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.
Highlights
Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly, affecting approximately 1 in 3 people over the age of 65 [1]
In order to determine whether PPADS can inhibit endothelial cell tube formation, we performed human umbilical vein endothelial cell (HUVEC) tube formation assays in the presence and absence of PPADS
We found that the ability of HUVECs to form master junctions, master segments and meshes was significantly reduced with increasing levels of PPADS in a concentration dependent manner (Figure 1A)
Summary
Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly, affecting approximately 1 in 3 people over the age of 65 [1]. AMD begins with the appearance of lipoproteinaceous deposits known as drusen between the retinal pigment epithelium (RPE) and Bruch’s membrane [2,3,4]. In approximately 10% of patients, dry AMD progresses to the ‘wet’ form of the disease, involving the growth of new blood vessels from the choroidal vasculature into the subretinal space (choroidal neovascularization, CNV). These immature new blood vessels leak fluid, leading to formation of a macular edema (reviewed in [2]). Wet AMD leads to degeneration of retinal tissues and eventually blindness
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