Topical application of PPADS inhibits complement activation and choroidal neovascularization in a model of age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia.

Figure 1. PPADS inhibits formation of tubes by HUVECs.

A) Representative phase contrast images of tubes formed in the absence of supplements (negative control), in the presence of vehicle (0.9% NaCl) or vehicle plus 1.04 µM PPADS or 4.17 µM PPADS. B) Tabular summary of mean values ± SEM of master junctions, master segments and meshes in cells incubated with the indicated concentration of PPADS and calculated IC₅₀ for each marker. C) Dose dependent PPADS mediated reduction of master junctions, master segments and meshes. The punctuated line indicates the extrapolated inhibition trend. IC₅₀ values are indicated as intercept points in the trend line. Asterisks in each inset mark an example of a measured junction, segment or mesh, respectively. Studies were performed a total of 4 times in triplicate.

Figure 2. PPADS Inhibits Complement Mediated Cell Lysis.

A) Representative flow cytometry plots indicating number of Hepa1c1c7 cells taking up propidium iodide (PI) when incubated in 1% normal human serum (NHS) and various concentrations of PPADS (blue lines) or 0.9% NaCl (red lines). B) Mean values ± SEM of PPADS mediated reduction of NHS mediated cell lysis from 5 independent studies. Significant differences are shown as capped lines between vehicle control (0.9% NaCl, 0 µM PPADS) and the various concentrations of PPADS (*** p<0.0005, ** p<0.005, * p<0.05).

Figure 3. PPADS Inhibits Formation of MAC in vitro.

A) Representative images of C5b9 labeling (red) of cells incubated with increasing concentrations of PPADS. Nuclei are counterstained with DAPI (blue). B) Mean values ± SEM of C5b9 staining intensity per cell at various concentrations of PPADS. Values represent cumulative data from 4 independent studies. Significant differences are indicated as capped lines between vehicle control (0.9% NaCl, 0 µM PPADS) and the various concentrations of PPADS (*** p<0.0005, ** p<0.005, * p<0.05).

Figure 4. PPADS Inhibits Formation of laser induced CNV.

A) Representative images of GSL-1 stained CNV at day 7 post laser in a mouse eye treated with 0.9% NaCl or 4.17 mM PPADS topically for 3 days post laser. B) Mean values ± SEM of CNV area of 0.9% NaCl treated eyes or 4.17 mM PPADS treated eyes at 7 days post laser. Studies were performed 3 times with 5 mice in each group, n = 15, NaCl = 91 laser spots, PPADS = 71 laser spots (*** p<0.0005).

Figure 5. PPADS Inhibits Deposition of MAC at the site of laser induced CNV.

A) Representative images of C5b9 labeling at 7 days post laser in eyes treated with 0.9% NaCl or 4.17 mM PPADS topically for 3 days. B) Mean values ± SEM of C5b9 intensity/area of 0.9% NaCl treated eyes and 4.17 mM PPADS treated eyes 7 days post lasering. Studies were performed 3 times with 5 mice in each group, n = 15, NaCl = 91 laser spots, PPADS = 71 laser spots (** p<0.005).