFs expressed approximately 70% less receptor than control shGFP MEFs. Cells were then starved and treated with 400 μL RV-PVR and/or 20 ng/mL PDGF-A, as indicated for 120 minutes at 37°C, then lysed and subjected to Western blot analysis using the indicated antibodies. Both immature and mature PDGFRα band intensities were normalized to RasGAP levels. These findings indicate that no more than 30% of the total pool of PDGFRα was required to trigger signaling events in response to RV-PVR. B: Near-confluent MEFs were serum starved overnight and treated with 400 μL DMEM or RV supplemented with an increasing concentration of PDGF-A. Cells were treated in parallel for 60 and 120 minutes, after which time they were harvested and TCLs were subjected to Western blot analysis using the indicated antibodies. Prolonged Akt phosphorylation and reduction in p53 levels (indicators of indirectly activated PDGFRα) were attenuated at saturating doses of PDGF-A, suggesting that PDGF-induced dimerization is a key component of PDGF-mediated attenuation of vitreous-driven indirect signaling of PDGFRα. C: R627 cells were serum starved overnight and either left alone or pre-incubated with 10 ng/mL PDGF-A for 30 minutes at 4°C (to ensure complete receptor dimerization on the cell surface), followed by treatment with or without 400 μL RV for 10 minutes at 4°C. After treatment, cells were lysed and subjected to Western blot analysis using anti–phospho-PDGFRα, followed by anti-pan PDGFRα, and normalized relative to the untreated control. Although indirect activation of PDGFRα still occurred at 4°C, receptor activation was approximately 2.5-fold lower compared with indirect receptor activation at 37°C (Supplemental Figure S4), demonstrating a correlation between dimerization and reduced capacity to undergo indirect activation, further suggesting that PDGF diminishes non–PDGF-mediated activation of PDGFRα by dimerizing PDGFRαs. D: Schematic showing the details and consequences of direct and indirect activation of PDGFRα. ROS, reactive oxygen species; SFK, Src-family kinases.

Figure 4

PDGF-dependent activation of PDGFRα was inhibited by VEGF-A, a heat-labile, PDGFRα-associated agent in human PVR vitreous. A: Patient PVR vitreous contains inhibitory activity against PDGF-mediated PDGFRα activation. ARPE-19α cells were grown to near confluence, serum starved overnight, and then treated with serum-free medium alone (—), 200 μL human patient PVR vitreous (HV-PVR), 10 ng/mL PDGF-A, or both HV-PVR and PDGF-A for 5 minutes at 37°C. Cells were lysed and subjected to anti–phospho-PDGFRα (p-PDGFRα) and then anti-PDGFRα Western blot analysis. The p-PDGFRα immunoblot signal was normalized to total PDGFRα (PAN) and is presented as fold induction over the non-stimulated control. Blots shown are representative of three independent experiments. HV-PVR reduced PDGF-mediated activation of PDGFRα by approximately 50%, suggesting that patient PVR vitreous contains an inhibitor of PDGF-mediated PDGFRα activation. B: Inhibitory activity in human patient PVR vitreous (HV-PVR) is labile to heat. In a manner similar to A, cells were starved and treated with serum-free medium without treatment (—), 200 μL HV-PVR, 10 ng/mL PDGF-A, or both HV-PVR and PDGF-A; some HV-PVR treatments were first heat treated to 90°C for 5 minutes and then rapidly cooled on ice. Cells were treated for 5 minutes at 37°C and lysed, and the resulting TCLs were subjected to the same Western blot analysis as in A. Although PDGF-A largely survived the heat treatment, nearly all PDGF-inhibitory activity was eliminated from HV-PVR. Moreover, there were enough endogenous PDGFs in heat-treated HV-PVR to elicit a 3.5-fold activation of PDGFRα (over the non-stimulated control). Thus, heat treatment unmasked the ability of HV-PVR to activate PDGFRα, suggesting the presence of a heat-labile inhibitor that blocks vitreal PDGFs from functioning. C: HV-PVR–mediated inhibition of PDGF-dependent PDGFRα could be overcome by increasing the concentration of PDGF. Cells were cultured and starved as described in A. The indicated amount of PDGF-A was added to either 200 μL DMEM or HV-PVR and then used to treat cells for 5 minutes at 37°C. Cells were lysed and subjected to Western blot analysis, and the results were quantified as in Figure 1. Results from three independent experiments revealed that HV-PVR significantly inhibited PDGFRα phosphorylation at low doses of PDGF-A: 1, 2.5, 5, and 10 ng/mL. ^∗P < 0.05 using a paired t-test. D: Cells preconditioned with patient PVR vitreous became resistant to subsequent treatment with PDGF-A. Cells were cultured and starved, as described in A, then pre-incubated for 15 minutes at 37°C with either DMEM or 200 μL HV-PVR. After incubation, the media/vitreous was removed and cells were extensively washed with PBS, after which they were treated with serum-free medium alone (—) or 10 ng/mL PDGF-A for 10 minutes at 37°C. Cells were subsequently lysed, and the resulting TCLs were subjected to Western blot analysis with the indicated antibodies. These data suggest that the inhibitor(s) present in HV-PVR acted at the level of cells. E: Preclearing HV-PVR vitreous with PDGF TRAP significantly reduced its ability to inhibit PDGFRα activation by exogenously added PDGF. HV-PVR (200 μL) was not manipulated or precleared with 2 μmol/L PDGF TRAP or an equimolar amount of a control IgG-Fc fragment (IgG-Fc). These clarified samples were then tested for their ability to block PDGFRα activation by exogenously added PDGF-A. To this end, cells were treated with clarified vitreous and 10 ng/mL PDGF-A for 10 minutes at 37°C. Serum-free media without treatment (—) and 10 ng/mL PDGF-A alone were used as negative and positive controls, respectively. Cells were lysed, and the resulting TCLs were subjected to the same Western blot analysis as used in A. The ability of PDGF TRAP to reduce PDGF-inhibitory activity from HV-PVR suggests that this inhibitor can associate with the extracellular domain of PDGFRα. F: Neutralizing VEGF-A in human PVR vitreous with ranibizumab enabled vitreal PDGFs to activate PDGFRα. Cells were serum starved overnight and either lysed immediately (—) or treated for 10 minutes at 37°C with 10 ng/mL PDGF-A, 10 μg/mL α-VEGF, or 200 μL HV-PVR supplemented with 10 μg/mL nonimmune IgG, 10 μg/mL α-VEGF, or a combination of 10 μg/mL α-VEGF and 2 μmol/L PDGF TRAP. After treatment, cells were lysed and the resulting TCLs were subjected to Western blot analysis using the indicated antibodies and quantified. Ratios representing normalized band intensities are shown under each immunoblot. Blots shown are representative of three independent experiments. These results show that neutralizing VEGF-A significantly enhanced the ability of vitreal PDGFs to activate PDGFRα.

Figure 5

Neutralizing VEGF-A in human PVR vitreous prevented PVR-associated signaling events and cellular outcomes in RPE cells isolated from a human PVR membrane. Experimental data shown in A–C were performed similarly to those of Figure 1, B–D, with the exception that, in these experiments, HV-PVR was used (instead of RV-PVR) to stimulate PVR membrane-derived RPE cells (instead of RCFs). D: Comparison of VEGF-A and PDGF levels in the vitreous of patients with or without PVR. Vitreous from patients with PVR or non-PVR retinal diseases (macular holes or macular puckers) was subjected to multiplex analysis to determine the concentration of VEGF-A and PDGFs (total of A, AB, and B isoforms). Although 94% of PVR samples had a detectable level of VEGF-A, the same was true for only 34% of non-PVR samples. Molar amounts of VEGF-A and PDGFs in each sample were frequently detected at similar levels (symbols labeled with the same letter are the same sample). Most had low levels of both, whereas when PDGFs were present, there was also a matched (samples A to C) or slightly elevated (samples D to H) amount of VEGF-A. These observations indicate that the ratio of vitreal VEGF-A/PDGF correlates with clinical PVR and is a potential biomarker for PVR susceptibility. *P < 0.05 using a paired t-test.