P2X7 Receptor Antagonist Attenuates Retinal In�ammation and Neovascularization Induced by Oxidized Low Density Lipoprotein

Background: In�ammation and neovascularization are two vital pathological phases of AMD. Recent evidence indicates that blocking P2X7 receptor may relieve in�ammation. Here, we investigated whether A740003, a P2X7 receptor antagonist, could prevent retinal in�ammation and neovascularization induced by ox-LDL and explored the underlying mechanisms. Methods: 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 in�ammatory cytokines. ROS assay was performed to detect the ROS generation. C57BL/6 mice were subretinally injected with ox-LDL to induce retinal in�ammation 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 �at mount and immuno�uorescence. Thickness of INL and ONL was measured to evaluate the retinal edema. Results: ox-LDL induced in�ammatory 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. In�ammatory cytokines including IL-1b and IL-18 were down-regulated in A740003 treated cells. A740003 suppressed ROS generation and inhibited the phosphorylation of IKBa signi�cantly. Whole �at mount of retinas and immuno�uorescence showed that A740003 inhibited the angiogenesis induced by ox-LDL in vivo. 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 band a-wave amplitudes of ERG were remarkably preserved in A740003 treated mice. Conclusions: A740003 signi�cantly reduced ocular in�ammatory responses and VEGF production in ARPE-19 cells. In addition, the P2X7R antagonist reduced retinal in�ammation, neovascularization, and protected retinal function in mice. The protective effects were associated with regulating of NLRP3 in�ammasome and NF-kB pathway, suppression of ROS generation, as well as inhibition of angiogenic factors.


Introduction
Age-related macular degeneration (AMD) is a blinding disease among people over age of 50 worldwide.
The prevalence of AMD is expected to increase dramatically as the global population ages [1].AMD is closely related with the chronic in ammation in retinal pigmented epithelial (RPE) cells, Bruch membrane and choroid membrane [2,3].There are two types of AMD according to the clinical characteristics, dry AMD and wet AMD.Dry AMD, also called non-exudative AMD, is characterized by retinal in ammation, which mainly happens in the early stage of AMD.Wet AMD, also called exudative AMD, is characterized by choroidal neovascularization and retinal angiogenesis, which usually happens in the late stage of AMD [4].Better understanding of the pathogenesis of AMD is crucial for seeking better and earlier treatment modalities.
The total cholesterol, triacylglycerol and low density lipoprotein (LDL) are obviously higher in AMD patients compared to normal people [5].Moreover, LDL is susceptible to oxidation, resulting in the formation of ox-LDL [6].ox-LDL can induce a large amount of reactive oxygen species (ROS) and oxidative stress injury.Chronic oxidative stress contributes to the pathogenesis of AMD.ox-LDL leads to lipid deposition, in ammation and pyroptosis when it is accumulated in cells.It is reported that high level of ox-LDL may lead oxidative stress and in ammation in glaucoma patients, which impedes wound healing after surgery [7].The RPE becomes vulnerable to apoptosis through a number of mechanisms including the high oxidative stress environment.Oxidized low density lipoproteins (ox-LDL) are also internalized by the RPE, and alter photoreceptor turnover and lysosomal function [8].The oxysterols in ox-LDL are cytotoxic to RPE cells [8,9].It is found that ox-LDL induces a pathologic response in RPE, which suggests that ox-LDL is one trigger for initiating early events in the pathogenesis of AMD [10].
Retinal angiogenesis may also relate with the overproduction of ox-LDL and ROS generation.Recently, it is reported that ox-LDL is immunohistochemically detected in surgically excised choroidal neovascular membranes from eyes with AMD [11].ox-LDL levels were higher in peripheral blood of AMD patients with choroidal neovascularization compared to normal individuals [12], which suggested a close relationship between ox-LDL and AMD.ox-LDL may also induce retinopathy through damaging the blood capillary [13].These researches suggest that ox-LDL may induce retinal in ammation and neovascularization.
Pyroptosis related with NLRP3 in ammasome activation is recently identi ed to be a novel type of programmed cell death.The canonical in ammasome complexes are assembled around protein members of the nod-like receptor (NLRs) and composed of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), converting the pro-Caspase-1 zymogen into a catalytically active enzyme [14].NLRP3 in ammasome senses and responds to a diversity of pathogens or danger-associated molecular patterns.Overproduction of ROS activates NLRP3, recruiting ASC and regulating auto-activation of pro-Caspase-1.After activation of pro-Caspase-1, pro-IL-1β and pro-IL-18 are cleaved into mature peptides and secreted outside to mediate the following in ammation [15].Activation of NF-κB pathway upregulates the transcription of NLRP3 and pro-IL-1β.Ox-LDL induces a large amount of ROS in macrophage cells and activate NLRP3 in ammasome, and then promote the secretion of in ammatory cytokines such as IL-1b and IL-18 [16].Beside, the ROS mediated by ox-LDL also leads the activation of NF-κB signaling pathway, which may strengthen the in ammation response [14].
The P2 × 7 receptor (P2 × 7R) is an ATP-gated ion channel which is a key player in oxidative stress under pathological conditions.P2 × 7R is expressed in the RPE and neural retina.Activation of P2 × 7R induces calcium ion in ux and leads the interaction of P2 × 7R and NLRP3 in ammasome and thus activates the NLRP3 signaling pathway.It was reported that P2 × 7R played a critical role in regulating the normal physiological functions of macrophages and monocytes and it may induce the synthesis and secretion of in ammatory cytokines [17].
P2 × 7R activation is always followed by the activation of NLRP3 in ammasome.A740003, an effective P2 × 7R antagonist, is commonly used in previous studies.Recently, it is reported that P2 × 7R antagonist protects retinal ganglion cells by inhibiting microglial activation [18].Besides, blocking of the P2 × 7R inhibits the activation of NF-κB pathways in the cartilage tissue [19].Therefore, we speculated that P2 × 7R inhibitor attenuated retinal in ammation induced by ox-LDL through regulating the activation of NF-κB pathway and NLRP3 in ammasome, and reduced retinal angiogenesis by downregulating the expression of VEGF and HIF-1α.

Preparation of ox-LDL and A740003
Human ox-LDL was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China).ARPE-19 cells were exposed to 100 µg/ml ox-LDL diluted with DMEM/F12 medium.Concentration of 3.0 mg/ml ox-LDL was used for subretinal injection of C57BL/6 mice.A740003 (ApexBio, Huston, TX, USA) was dissolved in 100% dimethyl sulfoxide (DMSO) and diluted with DMEM/F12 medium or PBS to a nal DMSO concentration no more than 1%.DMEM/F12 dilution was used for ARPE-19 treatment and PBS dilution was used for intraperitoneal injection of C57BL/6 mice.
Cell Culture And Treatment ARPE-19 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).ARPE-19 cells were grown in DMEM/F12 medium (Gibco, New York, NY, USA) supplemented with 10% FBS (TBD science, Tianjin, China), 1% penicillin and streptomycin (Solarbio) in 37 °C with 5% CO2 incubator.The optimal ox-LDL concentration for following studies was 100 µg/ml, which was chosen based on the mRNA expression levels of NLRP3 and VEGF.The optimal concentration is corresponding to the previous report [20].After 24 hours starvation, ARPE-19 cells were exposed to 100 µg/ml of ox-LDL for 24 hours with 2 hours pretreatment of 1 µM A740003.Besides, ARPE-19 cells were pretreated with 200 µM of A740003 for 2 hours, followed by stimulation of 100 µg/ml ox-LDL for 48 hours.The optimal concentrations of A740003 for 24/48 hours exposure to ox-LDL were screened by quantitative real-time PCR results.

Animal Care And Use
Male C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA).The animals were housed under 12-h light-dark cycle and given a standard chow diet.Animal care followed the guidelines formulated by the Association for Research in Vision and Ophthalmology (ARVO).Experiments and procedures involving animals were permitted by the Ethics Committee of Henan Eye Institute.Every effort was made to minimize animal discomfort and stress.

Subretinal Injection Of Ox-ldl
One µL ox-LDL (3.0 mg/ml) was injected subretinally into the right eyes of the mice, and the lateral eyes were not injected.Injection was performed according to a protocol described previously [21].Brie y, mice were anesthetized with intraperitoneal injection of 4% chloral hydrate (10 ml/kg).Pupils were dilated with tropicamide phenylephrine eye drops (Santen Pharmaceutical Co., Ltd, Osaka, Japan) 10 minutes prior to injection.An aperture within the dilated pupil area was made through the sclera, below the ora serrata with a 30-gauge needle.Then a blunt 32-gauge Hamilton syringe was inserted through the aperture, avoiding damage of the lens and penetrating the neuroretina.One µL ox-LDL was injected into subretinal space under the dissecting microscope.Successful delivery of ox-LDL was con rmed by viewing subretinal blebs demarcating the retinal detachment in the injected retinal area.Such detachments usually resolved within 1 to 2 days.All animals received antibiotic eyedrops to the cornea and were observed daily after operation.Only animals with minimal surgical complications and initial retinal blebs occupying more than 60% the retina were retained for further study.
Real-time Quantitative Pcr Analysis ARPE-19 cells were collected by digestion of 0.25% trypsin and centrifuging.Eyeballs of mice were enucleated at 2 weeks after ox-LDL subretinal injection.Retinas were dissected and homogenized for total RNA extraction.The mRNA levels of NLRP3, Caspase-1, P2 × 7R, VEGF, HIF-1α in ARPE-19 cells and retinas were detected by qPCR assay.Total RNA was extracted with Trizol reagent (ThermoFisher Scienti c, Waltham, MA, USA) from ARPE-19 cells and retinas according to the manufacturer's instructions.Complementary DNA (cDNA) was generated by using the PrimeScript® RT reagent kit (Takara Biotechnology, Dalian, China).qPCR was performed according to the manufacturer's instructions with the ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA).The ampli cation system used for qPCR was a volume of 20 µL PowerUp™ SYBR® Green Master Mix (ThermoFisher Scienti c).The cycling protocol comprised of 50 °C for 2 minutes, and then 95 °C for 2 minutes, followed by 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute.To determine the mRNA expression, which was normalized to the endogenous reference gene β-actin, all samples were detected in triple.Relative quanti cation was achieved by the comparative 2 −ΔΔCt method.The sequences of primers used for qPCR assay are shown in Table 1 and Table 2

Enzyme-linked Immunosorbent Assay
After pretreatment with 100 µg/ml A740003 for 2 hours, ARPE-19 cells were induced with ox-LDL for 24 hours.Then supernatants of all groups were collected and centrifuged to detect the concentrations of IL-1β (Raybiotech, Norcross, GA, USA) and IL-18 (R&D Systems, Minneapolis, CA, USA) by using human ELISA kits according to the manufacturers' protocols.The concentrations of IL-1β and IL-18 were calculated according to optical density measured at 450 nm by subtracting the optical density measured at 540 or 570 nm using a multifunction microplate reader (PerkinElmer, Waltham, MA, USA).

Electroretinogram (erg)
Retinal function was assessed with ERG following a previously described procedure [22].After overnight dark adaptation, mice were anesthetized with intraperitoneal injection of 4% chloral hydrate (10 ml/kg).The pupils were dilated with tropicamide eye drops 30 minutes prior to recording.Needle electrodes were subcutaneously inserted into the back and the tail as reference and ground leads respectively.Active electrodes were gently positioned on the center of cornea.All procedures were performed under dim red light.Full-eld ERGs were recorded with RetiMINER-C, a visual electrophysiology system (AiErXi Medical Equipment Co., Ltd., Chongqing, China).A series of stimulus intensities ranged from − 3 to 1 log cd-s/m 2 was applied for dark-adapted ERGs.After light adaptation of 5 minutes, light-adapted ERGs were recorded to strobe-ash stimuli (0 and 1 log cd-s/m 2 ) superimposed on the background light.Responses to brief ashes were analyzed by measuring the amplitudes of the a-and b-waves.

Whole Flat Mount Of Mouse Retinas
Retinal whole at mounts were prepared as previously described [23].Brie y, the eyeballs from C57BL/6 mice were removed and xed in 4% paraformaldehyde at room temperature for 1 hour.The cornea was cut an incision under dissecting microscope.At incision, the sclera was peeled towards optic nerve and then the lens and iris were removed.The retinas were extracted and permeabalized in 0.5%Tritonx-100 for 2 hours.The retinas were stained by isolectin-B4 for 30 minutes by gently shaking.After staining, the retinas were cut at 3, 6, 9, 12 o'clock for 4 incisions.Anti-uorescence mounting media was used for resistance to uorescence quenching before covering the retinas by coverslip.Flat-mounts were examined by uorescence microscopy (Olympus, Tokyo, Japan).

Immuno uorescent Staining
Immunohistochemistry was performed using methodology as previously described [22].Eyes were enucleated at 2 weeks after suretinal injection of ox-LDL and xed with 4% paraformaldehyde overnight.
After dehydration, the eyes were embedded in melting para n.Serial 4 µm para n sections were cut through cornea-optic nerve axis.Tissue sections were subsequently treated for antigen retrieval and blocked with 5% BSA for 30 minutes.Then sections were incubated with anti-VEGF antibody (1:200; Servicebio, Wuhan, China) overnight at 4℃.After washing, the slides were incubated with anti-rabbit secondary antibody for 50 minutes and DAPI for 10 minutes in dark.Anti-uorescence mounting media was used for resistance to uorescence quenching.Para n sections were examined under uorescence microscope.The average optical density of each section was measured using Image-Pro Plus 6.0 software.The thicknesses of inner nuclear layer (INL) and outer nuclear layer (ONL) in each retina were measured and summarized.

Statistical analysis
Results from experiment were expressed as mean ± standard error of mean (SEM).Statistical analysis was analyzed by the GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA).Experimental data were analyzed by one-way ANOVA or two-way ANOVA followed by Bonferroni correction.P value less than 0.05 was considered as statistically signi cant.

Results
A740003 inhibited the activation of NLRP3 in ammasome, phosphorylation of IKBα and decreased the expression of P2 × 7R in ARPE-19 cells ARPE-19 cells were cultured with 100 µg/ml of ox-LDL for 24 hours.Secretion of in ammatory cytokines was determined by ELISA.ELISA results showed that ox-LDL promoted the secretion of IL-1β (Fig. 1a) and IL-18 (Fig. 1b) signi cantly compared to control group.The up-regulated secretion of IL-1β and IL-18 indicated that more pro-IL-1β and pro-IL-18 were cleaved to mature in ammatory cytokines under the effects of mature Caspase-1.Pro-Caspase-1 turned into mature Caspase-1 when NLRP3 in ammasome was activated.Therefore, the ELISA results con rmed the activation of NLRP3 in ammasome in ARPE-19 cells exposed to ox-LDL.It indicated that ox-LDL induced the activation of NLRP3 in ammasome.Moreover, the qPCR assay was performed to determine the mRNA levels of NLRP3 and P2 × 7R.ox-LDL up-regulated the mRNA levels of NLRP3, P2 × 7R signi cantly (Fig. 1c, 1d).Western Blot was performed to evaluate the levels of proteins related with NLRP3 in ammasome.The results showed that the protein levels of NLRP3 (Fig. 1e, 1f), Caspase-1 (Fig. 1e, 1 g), P2 × 7R (Fig. 1e, 1h) and the phosphorylation of IKBα (Fig. 1e, 1i) in ARPE-19 cells increased obviously after exposure to ox-LDL.
In conclusion, the results suggested that the ox-LDL stimulation could up-regulate the secretion of in ammatory cytokines, which indicated the activation of NLRP3 in ammasome and pro-Caspase-1.However, P2 × 7R antagonist could inhibit the expression and secretion of in ammatory cytokines induced by ox-LDL.Moreover, the activation of of NLRP3 in ammasome and NF-κB signaling pathway induced by ox-LDL could be suppressed by A740003 treatment.
A740003 suppressed the up-regulation of angiogenic growth factors in ARPE-19 cells exposed to ox-LDL ARPE-19 cells were treated with 100 µg/ml of ox-LDL for 48 hours.The qPCR assay was performed to determine the mRNA levels of VEGF and HIF-1α.The resutls were shown in Fig. 2, mRNA levels of VEGF and HIF-1α increased signi cantly in ARPE-19 cells exposed to ox-LDL (Fig. 2a, 2b).VEGF and HIF-1α are primary angiogenic proteins contributing to neovascularization.Thus the protein levels of VEGF and HIF-1α are vital for evaluating the possibility of retinal neovascularization.Western Blot was performed to detect the levels of proteins related with angiogenesis.The results demonstrated that the protein levels of VEGF and HIF-1α up-regulated obviously in ARPE-19 cells incubated with ox-LDL (Fig. 2c, 2d, 2e).
In addition, another group of ARPE-19 cells was pretreated with 200 µM A740003 for 2 hours before ox-LDL incubation.The qPCR and Western Blot assay were performed to determine the mRNA and protein levels of HIF-1α and VEGF in ARPE-19 cells pretreated with A740003.The results manifested that mRNA levels of VEGF and HIF-1α decreased evidently in ARPE-19 cells pretreated with A740003 (Fig. 2a, 2b).
Furthermore, the protein levels of VEGF and HIF-1α were suppressed effectively by A740003 pretreatment in ARPE-19 cells (Fig. 2c, 2d, 2e).The results indicated that ox-LDL increased the mRNA and protein levles of angiogenic growth factors including VEGF and HIF-1α in ARPE-19 cells.Whereas, A740003 pretreatment could inhibited the HIF1α/VEGF axis effectively in ARPE-19 cells exposed to ox-LDL.A740003 might be effective to suppress the retinal neovascularization through inhibiting the HIF1α/VEGF axis.
A740003 suppressed the ROS generation induced by ox-LDL in ARPE-19 cells ROS generation in ARPE-19 cells was detected by DCFH-DA uorescent probe and microscope after 24 hours exposure to ox-LDL.The results showed that DCFH-DA uorescent intensity was signi cantly increased after ox-LDL incubation compared to control group (Fig. 3a).Image-Pro Plus 6.0 software was used to calculate the average optical density (AOD) of each group.The results showed that average optical density in ox-LDL incubated group was obviously higher than control group (Fig. 3b).It indicated that ROS generation was successfully induced by ox-LDL incubation in ARPE-19 cells.Over production of ROS could activate the NLRP3 in ammasome and result in in ammation.Moreover, compared to vehicletreated group, DCFH-DA uorescent density in A740003 treated group was signi cantly decreased, suggesting that ROS production was effectively suppressed by A740003 pretreatment (Fig. 3a, 3b).
A740003 inhibited the activation of NLRP3 in ammasome, phosphorylation of IKBα and decreased the over expression of P2 × 7R in vivo C57BL/6 mice were subretinally injected with 1 µL ox-LDL.A740003 (30 mg/kg/d) was intraperitoneally injected daily from 3 days before ox-LDL injection.Two weeks later, qPCR and Western Blot assay were performed to determine the mRNA and protein levels of NLRP3, P2 × 7R and Caspase-1.The protein level of IL-1β and phosphorylation of IKBα were also assessed by Western Blot.As shown in Fig. 4, mRNA levels of NLRP3, Caspase-1 and P2 × 7R were up-regulated signi cantly in mice injected with ox-LDL, whereas treatment with A740003 daily could inhibit the increase of NLRP3, Caspase-1 and P2 × 7R at mRNA level effectively compared to vehicle-treated group (Fig. 4a, 4b, 4c).Furthermore, Western Blot results demonstrated that protein levels of NLRP3, Caspase-1, IL-1β and P2 × 7R increased remarkably in mouse retinas injected with ox-LDL (Fig. 4d, 4e, 4f, 4 g, 4 h).It indicated that ox-LDL injection activated NLRP3 in ammasome.However, compared to vehicle-treated group, the protein levels of NLRP3, Caspase-1, IL-1β and P2 × 7R in mouse retinas of A740003 treated group decreased obviously (Fig. 4d, 4e, 4f, 4 g, 4 h).Subretinal injection of ox-LDL could increase the phosphorylation of IKBα and thus induce the activation of NF-κB signaling pathway (Fig. 4d, 4i).The Western Blot results also showed the ratio of p-IKBα to IKBα in mouse retinas of A740003 treated group signi cantly decreased compared to vehicletreated group (Fig. 4d, 4i).The results showed that ox-LDL could activate the NLRP3 in ammasome and NF-κB signaling pathway signi cantly.It indicated that A740003 treatment decreased the expression of P2 × 7R and suppressed the activation of NLRP3 in ammasome and NF-κB signaling pathway in vivo.
A740003 suppressed the expression of HIF-1α and VEGF in mice subretinally injected with ox-LDL As previously described, C57BL/6 mice were subretinally injected with 1 µL ox-LDL.A740003 (30 mg/kg/d) was intraperitoneally injected daily from 3 days before ox-LDL injection.Two weeks later, qPCR and Western Blot assay were performed to determine the mRNA and protein levels of VEGF and HIF-1α.As shown in Fig. 5, mRNA levels of VEGF and HIF-1α were up-regulated signi cantly in mice subretinally injected with ox-LDL (Fig. 5a, 5b).Western Blot results manifested that protein levels of HIF-1α and VEGF increased remarkably in mice subretinally injected with ox-LDL (Fig. 5c, 5d, 5e).It indicated that ox-LDL subretinal injection up-regulated the angiogenic factors including VEGF and HIF-1α intensively, which would contribute to the retinal angiogenesis.In addition, Western Blot results showed that protein levels of VEGF and HIF-1α decreased remarkably in mice treated with A740003 daily (Fig. 5c, 5d, 5e).Therefore, the results con rmed that the retinal angiogenic factors induced by ox-LDL were suppressed by A740003 treatment.
A740003 prevented the retinal function impairment induced by ox-LDL in vivo C57BL/6 mice were pretreated with A740003 daily from 3 days before ox-LDL injection.Two weeks after subretinal injection of ox-LDL, ERG was performed to evaluate the retinal function of mice injected with ox-LDL or PBS.Dark-and light-adapted ERGs showed that the amplitudes of a-wave and b-wave, which represented the functions of the photoreceptors and bipolar cells respectively, decreased signi cantly in mice subretinally injected with ox-LDL (Fig. 6a).However, the amplitudes of Dark-adapted a-wave and bwave in A740003 treated mouse retinas were higher than vehicle-treated mice (Fig. 6b, 6c).The amplitude of light-adapted b-wave was also preserved by A740003 treatment (Fig. 6d).It indicated that A740003 treatment could alleviate retinal function impairment induced by ox-LDL in mice.
A740003 inhibited retinal angiogenesis by down regulating VEGF expression and reduced the retinal edema induced by ox-LDL in vivo C57BL/6 mice were subretinally injected with ox-LDL and treated with A740003 daily from 3 days before ox-LDL injection.Two weeks later, immuno uorescent assay and retinal whole at mount were processed to investigate the retinal angiogenesis and retinal edema in C57BL/6 mice.The results of immuno uorescent assay showed that ox-LDL increased the VEGF level robustly in retinas after two weeks of ox-LDL subretinal injection (Fig. 7a, 7b).However, A740003 decreased the VEGF expression induced by ox-LDL signi cantly in mice compared to vehicle-treated group (Fig. 7a, 7b).Besides, the immuno uorescent results also showed that thickness of INL and ONL increased signi cantly in ox-LDL group (Fig. 7a, 7c).It indicated that ox-LDL resulted in the retinal edema in mice.Compared to vehicletreated group, the thickness of INL and ONL decreased obviously in A740003 treated group (Fig. 7a, 7c).The results suggested that A740003 inhibited the retinal edema induced by ox-LDL.The retinal whole at mount showed that ox-LDL successfully induced the retinal angiogenesis in mice (Fig. S1a, S1b).
However, A740003 decreased the number of new blood vessels in retinas signi cantly compared to vehicle-treated group (Fig. S1a, S1b).The results indicated that A740003 suppressed the retinal angiogenesis and retinal edema induced by ox-LDL successfully.

Discussion
Accumulating evidence indicates that chronic in ammation in response to ox-LDL is implicated in the pathogenesis of AMD [10,12,20,24].In this study, we investigated the effects of ox-LDL on proin ammatory responses and pro-angiogenesis in human ARPE-19 cells and in a novel mouse model presenting in ammatory responses and neovascularization.We found A740003, a P2 × 7 receptor antagonist, signi cantly inhibited ox-LDL induced retinal in ammatory responses, neovascularization, ROS generation and alleviated retinal function impairment in ox-LDL treated cells and injected mice.

ox-LDL induced pro-in ammatory responses and pro-angiogenesis factors both in vitro and in vivo
The results showed that ox-LDL promoted the secretion of IL-1β, IL-18, HIF-1α and VEGF in ARPE-19 cells, indicating ox-LDL induced pro-in ammatory responses and pro-angiogenesis.Besides, we further investigated the diverse effects of ox-LDL on retinal in ammation and angiogenesis in ARPE-19 cells.
Interestingly, we found 24 hours incubation with ox-LDL could promote the in ammatory cytokines signi cantly in ARPE-19 cells, while the expression of HIF-1α and VEGF showed no signi cance.However, 48 hours incubation with ox-LDL could promote the expression of HIF-1α and VEGF in ARPE-19 cells signi cantly.It suggested that pro-angiogenesis occurred after the in ammatory responses, which mimicked the development of AMD where in ammation usually precedes neovascularization.In addition, ox-LDL also induced production of ROS, which is involved in the process of AMD.
To con rm the role of ox-LDL in retina, we created a mouse model by subretinal injection of ox-LDL.
Similar to those observed in vitro, we found IL-1β, HIF-1α and VEGF levels were signi cantly increased in the mouse retinas 14 days after subretinal injection.Furthermore, retinal function was compromised and neovascularization was evident in ox-LDL injected eyes.Since ox-LDL is a trigger for initiating early events in AMD, and since the major pathologic processes including in ammatory responses and neovascularization were seen in the novel model, we suggested it might be an appropriate one for studying AMD.

Blocking of P2 × 7R reduced pro-in ammatory responses and pro-angiogenesis factors both in vitro and in vivo
To test whether P2 × 7R is involved in the pathogenesis of AMD and to test whether blocking P2 × 7R would prevent the pro-in ammatory responses and pro-angiogenesis induced by ox-LDL, A740003, a P2 × 7R antagonist was applied to block P2 × 7R in human RPE cells and mouse model.The results demonstrated that A740003 decreased the secretion of in ammatory cytokines including IL-1b and IL-18 obviously both in vitro and in vivo.Moreover, the protein levels of VEGF and HIF-1a in A740003 treated group down-regulated evidently compared to vehicle-treated group both in cells and mouse retinas.
Retinal immuno urescence and retinal at mount showed that A740003 alleviated the retinal edema and neovascularization in ox-LDL injected mice.It indicated that blocking P2 × 7R prevented the in ammatory responses and angiogenesis induced by ox-LDL effectively.
P2 × 7R is an ATP ligand-gated non-selective cation channel that is a member of the purine receptor family [25].It was veri ed that P2 × 7R involved in the activation of NLRP3 in ammasome.P2 × 7R activated channel protein pannexin-1, leading to the in ux of activating molecules and e ux of K + from cell, resulting in NLRP3 in ammasome activation in hepatic stellate cell [26].We detected the P2 × 7R  Besides, we also found that the subretinal injection of ox-LDL induced retinal edema in mice and A740003 alleviated the retinal edema to some extent.
We veri ed that a single subretinal injection of ox-LDL induced the activation of NLRP3 in ammasome and NF-κB signaling pathway in mice.Once NLRP3 in ammasome was activated, it would recruit ASC and mediate the auto-activation of pro-Caspase-1.The assembled NLRP3 in ammasome then turned itself into a cytokine processing platform by cleaving pro-IL1β/pro-IL-18 into mature peptides and releasing them into extracellular space for downstream in ammatory effects [14].brief, produced a large amount of ROS and thus activated NLRP3 in ammasome, and then promoted the secretion of in ammatory cytokines including IL-1β and IL-18.IL-1β and IL-18 are major in ammatory factors found in drusen of AMD patients [28].We found ox-LDL subretinal injection could promote the secretion of IL-1β signi cantly in mice.We also found that the subretinal injection of ox-LDL induced retinal edema.
Moreover, ROS generation induced by ox-LDL can also lead the activation of NF-κB signaling pathway.
NF-κB signaling pathway is also involved with in ammation response.NF-κB is a nuclear transcription factor that regulates expression of a large number of genes that are critical for the regulation of apoptosis and in ammation.The activation of NF-κB signaling pathway could promote the transcription of NLRP3.The various stimuli that activate NF-κB would cause phosphorylation of IκB, which is followed by its ubiquitination and subsequent degradation [14].
The results indicated that ox-LDL induced retinal in ammation and neovascularization through activation of NF-κB pathway and NLRP3 in ammasome, induction of ROS generation, and up-regulating the expression of HIF-1α and VEGF.
Although the study reveals strong evidence for the pathogenic roles of ox-LDL and therapeutic effects of A740003 in AMD, there are still some limitations that must be addressed.First, the direct interaction of P2 × 7R and NLRP3 needs further investigation, which may provide more details about the relationship between the activation of P2 × 7R and NLRP3 in ammasome.Moreover, further researches and trails need to be performed to testify the effectiveness and safety of P2 × 7R antagonist.There is still a long way to go before applying the P2 × 7R antagonist to AMD in clinic.

Conclusions
In conclusion, we demonstrated that A740003 signi cantly reduced in ammatory responses and angiogenic factors in ARPE-19 cells induced by ox-LDL.In addition, the intraperitoneal injection of

[ 14 ]
. However, A740003 treatment inhibited the ROS production induced by ox-LDL signi cantly.It manifested that blocking P2 × 7R reduced the ROS overproduction.The underlying mechanisms of decrease in ROS production remains unknown.Besides, ERG resutls showed that ox-LDL induced the retinal function impairment in mouse model.However, daily treatment of A740003 could preserve the retinal function obviously in mouse injected with ox-LDL.It showed that blocking P2 × 7R could rescue the retinal function to some extent.The protect effects was associated with the NLRP3 in ammasome the NF-κB signaling pathwayThe results showed that blocking P2 × 7R could decrease the protein levels of NLRP3, Capase-1, IL-1β and P2 × 7R signi cantly both in ARPE-19 cells and mice.It revealed that A740003 suppressed the activation of NLRP3 in ammasome and alleviated the in ammation induced by ox-LDL.Moreover, we found that blocking P2 × 7R inhibited the phosphorylation of IKBα induced by ox-LDL signi cantly both in cells and mouse retinas.It indicated that A740003 could inhibit the activation of NF-κB pathway effectively.

A740003 prevented retinal in
ammation and neovascularization, and preserved retinal function in C57BL/6 mice subretinally injected with ox-LDL.The P2 × 7R antagonist could reduce retinal in ammation, neovascularization, and protect retinal function by regulating NLRP3 in ammasome and NF-κB pathway, suppression of ROS generation, as well as inhibition of HIF-1α and VEGF.The results provide a new clue for therapeutic strategies to treat retinal in ammation and neovascularization.

Figures
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expression in ARPE-19 cells and27]use retinas and discovered the relationship between P2 × 7R and NLRP3 in ammasome activation.We screened several concentrations of A740003 for treatment in ARPE-19 cells.We found that 1 µM A740003 reduced the expression of NLRP3, Caspase-1 and P2 × 7R signi cantly in ARPE-19 cells exposed to ox-LDL for 24 hours.However, 200 µM A740003 was required to inhibit the expression of HIF-1α and VEGF increased with ox-LDL incubation for 48 hours.It suggested that it would more and more di cult to reverse the harmful effects induced by ox-LDL with development.The underlying mechanisms of this phenomenon need further investigation.We also found that A740003 pretreatment reduced the up-regulated protein level of P2 × 7R induced by ox-LDL.Ox-LDL exposure increased P2 × 7R expression signi cantly in ARPE-19 cells.However, the protein level of P2 × 7R decreased remarkably in A740003 pretreated cells.Similar regulatory effects of P2 × 7R antagonist on P2 × 7R expression were reported byJiang et al. and Wu et al. [26,27]..