Abstract
Age-related macular degeneration (AMD) is a common, blinding disease associated with increased complement system activity. Eyes with AMD show elevated accumulation of the membrane attack complex (MAC) in the choriocapillaris and degeneration of macular choriocapillaris endothelial cells (ECs). Thus, one could reasonably conclude that the endothelial cell death that occurs in AMD is due to injury by the MAC. We therefore sought to identify strategies for protecting ECs against MAC lysis. RF/6A endothelial cells were pre-incubated with a library of FDA-approved small molecules, followed by incubation with complement intact human serum quantification of cell death. Two closely related molecules identified in the screen, econazole nitrate and miconazole nitrate, were followed in validation and mechanistic studies. Both compounds reduced lysis of choroidal ECs treated with complement-intact serum, across a range of doses from 1 to 100 µM. Cell rescue was confirmed in mouse primary choroidal ECs. Both exosome release and cell surface roughness (assessed using a Holomonitor system) were reduced by drug pretreatment in RF/6A cells, whereas endosome formation increased with both drugs, consistent with imidazole-mediated alterations of cell surface dynamics. The results in the current study provide further proof of principle that small molecules can protect choroidal ECs from MAC-induced cell death and suggest that FDA approved compounds may be beneficial in reducing vascular loss and progression of AMD.
Highlights
Current therapies for Age-related macular degeneration (AMD) include over the counter vitamin supplements (Age Related Eye Disease Study, or “AREDS2”, vitamins) which have shown a 25% risk reduction in progression to the neovascular form of AMD patients over 5 years[7,8]
The finding that membrane attack complex (MAC) accumulates in aging choriocapillaris, and especially in AMD, in addition to morphometric studies showing loss of choriocapillaris endothelial cells in AMD prior to the loss of RPE or photoreceptor cells[14,15,16] led us to develop a model of EC injury in vitro
Econazole nitrate (Ecz) and miconazole nitrate (Mcz), belong to the same family of compounds with very similar structure, differing by the substitution of one chlorine atom (Fig. 1), and these were selected for further study
Summary
Current therapies for AMD include over the counter vitamin supplements (Age Related Eye Disease Study, or “AREDS2”, vitamins) which have shown a 25% risk reduction in progression to the neovascular form of AMD patients over 5 years[7,8]. The finding that MAC accumulates in aging choriocapillaris, and especially in AMD, in addition to morphometric studies showing loss of choriocapillaris endothelial cells in AMD prior to the loss of RPE or photoreceptor cells[14,15,16] led us to develop a model of EC injury in vitro. In this model, the nonhuman primate endothelial cell line RF/6A, incubated with human complement-deficient (i.e., heat inactivated, C5-depleted or cobra venom factor-depleted) or complement-intact serum, was evaluated in terms of global gene expression, MMP9 synthesis, monolayer permeability, and cell death. In the current study we describe a drug screen in which a library of FDA approved compounds was tested for its ability to protect RF/6A cells from complement-mediated lysis, and we describe two closely related compounds that show efficacy in rescuing ECs from cell death
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