P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low Density Lipoprotein

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background: </jats:bold>Inflammation and neovascularization are two vital pathological phases of AMD. Recent evidence indicates that blocking P2X7 receptor may relieve inflammation. Here, we investigated whether A740003, a P2X7 receptor antagonist, could prevent retinal inflammation and neovascularization induced by ox-LDL and explored the underlying mechanisms.<jats:bold>Methods: </jats:bold>ARPE-19 cells were pretreated with A740003 for 2 hours before ox-LDL exposure. Western Blot, ELISA and qPCR were used to detect the proteins and mRNA levels of NLRP3, Caspase-1, P2X7R, pIKBa, IKBa, angiogenic factors and inflammatory cytokines. ROS assay was performed to detect the ROS generation. C57BL/6 mice were subretinally injected with ox-LDL to induce retinal inflammation and neovascularization. A740003 was administrated intraperitoneally starting from day 3 before to day 14 after ox-LDL injection. Retinal function was assessed by dark- and light-adapted ERG. Retinal neovascularization was detected by retinal whole flat mount and immunofluorescence. Thickness of INL and ONL was measured to evaluate the retinal edema. <jats:bold>Results:</jats:bold> ox-LDL induced inflammatory responses and VEGF overproduction in human RPE cells and mouse retinas. However, A740003 decreased the expression of NLRP3, P2X7R, HIF-1a and VEGF in ARPE-19 cells. Inflammatory cytokines including IL-1b and IL-18 were down-regulated in A740003 treated cells. A740003 suppressed ROS generation and inhibited the phosphorylation of IKBa significantly. Whole flat mount of retinas and immunofluorescence showed that A740003 inhibited the angiogenesis induced by ox-LDL<jats:italic> in vivo</jats:italic>. The elevated mRNA and protein levels of NLRP3, Caspase-1, P2X7R, HIF-1a and VEGF decreased and IL-1b release was repressed in A740003 treated mice. Besides, the phosphorylation of IKBa was suppressed in the retinas of A740003 treated mice. The retinal edema was alleviated by A740003 treatment. The b- and a-wave amplitudes of ERG were remarkably preserved in A740003 treated mice. <jats:bold>Conclusions: </jats:bold>A740003 significantly reduced ocular inflammatory responses and VEGF production in ARPE-19 cells. In addition, the P2X7R antagonist reduced retinal inflammation, neovascularization, and protected retinal function in mice. The protective effects were associated with regulating of NLRP3 inflammasome and NF-kB pathway, suppression of ROS generation, as well as inhibition of angiogenic factors.</jats:p>

P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low‐Density Lipoprotein

“…In addition, the intraperitoneal injection of A740003 prevented retinal inflammation and neovascularization and preserved retinal function in C57BL/6 mice subretinally injected with ox-LDL. The P2X7R antagonist could reduce retinal inflammation and neovascularization and protect retinal function by regulation of the NLRP3 inflammasome and NF-κB pathway and suppression of ROS generation, as well as inhibition of HIF-1α and VEGF [38]. The results provide a new clue for therapeutic strategies to treat retinal inflammation and neovascularization.…”