The Adenosine A2A Receptor Antagonist KW6002 Mitigates Aldosterone-induced Central Serous Chorioretinopathy in Mice

The Adenosine A2A Receptor Antagonist KW6002 Mitigates Aldosterone-induced Central Serous Chorioretinopathy in Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Adenosine A 2A Receptor Antagonist KW6002 Mitigates Aldosterone-induced Central Serous Chorioretinopathy in Mice Qiaoli Liu, Zijun Yu, Wei-hang Tang, Jiasheng Yang, Jia-nan Que, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8526598/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Central serous chorioretinopathy (CSC), a prevalent disease characterized by choroidal vascular abnormalities, has extremely limited treatment options. This study investigates the effects of the selective adenosine A 2A receptor (A 2A R) antagonist KW6002 on choroidal vascular hyperpermeability and the blood-retinal barrier (BRB), and explores its therapeutic potential in experimental CSC. Methods We examined the expression of A 2A R in the retinal pigment epithelium (RPE)-choroid-sclera complex using quantitative real-time PCR (qPCR) and Western blotting (WB) in mice with an established aldosterone-induced acute CSC model. Before modeling, mice were administered 5 mg/kg KW6002 or a vehicle control via intraperitoneal injection. The retinal and choroidal thickness was assessed by optical coherence tomography (OCT) and hematoxylin-eosin(H&E) staining. We observed Müller cells activation, retinal microglia infiltration, and proinflammatory cytokine expression via immunofluorescence and qPCR. Next, we employed the effects of the A 2A R antagonist KW6002 and genetic A 2A R knockout on BRB integrity using immunofluorescence and WB. Finally, to clarify how A 2A R knockout confers therapeutic benefits in CSC, we assessed activation of the TNF-α/NF-κB-MMP2/9 signaling pathway. Results We found that A 2A R signaling was significantly upregulated in the RPE-choroid-sclera complex in CSC models, and both A 2A R antagonist KW6002 and A 2A R knockout significantly inhibited the aldosterone-induced central retinal and choroidal pathologic thickening. Moreover, KW6002 treatment decreased the activation of Müller cells and the proliferation of microglia, inhibited the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and ameliorated the retinal damage caused by aldosterone; in contrast, A 2A R knockout resulted in significant upregulation of key tight junction proteins (ZO-1, Claudin-1, and Claudin-5). In summary, these results suggest that the protective effects are likely due to A 2A R inhibiting TNF-α/NF-κB–MMP2/9 signaling axis. Conclusions Our findings show that the A 2A R antagonist KW6002, or A 2A R knockout, offers a protective effect in experimental CSC, reduces inflammation and maintains the integrity of the BRB, and mediates such protection through inhibiting TNF-α/NF-κB pathway. These findings present a new method for treating CSC, which will guide our future clinical strategy development. Adenosine A2A receptor blood-retinal barrier central serous chorioretinopathy TNF-α/NF-κB signaling pathway inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Central serous chorioretinopathy (CSC) ranks among the most prevalent vision-threatening retinal disorders, after age-related macular degeneration (AMD), diabetic retinopathy, and retinal branch vein occlusion(Wang et al. 2008 ). Furthermore, it shows great gender preference, as studies report more cases (approximately 10 cases per 100,000 people) happening in men compared to merely 2 cases per 100,000 in women(Kitzmann et al. 2008 ). The clinical hallmark of CSC is neurosensory retinal detachment resulting from the accumulation of serous subretinal fluid, which stems from retinal pigment epithelium (RPE) dysfunction and increased choroidal permeability and thickness(Daruich et al. 2015 ; Imamura et al. 2011 ; Yanagi 2020 ). Although retinal detachment resolves spontaneously in most CSC patients, 30%-50% develop chronic or recurrent disease, leading to permanent retinal tissue damage and vision loss(Gemenetzi et al. 2010 ; Nicholson et al. 2013 ; Salehi et al. 2015 ). Given the vision-threatening nature of CSC, early and effective intervention is critical. Current treatment strategies for CSC including intravitreal anti-vascular endothelial growth factor (anti-VEGF) injection, laser photocoagulation, and photodynamic therapy (PDT), are all associated with distinct clinical limitations. The first-line treatment, half-dose PDT, is constrained by the high cost of the photosensitizer verteporfin, reliance on specialized equipment and trained personnel, and a procedural complexity greater than that of intravitreal injection(Bae et al. 2011 ). Anti-VEGF therapy, on the other hand, presents challenges such as stringent sterile requirements, potential postoperative complications, and the burden of repeated injections(Meyer et al. 2011 ; Patel et al. 2022 ). Therefore, the development of safer, more convenient, and more effective treatment strategies remains an unmet clinical needed. Mounting evidence indicates that choroidal vascular abnormalities – including pathological dilation, hyperpermeability, and disruption of the RPE tight junctions and blood-retinal barrier (BRB) – play a pivotal role in the pathogenesis of CSC(Guyer et al. 1994 ; Imamura et al. 2011 ). Corticosteroids are a leading cause of CSC via the overactivation of mineralocorticoid receptors in the choroid(Bousquet et al. 2019 ). Corticosteroids bind to the mineralocorticoid receptors, leading to up-regulation of calcium-activated potassium channels in choroidal endothelial cells, causing smooth muscle relaxation, and subsequently, choroidal hyperpermeability. Corticosteroids also disrupt the RPE barrier function, cause dysregulation of choroidal hemodynamics, and interfere with ion transport via upregulation of adrenergic receptors. Aldosterone induces a mineralocorticoid/glucocorticoid receptor-dependent upregulation of epithelial sodium channel-α, potassium channels, and aquaporin-4 (AQP4) in retinal Müller cells, culminating in retinal swelling. Given the accumulating evidence implicating corticosteroids and mineralocorticoid receptors in disease pathogenesis, intravitreal aldosterone administration recapitulates key CSC pathological features, including choroidal vasodilation, vascular leakage, RPE dysfunction, and choroidal thickening, thereby establishing a mechanistic model of CSC development(Canonica et al. 2019 ; Yu et al. 2022 ). Extracellular adenosine signals through G protein-coupled receptors (A 1 , A 2A , A 2B , and A 3 ), serving a key regulatory function in multiple tissues, including the retina and choroid, in both normal and diseased conditions(Adair et al. 2005 ; Fredholm et al. 2001 ). Immunoreactivity for the adenosine A 2A receptor (A 2A R), has been detected in endothelial progenitor cells, vascular progenitor cells, and endothelial cells within the retina/choroid(Lutty and McLeod 2003 ). Our previous studies demonstrate that adenosine A 2A R antagonists significantly attenuate retinal inflammatory responses and suppress neovascularization during the hyperoxic phase in the oxygen-induced retinopathy model(Zhong et al. 2021 ; Zhou et al. 2018 ). Furthermore, A 2A R antagonists can inhibit choroidal neovascularization by reducing inflammatory mediator levels and suppressing angiogenic activity(Sorenson et al. 2021 ). Beyond their critical roles in mitigating inflammation and blocking pathological angiogenesis, A 2A R also play pivotal roles in regulating the blood-brain barrier (BBB). Specifically, activation of the A 2A R downregulates the expression of tight junction and adherent junction molecules in human and murine brain microvascular endothelial cells, thereby increasing BBB permeability. Conversely, endothelial cell-specific knockout of A 2A Rs effectively reduces BBB leakage and markedly suppresses immune cell infiltration across the BBB(Fernandez et al. 2023 ; Kim and Bynoe 2015 ). Likewise, A 2A R antagonists can normalize the BBB permeability in insulin-resistant mice(Yamamoto et al. 2019 ). Moreover, our recent study reveals that the selective A 2A R antagonist KW6002 increases the blood-cerebrospinal fluid barrier integrity to reduce T-cell migration across the choroid plexus (CP), consequently reducing pathological damage in experimental autoimmune encephalomyelitis(Zheng et al. 2022 ). These collective findings led us to propose that targeting the adenosine A 2A R signaling pathway may offer novel therapeutic strategies for disorders associated with central nervous system barrier dysfunction. This study sought to investigate the protective role of A 2A R antagonism in an aldosterone-induced CSC model and elucidate its underlying molecular mechanisms. We demonstrated that the A 2A R antagonist KW6002 attenuates aldosterone-induced choroidal thickening and vasodilation, suppresses macrophage/microglia infiltration and inflammatory cytokine production, and preserves BRB integrity. Furthermore, A 2A R knockout (A 2A R-KO) preserved blood-retinal barrier integrity by suppressing Matrix Metallopeptidase (MMP)-2/3/9 expression and maintaining tight junction proteins (ZO-1, Occludin, Claudin-1, and Claudin-5). The A 2A R-KO-mediated preservation of blood-retinal barrier integrity was associated with inhibition of the TNF-α/NF-κB pathway. Collectively, these findings support that targeting the A 2A R offers a novel therapeutic strategy for treating CSC. MATERIALS AND METHODS Animals Numerous neuroscience studies, including those from our laboratory and other research teams, have extensively characterized A 2A R-KO mice. Through crossbreeding of heterozygous A 2A R-KO mice (A 2A R-KO +/− ), we generated homozygous A 2A R-KO (A 2A R-KO +/+ ), A 2A R-KO +/− , and wild-type littermate controls (WT). All mice originated from an identical breeding lineage. Genotypic determination was executed via PCR analysis of genomic DNA extracted from tail biopsies, utilizing three primer sets targeting the Neo-AR cassette and adjacent A 2A R gene, as previously described(Chen et al. 1999 ). A 2A R-Trp Tomato transgenic mice were generated by crossing A 2A R-Trp Cre mice (MMRRC, Stock Number: 031168-UCD) and Tomato reporter mice (B6;129 S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Laboratory). In these A 2A R-Trp Tomato mice, Cre recombinase expression mediates the specific excision of the loxP-flanked STOP cassette in Tomato reporter mice, thereby activating tdTomato fluorescent protein expression(Zhong et al. 2021 ) (Fig. 1 A). To characterize the spatial distribution of A 2A R in the retina, we performed dual immunofluorescence colocalization assays combining endogenous tdTomato fluorescence with cell type-specific retinal markers. The C57BL/6J male mice aged eight weeks were purchased from Beijing Vital River Laboratory (Beijing, China). Experimental procedures were performed exclusively on the right eye of each mouse under a randomized design, with a group size of 5–6 animals. To control for potential litter effects, the A 2A R-KO and WT mice used in this study were sourced from 2–3 independent breeding litters. All animal procedures were conducted following protocols approved by the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou Medical University, China. Mice were housed 4–5 per cage under controlled environmental conditions (22 ± 1°C and 60 ± 2% relative humidity) with ad libitum access to food and water. Bedding material was changed every 5 days, and fresh bedding was provided immediately after surgery. CSC induction and drug administration For in vivo administration of the A 2A R antagonist KW6002 (GC11590, GLPBIO, Montclair, USA), the compound was freshly prepared before each administration by dissolving in a vehicle containing 15% dimethyl sulfoxide, 15% castor oil, and 70% physiological saline to achieve a final concentration of 5 mg/ml. Mice received daily intraperitoneal injections of KW6002 (5 mg/kg) or an equivalent volume of vehicle for 7 consecutive days. During model induction, animals were anesthetized via inhalation of 5% isoflurane (RWD Life Science, Shenzhen, China) and maintained under anesthesia with 1% isoflurane throughout the procedure. Pupils were dilated using tropicamide eye drops. Previous studies have indicated that approximately 10% of intravitreally administered glucocorticoids reach the retina(Chang-Lin et al. 2011 ). To ensure delivery of at least 10 nM aldosterone to the apical side of retinal pigment epithelial cells, an acute central serous chorioretinopathy phenotype was induced by intravitreal injection of 2 mM aldosterone (GC41390, GLPBIO, Montclair, USA) at a volume of 2 µL per eye using a Hamilton microsyringe (Hamilton Company, Reno, USA). Mice exhibiting intraoperative complications (e.g., vitreous hemorrhage, retinal detachment) were excluded. Twenty-four hours after modeling, mice received a final administration and were subsequently sacrificed for further experimentation. Experimental procedures were performed exclusively on the right eye of each mouse under a randomized design, with a group size of 5–6 animals. Optical coherence tomography (OCT) To evaluate the impact of aldosterone intravitreal injection on retinal thickness, we acquired images using spectral domain optical coherence tomography (Heidelberg Engineering, Dossenheim, Germany), a non-invasive method for visualizing the microstructure of live animal retinas, before and 24 hours after aldosterone administration. Prior to imaging, mice were anesthetized, and topical mydriatic eye drops were applied to induce pupil dilation. The eye-tracking function was employed to conduct continuous dynamic imaging along a 30° horizontal line, focusing on the optic disc region. The scan parameters were adjusted to achieve optimal signal intensity and contrast. Following imaging, the anesthesia mask was promptly removed, the mice were placed on a heating pad to maintain body temperature, and ophthalmic ointment was applied to both eyes to protect the corneas. Retinal thickness was measured perpendicular to the RPE layer at 500 µm from the optic disc in all obtained images using ImageJ software (version 1.47; NIH, MD), and average values were calculated. Fluorescein fundus angiography (FFA) To analyze fundus changes and retinal vascular morphological alterations in mice after model establishment, fluorescein fundus angiography (FFA) was performed prior to tissue collection. FFA imaging was performed using a rodent-specific fundus camera system (Eyemera FUNDUS, IIscience, Korea) with a spatial resolution of < 3 µm for mouse imaging. After anesthesia, tropicamide eye drops were administered to induce pupil dilation. Subsequently, 4% sodium fluorescein solution was administered intraperitoneally at a dose of 0.05 mL/20 g body weight. After a 4‑minute waiting period, the mouse was positioned in a prone position on a specialized imaging stage. Ofloxacin ophthalmic ointment was applied to the corneal surface as a contact medium. The integrated blue LED light source was adjusted to an appropriate intensity, and the objective lens was gently brought into contact with the cornea through the ointment medium before image acquisition. Post-procedure, mice were maintained on a heating pad, and ophthalmic ointment was applied to both eyes for corneal protection. Immunofluorescence staining For the globe frozen section, eyes were fixed in 4% PFA for 2 hours following PBS perfusion. Under a stereomicroscope, connective tissues were carefully trimmed, and the cornea, lens, and iris were removed to isolate the intact retina. Subsequently, retinas were dehydrated in a sucrose gradient and embedded in an optimal temperature compound. Specimens were sectioned at 14 µm thickness. Mouse retinal cryosections were processed for immunofluorescence labeling. Sections were washed three times with PBS and incubated for 2 hours in PBS containing 0.3% Triton X-100 and 0.5% BSA. The sections were then incubated overnight at 4 ℃ with the following primary antibodies: goat anti-CD31 (1:200, AF3628, R&D Systems, China), rabbit anti-IBA1 (1:400, 019-19741, Wako, Tokyo, Japan), rabbit anti-AQP4 (1:400, MABN2527, Sigma, Missouri, USA), mouse anti-GFAP (1:400, G3893, Sigma, Missouri, USA), rat anti-CD68 (1:200, MCA1957GA, Bio-Rad, California, USA), rabbit anti-RPE65 (1:200, 83861-1-RR, Proteintech, Illinois, USA), and rabbit anti-ZO-1 (1:200, 21773-1-AP, Proteintech, Illinois, USA). After washing with PBS (3 × 10 minutes), sections were incubated with secondary antibodies (donkey anti-goat Fluor 488, donkey anti-rabbit Fluor 488, donkey anti-goat Fluor IgG 594, or donkey anti-rat Fluor 594; all 1:1000, Invitrogen, California, USA) at room temperature for 2 hours to visualize the primary antibody signals. Following secondary antibody incubation, retinal sections were washed with PBS and counterstained with DAPI (1:1500, Beyotime, Shanghai, China) for 8 minutes. Images were acquired at 20× or 40× magnification using a confocal laser scanning microscope (LSM900, Carl Zeiss). Hematoxylin-eosin (H&E) staining For quantitative assessment of retinal and choroidal thickness variations, ocular tissues were processed for histological analysis. At 24 hours post-intravitreal aldosterone administration, mice were euthanized by intraperitoneal injection of pentobarbital sodium (2.5 mg/10 g body weight). Eyes were immediately enucleated and fixed in 4% PFA for 24 hours, followed by standard paraffin embedding. Serial sections (3 µm thickness) were cut and stained with hematoxylin and eosin (H&E) for morphological examination. Digital images of retinal sections were acquired and analyzed using ImageJ software (NIH, USA). Thickness measurements were performed at two standardized locations: (1) central retina (500 µm from the optic nerve head), and (2) peripheral retina (1 mm from the trabecular meshwork). Both retinal and choroidal thicknesses were measured and compared across experimental groups. Quantitative real-time PCR (qPCR) Total RNA was isolated from RPE-choroid-sclera complexes and retinal tissues using TRIZOL Reagent (Cat. No. 15596026, Thermo Fisher Scientific) according to the manufacturer's protocol. RNA concentration and purity were determined using a spectrophotometer (Beckman, LA, USA), with samples having an A260/A280 ratio between 1.8 and 2.0 considered acceptable. For cDNA synthesis, 1000 ng of total RNA was reverse-transcribed using the Vazyme cDNA Synthesis Kit (R323-01, Vazyme, Jiangsu, China), followed by appropriate dilution and storage at -20 ℃. Quantitative real-time PCR (qPCR) was performed on a CFX 96 real-time PCR system (Bio-Rad) using SYBR-Green Premix Ex Taq (Q712-03, Vazyme, Jiangsu, China) with technical triplicates for each sample. The following primers were used: PPIA (forward: 5′-AGC ATA CAG GTC CTG GCA TCT TGT − 3′, reverse: 5′-CAA AGA CCA CAT GCT TGC CAT CCA − 3′); A 2A R (forward: 5′-GCC ATC CCA TTC GCC ATC A′, reverse: 5′-GCA ATA GCC AAG AGG CTG AAG A -3′); IL-6 (forward: 5′-TCT GAA GGA CTC TGG CTT TG-3′, reverse: 5′-GAT GGA TGC TAC CAA ACT GGA-3′); IL-1β (forward: 5′-CCA AGC AAC GAC AAA ATA CC-3′, reverse: 5′-GTT GAA GAC AAA CCG TTT TTC C-3′); TNF‐α (forward: 5′-AAC TCG AGT GAC AAG CCC GTA G-3′, reverse: 5′-GTA CCA CCA GTT GGT TGT CTT TGA-3′). Relative gene expression was calculated using the 2 −ΔΔCt method with PPIA serving as the internal reference gene. Western blotting Retinal tissue or RPE-choroid-sclera complex was homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (B14001 & B15001, Bimake, Texas, USA). The lysate was centrifuged at 10,000 rpm for 10 minutes, and protein concentration in the supernatant was determined using a BCA protein assay kit (P0010, Beyotime, Shanghai, China). Samples were denatured at 95 ℃ for 10 minutes in 5× SDS loading buffer (P0015, Beyotime, Shanghai, China) and separated by SDS-PAGE. Proteins were transferred to 0.45 µm PVDF membranes (BS-PVDF-45-S, Biosharp, Hefei Ulife Biotechnology Co., Ltd.) in ice-cold transfer buffer at a constant current of 350 mA for 60 minutes. Membrane were blocked with 5% non-fat milk at room temperature for 2 hours and then incubated overnight at 4 ℃ with the following primary antibodies: β-actin (1:1000, 66009-1-Ig, Proteintech, Illinois, USA), ZO-1 (1:5000, 21773-1-AP, Proteintech, Illinois, USA), GAPDH (1:1000, GB15004, Servicebio, Hubei, China), A 2A R (1:200, FI-A2A-Go-Af700, Frontier Institute, Lausanne, Switzerland), Claudin-1(1:1000, 51-9000, Invitrogen, California, USA), Occludin (1:1000, 71-1500, Invitrogen, California, USA), Claudin-5 (1:1000, YP-Ab-16961, UpingBio, Shanghai, China), TNF-α (1:1000, 17590-1-AP, Proteintech, Illinois, USA), NF-κB p65 (1:1000, ET1603-12, Huabio Co., Ltd.), phosphorylated NF-κB p65 (1:1000; Cell Signal Technology, catalogue no. 3033), MMP2 (1:1000, YP-mAb-02682, UpingBio, Shanghai, China), MMP3 (1:1000, YP-Ab-02788, UpingBio, Shanghai, China), and MMP9 (1:1000, YP-Ab-02639, UpingBio, Shanghai, China). See Table 1 for antibody details. Table 1 Primary antibodies are used for immunofluorescence and Western blotting Following primary antibody incubation, membranes were washed and incubated with HRP-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Massachusetts, USA) for 2 hours at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence and detected with a Bio-Rad ChemiDoc XRS + imaging system. The band intensities were analyzed with ImageJ software (version 1.47; NIH, MD) and were normalized to β-actin or GAPDH. Relative protein expression was calculated with the control or WT group set to 1. Experiments were independently repeated at least three times. Statistical analysis Data are presented as mean ± SEM. Statistical comparisons between two groups were performed using an independent Student's t-test, while comparisons among three or more groups were conducted using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). Values of p < 0.05 were considered statistically significant. RESULTS A 2A R signaling is upregulated in the RPE-choroid-sclera complex of the CSC model Initial characterization of A 2A R expression was performed using A 2A R-tdTomato reporter mice, enabling cell type-specific identification of A 2A R-positive cells through tdTomato fluorescence (Fig. 1 A). Immunohistochemical analysis revealed distinct co-localization of A 2A R -tdTomato labeling with the two retinal vascular plexuses—the superficial capillary plexus (located in the nerve fiber and ganglion cell layers) and the deep capillary plexus (located in the inner nuclear layer)—as well as with the choroidal vasculature (including capillaries and larger vessels) (Fig. 1 B), indicating robust A 2A R expression in ocular vascular networks, and the A 2A R -tdTomato labeling is partially co-localized with glial cells. Then, we performed a western blotting and found the protein level of A 2A R was increased in the RPE-choroid-sclera complex 24 hours following CSC induction (Fig. 1 C-D, p < 0.05). Furthermore, qPCR revealed significant upregulation of A 2A R mRNA level in the RPE-choroid-sclera complex 24 hours after CSC induction (Fig. 1 E, p < 0.01). Pharmacological blockade of A 2A R attenuates aldosterone-induced retinal and choroidal thickening To investigate the therapeutic potential of the A 2A R antagonist KW6002 in CSC, we established a CSC model using intravitreal aldosterone injection. Mice received daily intraperitoneal KW6002 (5 mg/kg) or vehicle for seven days prior to CSC modeling. In vivo morphological assessment was performed 24 hours post-aldosterone injection, and mice were sacrificed after a final dose for subsequent histological analysis (Fig. 2 A). Fundus photography revealed that aldosterone administration induced irregular retinal swelling (yellow arrows), which was significantly attenuated by KW6002 pretreatment. FFA demonstrated pronounced vascular tortuosity in aldosterone-treated retinas compared to controls (red arrows), while KW6002 pretreatment substantially improved vascular morphology, indicating a protective effect against aldosterone-induced retinal edema and vascular abnormalities (Fig. 2 B). At 500 µm from the optic disc, OCT measurements indicated significantly greater retinal thickness in the aldosterone group relative to controls (p < 0.05), along with marked intraocular inflammation (yellow arrow). Disruption of the RPE layer was also observed (red arrow). KW6002 administration substantially reduced peri-papillary inflammation and demonstrated a trend toward normalization of retinal thickness (Fig. 2 C, F). Histological examination with H&E staining assessed retinal and choroidal thickness, with central measurements taken 500 µm from the optic disc and peripheral measurements obtained 1000 µm from the ciliary body (Fig. 2 D). Compared to controls, aldosterone administration significantly increased central retinal thickness ( p < 0.05), while KW6002 treatment could ameliorate the central retinal thickness (Fig. 2 H, p 0.05), corroborating the OCT findings. Notably, aldosterone induced significant thickening of the central choroid ( p < 0.01 versus control), which was effectively prevented by KW6002 treatment (Fig. 2 G, p 0.05). These findings demonstrate that KW6002 effectively prevents aldosterone-induced thickening in both retinal and choroidal tissues, particularly in central regions, while simultaneously ameliorating vascular tortuosity. Genetic deletion of A 2A R reduces retinal and choroidal thickening in the CSC model To further validate the protective role of A 2A R in CSC pathogenesis, we conducted comparative studies using A 2A R-KO mice and their wild-type littermates. Fundoscopic analysis demonstrated that A 2A R-KO mice exhibited significantly attenuated retinal elevation (yellow arrows) and reduced vascular tortuosity (red arrows) compared to WT (Fig. 3 A). Quantitative analysis of H&E-stained sections revealed that A 2A R-KO mice had significantly thinner central retina (Fig. 3 C, p < 0.05) and central choroid (Fig. 3 D, p 0.05). Quantitative OCT measurements confirmed these findings, revealing a marked reduction in retinal thickness in A 2A R-KO mice relative to WT animals (Fig. 3 G-H, p < 0.05). These results provide compelling evidence that genetic ablation of A 2A R confers protection against aldosterone-induced retinal edema and vascular abnormalities. A 2A R antagonist KW6002 attenuates retinal inflammation and Müller cell activation in the CSC model As aldosterone could upregulate ion and water channels in Müller glial cells to promote retinal edema (Zhao et al. 2010 ), we investigated the effects of A 2A R antagonism on glial cell activation. Immunofluorescence staining of Müller cells (GFAP + ) and AQP4 in retinal tissues revealed that aldosterone administration significantly increased GFAP fluorescence intensity compared to controls ( p < 0.01), indicating Müller cell activation, along with elevated AQP4 expression ( p < 0.05). Prophylactic treatment with KW6002 effectively attenuated these pathological changes (Fig. 4 A-C, p < 0.05). Given that aldosterone induces intraocular inflammation, as evidenced by inflammatory cell infiltration and posterior segment alterations on OCT imaging, we decided to investigate the aldosterone-induced microglia/macrophage activation through staining with IBA1. The aldosterone group exhibited a marked increase in IBA1 + cells compared to controls (Fig. 4 D-E, p < 0.0001), while KW6002 pretreatment significantly reduced this inflammatory response (Fig. 4 D-E), demonstrating its ability to mitigate aldosterone-induced microglial/macrophage infiltration. In addition, qPCR analysis of retinal tissues showed that mRNA levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly upregulated in the Aldo-induced model group compared with the control group (Fig. 4 F-H, p < 0.05). Following the KW6002 intervention, these cytokine expressions were significantly reduced relative to the Aldo model group (Fig. 4 F-H, p < 0.05). Collectively, these findings demonstrate that A 2A R antagonism effectively ameliorates both aldosterone-induced retinal inflammation and Müller cell activation in this CSC model. A 2A R inhibition preserves BRB integrity by preventing aldosterone-induced tight junction disruption in the RPE-choroid-sclera complex To elucidate the protective mechanisms of the A 2A R antagonist KW6002 on BRB integrity, we systematically evaluated the expression and distribution of the tight junction (TJs) protein ZO-1 and RPE marker RPE65 using immunofluorescence staining and western blot analysis. Our investigations revealed three key findings: First, immunofluorescence analysis demonstrated that 24hours aldosterone treatment significantly disrupted the continuous distribution of RPE65 and ZO-1 signals ( p < 0.01 and p < 0.0001, respectively), with evident TJs structural damage (Fig. 5 A, B, D, E). These morphological alterations strongly indicate aldosterone-induced BRB dysfunction. Notably, KW6002 pretreatment markedly attenuated these pathological changes ( p < 0.05 and p < 0.01, respectively). Further confirmation via immunostaining for CD31, a specific marker of vascular endothelial cells, demonstrated that KW6002 could partially reverse aldosterone-induced vascular endothelial cell damage (Figs. 5 C, F, p < 0.05). Second, in the A 2A R-KO mouse model, we observed significantly higher protein levels of ZO-1, Claudin-1, and Claudin-5 in the RPE-choroid-sclera complex of aldosterone-treated A 2A R-KO mice compared to WT (Fig. 5 I, J, M, N, O, P, p < 0.01, p < 0.05, and p 0.05). Collectively, our results demonstrate that KW6002 protects BRB structural and functional integrity through A 2A R signaling inhibition, primarily by maintaining TJs protein stability. This protective mechanism is evident at both pharmacological (KW6002 treatment) and genetic (A 2A R-KO) levels, providing compelling evidence for A 2A R inhibition as a therapeutic strategy for BRB protection. A 2A R inactivation suppresses TNF-α/NF-κB-MMP2/9-mediated tight junction disruption in the CSC model Previous studies have demonstrated that RNA sequencing analysis of the RPE-choroid-sclera complex in acute CSC rat models revealed upregulation of the TNF-α/NF-κB signaling pathway, which mediates inflammatory responses(Canonica et al. 2019 ). To further elucidate the molecular mechanisms underlying the protective effects of A 2A R inhibition on aldosterone-induced choroidal thickening, vasodilation, and BRB integrity disruption, we performed western blotting analysis to examine the expression of proteins in the TNF-α/NF-κB signaling pathway in A 2A R-KO mice within the CSC model. Compared to WT, A 2A R-KO mice exhibited significantly reduced expression of TNF-α and phosphorylated NF-κB p65 (p-NF-κB p65) (Fig. 6 A-B, D, p < 0.05 and p 0.05), the p-p65/p65 ratio was significantly lower in the A 2A R-KO group compared to WT controls (Fig. 6 E, p < 0.05). These results demonstrate that A 2A R inhibition effectively suppresses TNF-α/NF-κB signaling pathway in the CSC model. Transcriptomic analyses of both acute and chronic CSC models revealed consistent upregulation of MMP3 and MMP9(Canonica et al. 2019 ). Based on the above data, we investigated the expression profiles of MMP2, MMP3, and MMP9 – key effector molecules implicated in BRB disruption. Our results showed significantly reduced expression of these molecules in the A 2A R-KO group compared to WT (Fig. 6 A, F-H, p < 0.05). Collectively, these findings suggest that A 2A R inhibition exerts protective effects by modulating the TNF-α/NF-κB-MMP2/3/9 signaling pathway, thereby alleviating inflammation and preserving RPE-choroid-sclera complex barrier integrity in the CSC model. DISCUSSION Our findings identify the A 2A R as a promising therapeutic target for CSC, supported by the following key evidences: (1) The A 2A R was expressed in both choroidal and retinal vascular endothelial cells, with selective upregulation in the RPE complex of the aldosterone-induced CSC model; (2) Treatment with the A 2A R antagonist KW6002 and genetic knockout of A 2A R effectively reversed aldosterone-induced pathological features of CSC; (3) KW6002 treatment suppressed aldosterone-induced activation of retinal glial cells and retinal inflammatory responses; (4) A 2A R deletion preserves BRB integrity by preventing downregulation of TJs proteins; (5) Mechanistically, A 2A R contributes to the disruption of both choroidal vascular endothelial and RPE barriers in CSC via the TNF-α/NF-κB-MMP2/9 signaling axis. Collectively, these results highlight the critical role of the A 2A R in CSC pathogenesis and provide experimental support for its potential as a therapeutic target. All experimental results are schematically summarized in Fig. 7 . A 2A R signaling as a critical mediator of barrier integrity in central serous chorioretinopathy The integrity of both the choroidal vascular system and RPE serves as a critical defensive mechanism against the pathological progression of CSC(Kaye et al. 2020 ). In the present study, we observed a significant increase in adenosine A 2A R expression within the RPE-choroid-sclera complex under CSC pathological conditions, indicating that the aberrant adenosine A 2A R signaling may be a valid therapeutic target. Indeed, we found that A 2A R-KO attenuated aldosterone-induced choroidal thickening and vasodilation, and increased the choroidal-retinal barrier integrity by preventing the reduction of TJs protein levels. This finding aligns with our finding that knockout of A 2A R in the choroid plexus epithelium delayed experimental autoimmune encephalomyelitis onset and alleviated pathological damages by preventing T-cell infiltration into the choroid plexus region(Zheng et al. 2022 ). The protection against aldosterone-induced CSC pathology was also consistent with previously reported protective effects of A 2A R antagonists on the blood-brain barrier(Fernandez et al. 2023 ; Kim and Bynoe 2015 ; Yamamoto et al. 2019 ). Based on these collective findings, we propose that A 2A R plays a crucial role in CSC pathogenesis, with its involvement closely related to choroidal-retinal barrier dysfunction and CSC pathological development. How does the A 2A R exert its control over CSC pathology? The pathogenesis of CSC with pathological accumulation of subretinal fluid involves the well-established core pathological components: enhanced permeability of larger choroidal vessels and disruption of the RPE barrier(Lee et al. 2018 ). One of the key mechanisms is the ability of the A 2A R antagonist to protect against disruption of the RPE barrier, the core of CSC pathogenesis. Increased permeability of choroidal capillaries leads to elevated hydrostatic pressure within the choroid, which subsequently results in RPE dysfunction(Gass 1967 ). Specifically, cadherin-5, a key regulator of cell-cell adhesion, is essential for maintaining vascular integrity in the choroid. Dysfunction of cadherin-5 compromises vascular permeability and weakens intercellular connections in large choroidal vessels, potentially underlying the vascular abnormalities observed in CSC(Schubert et al. 2014 ). Indeed, our analysis reveals that 24hours aldosterone treatment significantly disrupted the continuous distribution of RPE65 and ZO-1 signals, with evident TJs structural damage. Importantly, we found that KW6002 treatment markedly attenuated these pathological changes. Similarly, genetic deletion of A 2A R-KO increased protein expression of ZO-1, Claudin-1, and claudin-5 in the RPE-choroid-sclera complex in aldosterone-treated mice. Thus, KW6002 protects BRB structural and functional integrity through A 2A R signaling inhibition, primarily by maintaining TJs protein stability, providing compelling evidence for A 2A R inhibition as a therapeutic strategy for BRB protection. Another pathological component that A 2A R antagonists target to control CSC pathology is inflammation, an established as a critical pathogenic factor in CSC to disrupt the integrity of both the choroidal vascular system and RPE(Shen et al. 2024 ). The characteristic choroidal vascular hyperpermeability in CSC appears to result from the synergistic effects of inflammatory mediators and oxidative stress(Nicolò et al. 2020 ). Notably, breakdown of the BRB may trigger a cascade of secondary inflammatory responses, leading to prolonged disease duration and neurosensory retinal detachment. Recent studies have investigated serum-based inflammatory biomarkers as potential predictors of CSC progression or severity. Alterations in plasma cytokine levels have been observed in CSC patients, characterized by elevated expression of VEGF, IL-6, IL-10, and IL-12(Karska-Basta et al. 2021 ). Matet et al(Matet et al. 2020 ) reported decreased lipocalin-2 levels in acute CSC patients compared to healthy controls. Lipocalin-2, an acute-phase reactant, exhibits both anti-inflammatory and pro-inflammatory properties. Recent investigations have demonstrated that systemic inflammatory markers, including the neutrophil-to-lymphocyte ratio and monocyte-to-high-density lipoprotein ratio, are significantly elevated in patients with acute CSC(Erol et al. 2017 ; Sirakaya et al. 2020 ). Several studies have demonstrated the efficacy of topical NSAIDs in acute CSC. For example, Alkin Z et al(Alkin et al. 2013 ) reported that 0.1% nepafenac significantly enhanced subretinal fluid absorption and improved best-corrected visual acuity (BCVA) in acute CSC patients. Subsequent investigations by Bahadorani S et al. (Bahadorani et al. 2019 ) Consistent with this, we demonstrated that aldosterone administration induced intraocular inflammation accompanied by concurrent upregulation of adenosine A 2A R expression. Importantly, the pharmacological blockade of A 2A R with KW6002 effectively mitigated retinal inflammation and attenuated both choroidal and retinal edema, consistent with previous reports. This also collaborates with the previous report that intravitreal administration of the A 2A R antagonist ZM 241385 in ocular hypertensive models effectively suppresses microglial activation and downregulates pro-inflammatory cytokine expression(Liu et al. 2016 ). Similarly, during ischemic injury, KW6002 treatment attenuates microglial reactivity and reduces IL-1β levels, conferring neuroprotective effects on retinal tissue(Boia et al. 2016 ). Furthermore, the A 2A R antagonist SCH 58261 has been shown to inhibit pathological overactivation of retinal microglia while promoting retinal cell survival(Aires et al. 2019 ). Early studies with transcriptomic analyses reveal that excessive aldosterone significantly upregulates genes encoding inflammatory response proteins, neutrophil chemotaxis, responsiveness to IL-1, IL-6, IL-12, and TNF-α, as well as other inflammatory genes involved in the NF-κB and TNF-α pathways, CCL3, CXCL1, and the CCL2/CCR2 axis(Canonica et al. 2019 ; Sanz-Rosa et al. 2005 ). Activation of NF-κB has also been shown to trigger inflammation in age-related macular degeneration(Ghosh et al. 2017 ). Our analysis confirmed that the TNF-α/NF-κB signaling pathway was activated in the CSC model. Furthermore, inhibition of the NF-κB pathway can effectively prevent vascular leakage and inflammatory cell infiltration, reduce the expression of pro-inflammatory mediators and leukocyte adhesion molecules, and regulate vascular tension and permeability(Reddy et al. 2020 ; Sanz-Rosa et al. 2005 ). Thus, aldosterone may induce CSC pathology via the TNF-α/NF-κB signaling pathway. Indeed, our results revealed that A 2A R-KO significantly inhibited TNF-α, p65, and p-p65 levels, indicating that A 2A R antagonism can block the expression of these inflammatory mediators. This finding further collaborates with our early findings that A 2A R antagonists or A 2A R-KO can modulate microglial reactivity, inhibit the expression of inflammatory factors TNF-α and IL-1β, and protect the retina from transient ischemic injury(Boia et al. 2017 ). Furthermore, the A 2A R antagonist KW6002 is a potential therapeutic option for CSC. While half-dose PDT remains the first-line option for chronic CSC, its broad clinical application faces significant limitations(Radke et al. 2025 ). The treatment's dependence on the costly photosensitizer verteporfin, the requirement for specialized laser equipment and trained personnel, and its relatively complex procedure compared to intravitreal injections all hinder its routine use. Currently, the most used drugs in clinical treatment are anti-VEGF agents (such as ranibizumab and aflibercept). These anti-VEGF drugs need to be administered through intravitreal injection (due to poor permeability of the blood-ocular barrier), which requires strict aseptic conditions and high professional requirements for medical resources. Although intravitreal injections have the advantages of high local drug concentration, wide applicability, and small incision, this operation also has multiple potential risks of complications(Patel et al. 2022 ). Clinical research data show that after the operation, various potential complications may occur, such as ocular pain, subconjunctival hemorrhage (occurrence rate 23%-74%)(Fasih et al. 2013 ; Wu et al. 2008 ), temporary high intraocular pressure(de Vries et al. 2020 ; Gismondi et al. 2009 ), retinal detachment (0.013%-0.067%)(Meyer et al. 2011 ; Storey et al. 2019 ; Tolentino 2011 ), endophthalmitis (0.019%-1.6%)(McCannel 2011 ; Scott and Flynn 2007 ) and lens damage. Moreover, repeated intravitreal injections may accelerate lens opacification and increase the incidence of complications in cataract surgery(HahnJiramongkolchai et al. 2016 ; HahnYashkin et al. 2016 ). Compared with the currently widely used anti-VEGF drugs, KW6002 has unique advantages in drug administration: KW6002 can better cross the blood-ocular barrier. Our research group previously demonstrated in the study of retinopathy of prematurity (ROP) that intraperitoneal injection of KW6002 could effectively protect retinal vascular regeneration: this drug selectively inhibit pathological cell proliferation, effectively reduce the area without blood vessels and the formation of new blood vessels, while not affecting the normal physiological development of blood vessels(Zhong et al. 2021 ; Zhou et al. 2018 ), showing similar therapeutic effects to anti-VEGF drugs. Additionally, as an FDA-approved drug, the safety of KW6002 has been fully verified by a large amount of preclinical and clinical data(Chen and Cunha 2020 ). These studies provide important theoretical support for the use of KW6002 in the treatment of ocular diseases. In summary, our findings indicate that adenosine A 2A R antagonists can significantly reduce aldosterone-induced choroidal thickening and vasodilation, maintain BRB integrity by preventing the reduction of TJs protein levels, and decrease the levels of inflammatory cytokines, chemokines, and MMPs. These effects are likely achieved through inhibition of the TNF-α/NF-κB signaling pathway. This study provided a directly evidence that the A 2A R is an effective therapeutic target to prevent CSC development. Limitations of the study This study mainly used the aldosterone-induced model, which was generally accepted as the model representing the acute CSC, but cannot mimic the progressive forms of CSC, such as subretinal fluid accumulation or serous retinal detachment. Thus, further investigations based on improved CSC animal models for elucidating the specific mechanisms underlying CSC are essential. Additionally, our investigation focused mainly on acute CSC models. Further studies are needed to evaluate the efficacy of A 2A R antagonists in chronic and recurrent forms of CSC. Conclusions The results of this study preliminarily confirm the protective effect of the A 2A R antagonist KW6002 on acute CSC, which is achieved by reducing inflammation, maintaining BRB integrity, and simultaneously inhibiting the TNF-α/NF-κB pathway. This study provides a new insight into the treatment of CSC and lays a foundation for future clinical strategies. Abbreviations CSC: Central serous chorioretinopathy, A 2A R: A 2A receptor, AMD: age-related macular degeneration, RPE: retinal pigment epithelium, BRB: blood-retinal barrier, AQP4: aquaporin-4, BBB: blood-brain barrier, CP: choroid plexus, MMP2: Matrix metallopeptidase 2, MMP3: Matrix metallopeptidase 3, MMP9: Matrix metallopeptidase 9, A 2A R -KO: A 2A R knockout, WT: wild-type, OCT: Optical coherence tomography, FFA: Fluorescein fundus angiography, H&E: Hematoxylin-eosin, qPCR: Quantitative real-time PCR, TJs: tight junction, BCVA: best-corrected visual acuity, ROP: retinopathy of prematurity Declarations Acknowledgements Not applicable. Author’s contributions Guarantor of integrity of the entire study: Jiang-fan Chen Study concepts; Qiaoli Liu, Jia Qu, Jiang-fan Chen, Wu Zheng Study design: Qiaoli Liu, Zijun Yu, Wei-hang Tang, Jia-nan Que, Jiaqi Li Data acquisition; Qiaoli Liu, Jiasheng Yang, Kailang Xu Data analysis; Qiaoli Liu Manuscript preparation; Jiang-fan Chen, Wu Zheng Manuscript editing; Jiang-fan Chen Manuscript review. All authors reviewed and approved the final manuscript. Funding This research was funded by the National Natural Science Foundation of China (Grant No. 31800903 and No. 82150710558) and the Natural Science Foundation of Zhejiang province, China (Grant No. LMS25H090008 and No. LQ18H090007). Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The animal experiments conducted in this study received approval from the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou University, China. Consent for publication Not applicable. Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. References Adair TH, Cotten R, Gu JW, Pryor JS, Bennett KR, McMullan MR. McDonnell P and Montani JP. Adenosine infusion increases plasma levels of VEGF in humans. BMC Physiol. 2005;5:10. Aires ID, Boia R, Rodrigues-Neves AC, Madeira MH, Marques C, Ambrósio AF, Santiago AR. Blockade of microglial adenosine A(2A) receptor suppresses elevated pressure-induced inflammation, oxidative stress, and cell death in retinal cells. Glia. 2019;67:896–914. Alkin Z, Osmanbasoglu OA, Ozkaya A, Karatas G, Yazici AT. 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07:57:38","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":188604,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/d17ac7137cf66add4035c513.html"},{"id":100367777,"identity":"eac302e4-3e31-4441-8ada-7452667be5b6","added_by":"auto","created_at":"2026-01-16 07:57:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":599662,"visible":true,"origin":"","legend":"\u003cp\u003eCellular localization of adenosine A\u003csub\u003e2A\u003c/sub\u003e receptor (A\u003csub\u003e2A\u003c/sub\u003eR) in retinal/choroidal vasculature and its dysregulation in the CSC model. (A) Targeted knock-in strategy for generating A\u003csub\u003e2A\u003c/sub\u003eR-Cre; Ai9 reporter mouse line. (B) Retinal immunofluorescence showing A\u003csub\u003e2A\u003c/sub\u003eR-tdTomato reporter (red) colocalization with endothelial marker CD31 (green) and glial cell marker GFAP (green). The merged channel reveals prominent A\u003csub\u003e2A\u003c/sub\u003eR expression within retinal vascular structures, particularly in both the superficial capillary plexus (located in the nerve fiber and ganglion cell layers) and the deep capillary plexus (located in the inner nuclear layer). Scale bar = 100 μm. High-resolution confocal images of choroidal immunofluorescence showing A\u003csub\u003e2A\u003c/sub\u003eR-tdTomato/CD31/DAPI triple labeling, confirming vascular endothelial expression in choroidal capillaries (red arrow) and larger vessels (white arrow). Scale bars = 50 μm. (C) Western blotting analysis of A\u003csub\u003e2A\u003c/sub\u003eR expression in control and aldosterone-treated groups (n\u0026nbsp;=\u0026nbsp;4/group). (D) Quantitation of A\u003csub\u003e2A\u003c/sub\u003eR protein level in panel C (n\u0026nbsp;=\u0026nbsp;4/group). (E) Quantitative RT-PCR analysis of A\u003csub\u003e2A\u003c/sub\u003eR mRNA levels in the \u003cstrong\u003eRPE-choroid-sclera complex \u003c/strong\u003ein control and aldosterone-treated groups (n\u0026nbsp;=\u0026nbsp;4/group). Data are presented as mean\u0026nbsp;±\u0026nbsp;SEM, independent Student's t-test. * \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/8dd9a610679d4fc2dc4cf9af.png"},{"id":100149243,"identity":"3fd595f5-28a5-4429-807e-1bcce92af882","added_by":"auto","created_at":"2026-01-13 13:03:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":899807,"visible":true,"origin":"","legend":"\u003cp\u003eKW6002 attenuates aldosterone‐induced choroidal thickening, vascular abnormalities, and retinal edema in a mouse model of CSC. (A) Schematic timeline of the experimental design. (B) Representative FFA images from control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups acquired following intraperitoneal fluorescein sodium injection. Yellow arrows indicate retinal edema; red arrows highlight vascular tortuosity. (C) Representative fundus images with corresponding OCT scans demonstrating retinal thickness across treatment groups. Green lines indicate measurement positions 500 μm from the optic disc; red line denotes full retinal thickness; yellow arrow indicates inflammatory cells in the vitreous cavity; red arrow indicates structural abnormalities in the retinal pigment epithelium (RPE). Scale bar: 500 μm. (D) Schematic representation of retinal histopathological assessment. (E) Representative hematoxylin-eosin (H\u0026amp;E) stained sections from all experimental groups. The higher-magnification inset below reveals choroidal details, including choroidal veins (red arrow) and capillaries (yellow arrow).. Scale bar: 100 μm. (F) Quantitative OCT analysis of mean retinal thickness across all groups (n = 6/group). (G-J) Quantitative analysis of choroidal and retinal thickness in both central and peripheral regions across all groups based on H\u0026amp;E staining (n = 6/group). Data represent mean ± SEM, analyzed by one ­ way ANOVA with Tukey's multiple comparisons test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/a50d41e6070e4d9363512399.png"},{"id":100149246,"identity":"46cdb27d-cd6a-48d2-b7ba-43005d83bbf8","added_by":"auto","created_at":"2026-01-13 13:03:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":795610,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003e receptor knockout (A\u003csub\u003e2A\u003c/sub\u003eR-KO) attenuates aldosterone-induced retinal and choroidal thickening. (A) Representative FFA images of central and peripheral retinal areas from WT and A\u003csub\u003e2A\u003c/sub\u003eR-KO mice following intraperitoneal fluorescein sodium injection. Yellow arrows indicate retinal edema; red arrows highlight vascular distortion. (B) Representative H\u0026amp;E-stained sections from WT and A\u003csub\u003e2A\u003c/sub\u003eR-KO groups. Scale bar: 100 μm. (C-F) Quantitative analysis of central and peripheral choroidal and retinal thickness in each experimental group (n\u0026nbsp;=\u0026nbsp;5/group). (G) Representative fundus images with corresponding OCT scans demonstrating retinal thickness in WT and A\u003csub\u003e2A\u003c/sub\u003eR-KO mice. Green lines indicate measurement positions 500 μm from the optic disc; red lines denote full retinal thickness. Scale bar: 500 μm. (H) Quantitative OCT analysis of mean retinal thickness (n\u0026nbsp;=\u0026nbsp;5/group). Data represent mean ± SEM, analyzed by an independent Student's t-test. * \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01, ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/f949acfc6d1eb3e70b26f4ce.png"},{"id":100149247,"identity":"f4b33fd3-6bd2-4caa-b862-7987addbbaea","added_by":"auto","created_at":"2026-01-13 13:03:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1039447,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003eR Inhibition suppresses glial cell activation in aldosterone-induced central serous chorioretinopathy. (A) Representative confocal images of retinal sections showing GFAP (red) and AQP4 (green) immunoreactivity in control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Insets show higher magnification of boxed regions. Scale bars: 100 μm. (B-C) Quantification of relative fluorescence intensity of GFAP and APQ4 (n\u0026nbsp;=\u0026nbsp;5/group). (D) Representative confocal images of retinal sections showing IBA1-positive microglia/macrophages (red) in control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Insets show higher magnification of boxed regions. Scale bars: 100 μm. (E) Quantification of IBA1\u003csup\u003e+\u003c/sup\u003e cell number per field (n\u0026nbsp;=5/group). (F-H) Relative mRNA expression of TNF-α, IL-6, and IL-1β in control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Data represented as mean ± SEM. Statistical analysis: one ­ way ANOVA with Tukey's multiple comparisons test of independent Student's t-test. * \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001, ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/1849137c40305c3ae8c888d8.png"},{"id":100367790,"identity":"9c7e48de-3415-466a-bbfa-09dec29aa30d","added_by":"auto","created_at":"2026-01-16 07:57:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":776150,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003eR inhibition protects against blood-retinal barrier disruption in the CSC model. (A) Confocal images of retinal frozen sections showing RPE65 immunostaining (green) in mice from control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Scale bar: 100 μm. (B) Frozen sections of mouse eyes immunostained for ZO-1 (red) from control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Scale bar: 50 μm. (C) Representative CD31 immunostaining (red) in mouse eyes from control, aldosterone, aldosterone + vehicle, and aldosterone + KW6002 groups. Scale bar: 50 μm. (D-F) Quantification of relative fluorescence intensity of RPE65, ZO-1, and CD31 (n\u0026nbsp;=\u0026nbsp;5/group). (G) Quantitative analysis of RPE65 expression relative to β-actin. (H) Western blotting analysis of RPE65 expression across the four experimental groups with β-actin as a loading control. (I-P) Western blot analysis and quantitative analysis of ZO-1, Occludin, Claudin-1, and Claudin-5 expression between WT and A\u003csub\u003e2A\u003c/sub\u003eR-KO groups with β-actin as loading control (n\u0026nbsp;=\u0026nbsp;5/group). Data are presented as mean\u0026nbsp;±\u0026nbsp;SEM. Statistical analysis: one ­ way ANOVA with Tukey's multiple comparisons test or independent Student's t-test. * \u003cem\u003ep \u003c/em\u003e\u0026lt;\u0026nbsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, **** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001; ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/8bc1629465a086c50f075091.png"},{"id":100368692,"identity":"0ecb43ab-c114-463d-9d2c-3422e7ef213d","added_by":"auto","created_at":"2026-01-16 07:58:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":760756,"visible":true,"origin":"","legend":"\u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003eR inactivation suppresses inflammatory signaling pathway activation in the CSC model. (A) Western blotting analysis of TNF-α, NF-κB p65, phosphorylated NF-κB p65 (p-p65), MMP‐2, MMP‐3, and MMP‐9 expression WT and A\u003csub\u003e2A\u003c/sub\u003eR-KO groups. β-actin or GAPDH served as the loading control. (B-D) Quantification of TNF-α, NF-κB p65, and phosphorylated NF-κB p65 protein levels relative to β-actin (n\u0026nbsp;=\u0026nbsp;5/group). (E) Quantification of the p‐p65/p65 ratio (n\u0026nbsp;=\u0026nbsp;5/group). (F-H) Quantification of MMP‐2 and MMP‐9 protein levels relative to β-actin, and MMP‐3 relative to GAPDH (n\u0026nbsp;=\u0026nbsp;5/group). Data are represented as mean\u0026nbsp;±\u0026nbsp;SEM. Statistical analysis: independent Student's t-test. * \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01; ns: not significant.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/da3cd28f2ed6b82d459dbe78.png"},{"id":100367251,"identity":"20927214-ed82-4f3b-95c9-658c032f454d","added_by":"auto","created_at":"2026-01-16 07:56:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":985913,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic schematic illustrating how A\u003csub\u003e2A\u003c/sub\u003eR inhibition prevents central serous chorioretinopathy by attenuating retinal inflammation and restoring blood-retinal barrier integrity.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/c82ec73d864424c56e453b86.png"},{"id":106994051,"identity":"b754ab9c-4ce0-4247-9f08-a4b251ddaca2","added_by":"auto","created_at":"2026-04-15 15:03:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7008649,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/54102279-d07e-4563-b8f1-8db96c52641c.pdf"},{"id":100149276,"identity":"a87dee8f-e235-46d7-9e0f-4e3638a15ffa","added_by":"auto","created_at":"2026-01-13 13:03:08","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32659330,"visible":true,"origin":"","legend":"","description":"","filename":"WB.zip","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/2d3411f09b32ef0e55404b0c.zip"},{"id":100367808,"identity":"7ccc0cd3-a4f9-40da-8848-9c6191cb90f8","added_by":"auto","created_at":"2026-01-16 07:57:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17181,"visible":true,"origin":"","legend":"","description":"","filename":"TABLE1PrimaryantibodiesareusedforimmunofluorescenceandWesternblotting.docx","url":"https://assets-eu.researchsquare.com/files/rs-8526598/v1/d027f27becf96a7829b59b25.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eThe Adenosine A\u003csub\u003e2A\u003c/sub\u003e Receptor Antagonist KW6002 Mitigates Aldosterone-induced Central Serous Chorioretinopathy in Mice\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCentral serous chorioretinopathy (CSC) ranks among the most prevalent vision-threatening retinal disorders, after age-related macular degeneration (AMD), diabetic retinopathy, and retinal branch vein occlusion(Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Furthermore, it shows great gender preference, as studies report more cases (approximately 10 cases per 100,000 people) happening in men compared to merely 2 cases per 100,000 in women(Kitzmann et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The clinical hallmark of CSC is neurosensory retinal detachment resulting from the accumulation of serous subretinal fluid, which stems from retinal pigment epithelium (RPE) dysfunction and increased choroidal permeability and thickness(Daruich et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Imamura et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yanagi \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although retinal detachment resolves spontaneously in most CSC patients, 30%-50% develop chronic or recurrent disease, leading to permanent retinal tissue damage and vision loss(Gemenetzi et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nicholson et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Salehi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Given the vision-threatening nature of CSC, early and effective intervention is critical. Current treatment strategies for CSC including intravitreal anti-vascular endothelial growth factor (anti-VEGF) injection, laser photocoagulation, and photodynamic therapy (PDT), are all associated with distinct clinical limitations. The first-line treatment, half-dose PDT, is constrained by the high cost of the photosensitizer verteporfin, reliance on specialized equipment and trained personnel, and a procedural complexity greater than that of intravitreal injection(Bae et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Anti-VEGF therapy, on the other hand, presents challenges such as stringent sterile requirements, potential postoperative complications, and the burden of repeated injections(Meyer et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the development of safer, more convenient, and more effective treatment strategies remains an unmet clinical needed.\u003c/p\u003e \u003cp\u003eMounting evidence indicates that choroidal vascular abnormalities \u0026ndash; including pathological dilation, hyperpermeability, and disruption of the RPE tight junctions and blood-retinal barrier (BRB) \u0026ndash; play a pivotal role in the pathogenesis of CSC(Guyer et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Imamura et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Corticosteroids are a leading cause of CSC via the overactivation of mineralocorticoid receptors in the choroid(Bousquet et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Corticosteroids bind to the mineralocorticoid receptors, leading to up-regulation of calcium-activated potassium channels in choroidal endothelial cells, causing smooth muscle relaxation, and subsequently, choroidal hyperpermeability. Corticosteroids also disrupt the RPE barrier function, cause dysregulation of choroidal hemodynamics, and interfere with ion transport via upregulation of adrenergic receptors. Aldosterone induces a mineralocorticoid/glucocorticoid receptor-dependent upregulation of epithelial sodium channel-α, potassium channels, and aquaporin-4 (AQP4) in retinal M\u0026uuml;ller cells, culminating in retinal swelling. Given the accumulating evidence implicating corticosteroids and mineralocorticoid receptors in disease pathogenesis, intravitreal aldosterone administration recapitulates key CSC pathological features, including choroidal vasodilation, vascular leakage, RPE dysfunction, and choroidal thickening, thereby establishing a mechanistic model of CSC development(Canonica et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExtracellular adenosine signals through G protein-coupled receptors (A\u003csub\u003e1\u003c/sub\u003e, A\u003csub\u003e2A\u003c/sub\u003e, A\u003csub\u003e2B\u003c/sub\u003e, and A\u003csub\u003e3\u003c/sub\u003e), serving a key regulatory function in multiple tissues, including the retina and choroid, in both normal and diseased conditions(Adair et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Fredholm et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Immunoreactivity for the adenosine A\u003csub\u003e2A\u003c/sub\u003e receptor (A\u003csub\u003e2A\u003c/sub\u003eR), has been detected in endothelial progenitor cells, vascular progenitor cells, and endothelial cells within the retina/choroid(Lutty and McLeod \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Our previous studies demonstrate that adenosine A\u003csub\u003e2A\u003c/sub\u003eR antagonists significantly attenuate retinal inflammatory responses and suppress neovascularization during the hyperoxic phase in the oxygen-induced retinopathy model(Zhong et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, A\u003csub\u003e2A\u003c/sub\u003eR antagonists can inhibit choroidal neovascularization by reducing inflammatory mediator levels and suppressing angiogenic activity(Sorenson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Beyond their critical roles in mitigating inflammation and blocking pathological angiogenesis, A\u003csub\u003e2A\u003c/sub\u003eR also play pivotal roles in regulating the blood-brain barrier (BBB). Specifically, activation of the A\u003csub\u003e2A\u003c/sub\u003eR downregulates the expression of tight junction and adherent junction molecules in human and murine brain microvascular endothelial cells, thereby increasing BBB permeability. Conversely, endothelial cell-specific knockout of A\u003csub\u003e2A\u003c/sub\u003eRs effectively reduces BBB leakage and markedly suppresses immune cell infiltration across the BBB(Fernandez et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kim and Bynoe \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Likewise, A\u003csub\u003e2A\u003c/sub\u003eR antagonists can normalize the BBB permeability in insulin-resistant mice(Yamamoto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, our recent study reveals that the selective A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 increases the blood-cerebrospinal fluid barrier integrity to reduce T-cell migration across the choroid plexus (CP), consequently reducing pathological damage in experimental autoimmune encephalomyelitis(Zheng et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These collective findings led us to propose that targeting the adenosine A\u003csub\u003e2A\u003c/sub\u003eR signaling pathway may offer novel therapeutic strategies for disorders associated with central nervous system barrier dysfunction.\u003c/p\u003e \u003cp\u003eThis study sought to investigate the protective role of A\u003csub\u003e2A\u003c/sub\u003eR antagonism in an aldosterone-induced CSC model and elucidate its underlying molecular mechanisms. We demonstrated that the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 attenuates aldosterone-induced choroidal thickening and vasodilation, suppresses macrophage/microglia infiltration and inflammatory cytokine production, and preserves BRB integrity. Furthermore, A\u003csub\u003e2A\u003c/sub\u003eR knockout (A\u003csub\u003e2A\u003c/sub\u003eR-KO) preserved blood-retinal barrier integrity by suppressing Matrix Metallopeptidase (MMP)-2/3/9 expression and maintaining tight junction proteins (ZO-1, Occludin, Claudin-1, and Claudin-5). The A\u003csub\u003e2A\u003c/sub\u003eR-KO-mediated preservation of blood-retinal barrier integrity was associated with inhibition of the TNF-α/NF-κB pathway. Collectively, these findings support that targeting the A\u003csub\u003e2A\u003c/sub\u003eR offers a novel therapeutic strategy for treating CSC.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eNumerous neuroscience studies, including those from our laboratory and other research teams, have extensively characterized A\u003csub\u003e2A\u003c/sub\u003eR-KO mice. Through crossbreeding of heterozygous A\u003csub\u003e2A\u003c/sub\u003eR-KO mice (A\u003csub\u003e2A\u003c/sub\u003eR-KO\u003csup\u003e+/\u0026minus;\u003c/sup\u003e), we generated homozygous A\u003csub\u003e2A\u003c/sub\u003eR-KO (A\u003csub\u003e2A\u003c/sub\u003eR-KO\u003csup\u003e+/+\u003c/sup\u003e), A\u003csub\u003e2A\u003c/sub\u003eR-KO\u003csup\u003e+/\u0026minus;\u003c/sup\u003e, and wild-type littermate controls (WT). All mice originated from an identical breeding lineage. Genotypic determination was executed via PCR analysis of genomic DNA extracted from tail biopsies, utilizing three primer sets targeting the Neo-AR cassette and adjacent A\u003csub\u003e2A\u003c/sub\u003eR gene, as previously described(Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA\u003csub\u003e2A\u003c/sub\u003eR-Trp Tomato transgenic mice were generated by crossing A\u003csub\u003e2A\u003c/sub\u003eR-Trp Cre mice (MMRRC, Stock Number: 031168-UCD) and Tomato reporter mice (B6;129 S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, Jackson Laboratory). In these A\u003csub\u003e2A\u003c/sub\u003eR-Trp Tomato mice, Cre recombinase expression mediates the specific excision of the loxP-flanked STOP cassette in Tomato reporter mice, thereby activating tdTomato fluorescent protein expression(Zhong et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To characterize the spatial distribution of A\u003csub\u003e2A\u003c/sub\u003eR in the retina, we performed dual immunofluorescence colocalization assays combining endogenous tdTomato fluorescence with cell type-specific retinal markers.\u003c/p\u003e \u003cp\u003eThe C57BL/6J male mice aged eight weeks were purchased from Beijing Vital River Laboratory (Beijing, China). Experimental procedures were performed exclusively on the right eye of each mouse under a randomized design, with a group size of 5\u0026ndash;6 animals. To control for potential litter effects, the A\u003csub\u003e2A\u003c/sub\u003eR-KO and WT mice used in this study were sourced from 2\u0026ndash;3 independent breeding litters. All animal procedures were conducted following protocols approved by the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou Medical University, China. Mice were housed 4\u0026ndash;5 per cage under controlled environmental conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;2% relative humidity) with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Bedding material was changed every 5 days, and fresh bedding was provided immediately after surgery.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCSC induction and drug administration\u003c/h3\u003e\n\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e administration of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 (GC11590, GLPBIO, Montclair, USA), the compound was freshly prepared before each administration by dissolving in a vehicle containing 15% dimethyl sulfoxide, 15% castor oil, and 70% physiological saline to achieve a final concentration of 5 mg/ml. Mice received daily intraperitoneal injections of KW6002 (5 mg/kg) or an equivalent volume of vehicle for 7 consecutive days. During model induction, animals were anesthetized via inhalation of 5% isoflurane (RWD Life Science, Shenzhen, China) and maintained under anesthesia with 1% isoflurane throughout the procedure. Pupils were dilated using tropicamide eye drops. Previous studies have indicated that approximately 10% of intravitreally administered glucocorticoids reach the retina(Chang-Lin et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To ensure delivery of at least 10 nM aldosterone to the apical side of retinal pigment epithelial cells, an acute central serous chorioretinopathy phenotype was induced by intravitreal injection of 2 mM aldosterone (GC41390, GLPBIO, Montclair, USA) at a volume of 2 \u0026micro;L per eye using a Hamilton microsyringe (Hamilton Company, Reno, USA). Mice exhibiting intraoperative complications (e.g., vitreous hemorrhage, retinal detachment) were excluded. Twenty-four hours after modeling, mice received a final administration and were subsequently sacrificed for further experimentation. Experimental procedures were performed exclusively on the right eye of each mouse under a randomized design, with a group size of 5\u0026ndash;6 animals.\u003c/p\u003e\n\u003ch3\u003eOptical coherence tomography (OCT)\u003c/h3\u003e\n\u003cp\u003eTo evaluate the impact of aldosterone intravitreal injection on retinal thickness, we acquired images using spectral domain optical coherence tomography (Heidelberg Engineering, Dossenheim, Germany), a non-invasive method for visualizing the microstructure of live animal retinas, before and 24 hours after aldosterone administration. Prior to imaging, mice were anesthetized, and topical mydriatic eye drops were applied to induce pupil dilation. The eye-tracking function was employed to conduct continuous dynamic imaging along a 30\u0026deg; horizontal line, focusing on the optic disc region. The scan parameters were adjusted to achieve optimal signal intensity and contrast. Following imaging, the anesthesia mask was promptly removed, the mice were placed on a heating pad to maintain body temperature, and ophthalmic ointment was applied to both eyes to protect the corneas. Retinal thickness was measured perpendicular to the RPE layer at 500 \u0026micro;m from the optic disc in all obtained images using ImageJ software (version 1.47; NIH, MD), and average values were calculated.\u003c/p\u003e\n\u003ch3\u003eFluorescein fundus angiography (FFA)\u003c/h3\u003e\n\u003cp\u003eTo analyze fundus changes and retinal vascular morphological alterations in mice after model establishment, fluorescein fundus angiography (FFA) was performed prior to tissue collection. FFA imaging was performed using a rodent-specific fundus camera system (Eyemera FUNDUS, IIscience, Korea) with a spatial resolution of \u0026lt;\u0026thinsp;3 \u0026micro;m for mouse imaging. After anesthesia, tropicamide eye drops were administered to induce pupil dilation. Subsequently, 4% sodium fluorescein solution was administered intraperitoneally at a dose of 0.05 mL/20 g body weight. After a 4‑minute waiting period, the mouse was positioned in a prone position on a specialized imaging stage. Ofloxacin ophthalmic ointment was applied to the corneal surface as a contact medium. The integrated blue LED light source was adjusted to an appropriate intensity, and the objective lens was gently brought into contact with the cornea through the ointment medium before image acquisition. Post-procedure, mice were maintained on a heating pad, and ophthalmic ointment was applied to both eyes for corneal protection.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining\u003c/h3\u003e\n\u003cp\u003eFor the globe frozen section, eyes were fixed in 4% PFA for 2 hours following PBS perfusion. Under a stereomicroscope, connective tissues were carefully trimmed, and the cornea, lens, and iris were removed to isolate the intact retina. Subsequently, retinas were dehydrated in a sucrose gradient and embedded in an optimal temperature compound. Specimens were sectioned at 14 \u0026micro;m thickness.\u003c/p\u003e \u003cp\u003eMouse retinal cryosections were processed for immunofluorescence labeling. Sections were washed three times with PBS and incubated for 2 hours in PBS containing 0.3% Triton X-100 and 0.5% BSA. The sections were then incubated overnight at 4 ℃ with the following primary antibodies: goat anti-CD31 (1:200, AF3628, R\u0026amp;D Systems, China), rabbit anti-IBA1 (1:400, 019-19741, Wako, Tokyo, Japan), rabbit anti-AQP4 (1:400, MABN2527, Sigma, Missouri, USA), mouse anti-GFAP (1:400, G3893, Sigma, Missouri, USA), rat anti-CD68 (1:200, MCA1957GA, Bio-Rad, California, USA), rabbit anti-RPE65 (1:200, 83861-1-RR, Proteintech, Illinois, USA), and rabbit anti-ZO-1 (1:200, 21773-1-AP, Proteintech, Illinois, USA). After washing with PBS (3 \u0026times; 10 minutes), sections were incubated with secondary antibodies (donkey anti-goat Fluor 488, donkey anti-rabbit Fluor 488, donkey anti-goat Fluor IgG 594, or donkey anti-rat Fluor 594; all 1:1000, Invitrogen, California, USA) at room temperature for 2 hours to visualize the primary antibody signals.\u003c/p\u003e \u003cp\u003eFollowing secondary antibody incubation, retinal sections were washed with PBS and counterstained with DAPI (1:1500, Beyotime, Shanghai, China) for 8 minutes. Images were acquired at 20\u0026times; or 40\u0026times; magnification using a confocal laser scanning microscope (LSM900, Carl Zeiss).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHematoxylin-eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eFor quantitative assessment of retinal and choroidal thickness variations, ocular tissues were processed for histological analysis. At 24 hours post-intravitreal aldosterone administration, mice were euthanized by intraperitoneal injection of pentobarbital sodium (2.5 mg/10 g body weight). Eyes were immediately enucleated and fixed in 4% PFA for 24 hours, followed by standard paraffin embedding. Serial sections (3 \u0026micro;m thickness) were cut and stained with hematoxylin and eosin (H\u0026amp;E) for morphological examination. Digital images of retinal sections were acquired and analyzed using ImageJ software (NIH, USA). Thickness measurements were performed at two standardized locations: (1) central retina (500 \u0026micro;m from the optic nerve head), and (2) peripheral retina (1 mm from the trabecular meshwork). Both retinal and choroidal thicknesses were measured and compared across experimental groups.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative real-time PCR (qPCR)\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from RPE-choroid-sclera complexes and retinal tissues using TRIZOL Reagent (Cat. No. 15596026, Thermo Fisher Scientific) according to the manufacturer's protocol. RNA concentration and purity were determined using a spectrophotometer (Beckman, LA, USA), with samples having an A260/A280 ratio between 1.8 and 2.0 considered acceptable. For cDNA synthesis, 1000 ng of total RNA was reverse-transcribed using the Vazyme cDNA Synthesis Kit (R323-01, Vazyme, Jiangsu, China), followed by appropriate dilution and storage at -20 ℃.\u003c/p\u003e \u003cp\u003eQuantitative real-time PCR (qPCR) was performed on a CFX 96 real-time PCR system (Bio-Rad) using SYBR-Green Premix Ex Taq (Q712-03, Vazyme, Jiangsu, China) with technical triplicates for each sample. The following primers were used: PPIA (forward: 5\u0026prime;-AGC ATA CAG GTC CTG GCA TCT TGT \u0026minus;\u0026thinsp;3\u0026prime;, reverse: 5\u0026prime;-CAA AGA CCA CAT GCT TGC CAT CCA \u0026minus;\u0026thinsp;3\u0026prime;); A\u003csub\u003e2A\u003c/sub\u003eR (forward: 5\u0026prime;-GCC ATC CCA TTC GCC ATC A\u0026prime;, reverse: 5\u0026prime;-GCA ATA GCC AAG AGG CTG AAG A -3\u0026prime;); IL-6 (forward: 5\u0026prime;-TCT GAA GGA CTC TGG CTT TG-3\u0026prime;, reverse: 5\u0026prime;-GAT GGA TGC TAC CAA ACT GGA-3\u0026prime;); IL-1β (forward: 5\u0026prime;-CCA AGC AAC GAC AAA ATA CC-3\u0026prime;, reverse: 5\u0026prime;-GTT GAA GAC AAA CCG TTT TTC C-3\u0026prime;); TNF‐α (forward: 5\u0026prime;-AAC TCG AGT GAC AAG CCC GTA G-3\u0026prime;, reverse: 5\u0026prime;-GTA CCA CCA GTT GGT TGT CTT TGA-3\u0026prime;). Relative gene expression was calculated using the 2 \u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method with PPIA serving as the internal reference gene.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eRetinal tissue or RPE-choroid-sclera complex was homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (B14001 \u0026amp; B15001, Bimake, Texas, USA). The lysate was centrifuged at 10,000 rpm for 10 minutes, and protein concentration in the supernatant was determined using a BCA protein assay kit (P0010, Beyotime, Shanghai, China). Samples were denatured at 95 ℃ for 10 minutes in 5\u0026times; SDS loading buffer (P0015, Beyotime, Shanghai, China) and separated by SDS-PAGE. Proteins were transferred to 0.45 \u0026micro;m PVDF membranes (BS-PVDF-45-S, Biosharp, Hefei Ulife Biotechnology Co., Ltd.) in ice-cold transfer buffer at a constant current of 350 mA for 60 minutes.\u003c/p\u003e \u003cp\u003eMembrane were blocked with 5% non-fat milk at room temperature for 2 hours and then incubated overnight at 4 ℃ with the following primary antibodies: β-actin (1:1000, 66009-1-Ig, Proteintech, Illinois, USA), ZO-1 (1:5000, 21773-1-AP, Proteintech, Illinois, USA), GAPDH (1:1000, GB15004, Servicebio, Hubei, China), A\u003csub\u003e2A\u003c/sub\u003eR (1:200, FI-A2A-Go-Af700, Frontier Institute, Lausanne, Switzerland), Claudin-1(1:1000, 51-9000, Invitrogen, California, USA), Occludin (1:1000, 71-1500, Invitrogen, California, USA), Claudin-5 (1:1000, YP-Ab-16961, UpingBio, Shanghai, China), TNF-α (1:1000, 17590-1-AP, Proteintech, Illinois, USA), NF-κB p65 (1:1000, ET1603-12, Huabio Co., Ltd.), phosphorylated NF-κB p65 (1:1000; Cell Signal Technology, catalogue no. 3033), MMP2 (1:1000, YP-mAb-02682, UpingBio, Shanghai, China), MMP3 (1:1000, YP-Ab-02788, UpingBio, Shanghai, China), and MMP9 (1:1000, YP-Ab-02639, UpingBio, Shanghai, China). See Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for antibody details.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary antibodies are used for immunofluorescence and Western blotting\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFollowing primary antibody incubation, membranes were washed and incubated with HRP-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Massachusetts, USA) for 2 hours at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence and detected with a Bio-Rad ChemiDoc XRS\u0026thinsp;+\u0026thinsp;imaging system. The band intensities were analyzed with ImageJ software (version 1.47; NIH, MD) and were normalized to β-actin or GAPDH. Relative protein expression was calculated with the control or WT group set to 1. Experiments were independently repeated at least three times.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical comparisons between two groups were performed using an independent Student's t-test, while comparisons among three or more groups were conducted using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). Values of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eA\u003csub\u003e2A\u003c/sub\u003eR signaling is upregulated in the RPE-choroid-sclera complex of the CSC model\u003c/h2\u003e \u003cp\u003eInitial characterization of A\u003csub\u003e2A\u003c/sub\u003eR expression was performed using A\u003csub\u003e2A\u003c/sub\u003eR-tdTomato reporter mice, enabling cell type-specific identification of A\u003csub\u003e2A\u003c/sub\u003eR-positive cells through tdTomato fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunohistochemical analysis revealed distinct co-localization of A\u003csub\u003e2A\u003c/sub\u003eR -tdTomato labeling with the two retinal vascular plexuses\u0026mdash;the superficial capillary plexus (located in the nerve fiber and ganglion cell layers) and the deep capillary plexus (located in the inner nuclear layer)\u0026mdash;as well as with the choroidal vasculature (including capillaries and larger vessels) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating robust A\u003csub\u003e2A\u003c/sub\u003eR expression in ocular vascular networks, and the A\u003csub\u003e2A\u003c/sub\u003eR -tdTomato labeling is partially co-localized with glial cells. Then, we performed a western blotting and found the protein level of A\u003csub\u003e2A\u003c/sub\u003eR was increased in the RPE-choroid-sclera complex 24 hours following CSC induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-D, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, qPCR revealed significant upregulation of A\u003csub\u003e2A\u003c/sub\u003eR mRNA level in the RPE-choroid-sclera complex 24 hours after CSC induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePharmacological blockade of A\u003csub\u003e2A\u003c/sub\u003eR attenuates aldosterone-induced retinal and choroidal thickening\u003c/h2\u003e \u003cp\u003eTo investigate the therapeutic potential of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 in CSC, we established a CSC model using intravitreal aldosterone injection. Mice received daily intraperitoneal KW6002 (5 mg/kg) or vehicle for seven days prior to CSC modeling. \u003cem\u003eIn vivo\u003c/em\u003e morphological assessment was performed 24 hours post-aldosterone injection, and mice were sacrificed after a final dose for subsequent histological analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eFundus photography revealed that aldosterone administration induced irregular retinal swelling (yellow arrows), which was significantly attenuated by KW6002 pretreatment. FFA demonstrated pronounced vascular tortuosity in aldosterone-treated retinas compared to controls (red arrows), while KW6002 pretreatment substantially improved vascular morphology, indicating a protective effect against aldosterone-induced retinal edema and vascular abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAt 500 \u0026micro;m from the optic disc, OCT measurements indicated significantly greater retinal thickness in the aldosterone group relative to controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), along with marked intraocular inflammation (yellow arrow). Disruption of the RPE layer was also observed (red arrow). KW6002 administration substantially reduced peri-papillary inflammation and demonstrated a trend toward normalization of retinal thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, F).\u003c/p\u003e \u003cp\u003eHistological examination with H\u0026amp;E staining assessed retinal and choroidal thickness, with central measurements taken 500 \u0026micro;m from the optic disc and peripheral measurements obtained 1000 \u0026micro;m from the ciliary body (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Compared to controls, aldosterone administration significantly increased central retinal thickness (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while KW6002 treatment could ameliorate the central retinal thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Peripheral retinal thickness remained consistent across all experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), corroborating the OCT findings.\u003c/p\u003e \u003cp\u003eNotably, aldosterone induced significant thickening of the central choroid (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 versus control), which was effectively prevented by KW6002 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Peripheral choroidal thickness exhibited minimal variations among groups without reaching statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These findings demonstrate that KW6002 effectively prevents aldosterone-induced thickening in both retinal and choroidal tissues, particularly in central regions, while simultaneously ameliorating vascular tortuosity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGenetic deletion of A\u003csub\u003e2A\u003c/sub\u003eR reduces retinal and choroidal thickening in the CSC model\u003c/h2\u003e \u003cp\u003eTo further validate the protective role of A\u003csub\u003e2A\u003c/sub\u003eR in CSC pathogenesis, we conducted comparative studies using A\u003csub\u003e2A\u003c/sub\u003eR-KO mice and their wild-type littermates. Fundoscopic analysis demonstrated that A\u003csub\u003e2A\u003c/sub\u003eR-KO mice exhibited significantly attenuated retinal elevation (yellow arrows) and reduced vascular tortuosity (red arrows) compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Quantitative analysis of H\u0026amp;E-stained sections revealed that A\u003csub\u003e2A\u003c/sub\u003eR-KO mice had significantly thinner central retina (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and central choroid (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), relative to WT, with a non-significant trend toward reduced peripheral retinal or choroidal thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Quantitative OCT measurements confirmed these findings, revealing a marked reduction in retinal thickness in A\u003csub\u003e2A\u003c/sub\u003eR-KO mice relative to WT animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results provide compelling evidence that genetic ablation of A\u003csub\u003e2A\u003c/sub\u003eR confers protection against aldosterone-induced retinal edema and vascular abnormalities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eA\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 attenuates retinal inflammation and M\u0026uuml;ller cell activation in the CSC model\u003c/h2\u003e \u003cp\u003eAs aldosterone could upregulate ion and water channels in M\u0026uuml;ller glial cells to promote retinal edema (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), we investigated the effects of A\u003csub\u003e2A\u003c/sub\u003eR antagonism on glial cell activation. Immunofluorescence staining of M\u0026uuml;ller cells (GFAP\u003csup\u003e+\u003c/sup\u003e) and AQP4 in retinal tissues revealed that aldosterone administration significantly increased GFAP fluorescence intensity compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating M\u0026uuml;ller cell activation, along with elevated AQP4 expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Prophylactic treatment with KW6002 effectively attenuated these pathological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eGiven that aldosterone induces intraocular inflammation, as evidenced by inflammatory cell infiltration and posterior segment alterations on OCT imaging, we decided to investigate the aldosterone-induced microglia/macrophage activation through staining with IBA1. The aldosterone group exhibited a marked increase in IBA1\u003csup\u003e+\u003c/sup\u003e cells compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while KW6002 pretreatment significantly reduced this inflammatory response (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E), demonstrating its ability to mitigate aldosterone-induced microglial/macrophage infiltration. In addition, qPCR analysis of retinal tissues showed that mRNA levels of proinflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly upregulated in the Aldo-induced model group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following the KW6002 intervention, these cytokine expressions were significantly reduced relative to the Aldo model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-H, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, these findings demonstrate that A\u003csub\u003e2A\u003c/sub\u003eR antagonism effectively ameliorates both aldosterone-induced retinal inflammation and M\u0026uuml;ller cell activation in this CSC model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eA\u003csub\u003e2A\u003c/sub\u003eR inhibition preserves BRB integrity by preventing aldosterone-induced tight junction disruption in the RPE-choroid-sclera complex\u003c/h2\u003e \u003cp\u003eTo elucidate the protective mechanisms of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 on BRB integrity, we systematically evaluated the expression and distribution of the tight junction (TJs) protein ZO-1 and RPE marker RPE65 using immunofluorescence staining and western blot analysis. Our investigations revealed three key findings: First, immunofluorescence analysis demonstrated that 24hours aldosterone treatment significantly disrupted the continuous distribution of RPE65 and ZO-1 signals (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), with evident TJs structural damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, D, E). These morphological alterations strongly indicate aldosterone-induced BRB dysfunction. Notably, KW6002 pretreatment markedly attenuated these pathological changes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). Further confirmation via immunostaining for CD31, a specific marker of vascular endothelial cells, demonstrated that KW6002 could partially reverse aldosterone-induced vascular endothelial cell damage (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, F, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Second, in the A\u003csub\u003e2A\u003c/sub\u003eR-KO mouse model, we observed significantly higher protein levels of ZO-1, Claudin-1, and Claudin-5 in the RPE-choroid-sclera complex of aldosterone-treated A\u003csub\u003e2A\u003c/sub\u003eR-KO mice compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J, M, N, O, P, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). While Occludin expression showed an increasing trend in A\u003csub\u003e2A\u003c/sub\u003eR-KO mice, this difference did not reach statistical significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK, L, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Collectively, our results demonstrate that KW6002 protects BRB structural and functional integrity through A\u003csub\u003e2A\u003c/sub\u003eR signaling inhibition, primarily by maintaining TJs protein stability. This protective mechanism is evident at both pharmacological (KW6002 treatment) and genetic (A\u003csub\u003e2A\u003c/sub\u003eR-KO) levels, providing compelling evidence for A\u003csub\u003e2A\u003c/sub\u003eR inhibition as a therapeutic strategy for BRB protection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eA\u003csub\u003e2A\u003c/sub\u003eR inactivation suppresses TNF-α/NF-κB-MMP2/9-mediated tight junction disruption in the CSC model\u003c/h2\u003e \u003cp\u003ePrevious studies have demonstrated that RNA sequencing analysis of the RPE-choroid-sclera complex in acute CSC rat models revealed upregulation of the TNF-α/NF-κB signaling pathway, which mediates inflammatory responses(Canonica et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To further elucidate the molecular mechanisms underlying the protective effects of A\u003csub\u003e2A\u003c/sub\u003eR inhibition on aldosterone-induced choroidal thickening, vasodilation, and BRB integrity disruption, we performed western blotting analysis to examine the expression of proteins in the TNF-α/NF-κB signaling pathway in A\u003csub\u003e2A\u003c/sub\u003eR-KO mice within the CSC model. Compared to WT, A\u003csub\u003e2A\u003c/sub\u003eR-KO mice exhibited significantly reduced expression of TNF-α and phosphorylated NF-κB p65 (p-NF-κB p65) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B, D, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). Although total NF-κB p65 expression showed no statistically significant differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), the p-p65/p65 ratio was significantly lower in the A\u003csub\u003e2A\u003c/sub\u003eR-KO group compared to WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These results demonstrate that A\u003csub\u003e2A\u003c/sub\u003eR inhibition effectively suppresses TNF-α/NF-κB signaling pathway in the CSC model.\u003c/p\u003e \u003cp\u003eTranscriptomic analyses of both acute and chronic CSC models revealed consistent upregulation of MMP3 and MMP9(Canonica et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on the above data, we investigated the expression profiles of MMP2, MMP3, and MMP9 \u0026ndash; key effector molecules implicated in BRB disruption. Our results showed significantly reduced expression of these molecules in the A\u003csub\u003e2A\u003c/sub\u003eR-KO group compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, F-H, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Collectively, these findings suggest that A\u003csub\u003e2A\u003c/sub\u003eR inhibition exerts protective effects by modulating the TNF-α/NF-κB-MMP2/3/9 signaling pathway, thereby alleviating inflammation and preserving RPE-choroid-sclera complex barrier integrity in the CSC model.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eOur findings identify the A\u003csub\u003e2A\u003c/sub\u003eR as a promising therapeutic target for CSC, supported by the following key evidences: (1) The A\u003csub\u003e2A\u003c/sub\u003eR was expressed in both choroidal and retinal vascular endothelial cells, with selective upregulation in the RPE complex of the aldosterone-induced CSC model; (2) Treatment with the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 and genetic knockout of A\u003csub\u003e2A\u003c/sub\u003eR effectively reversed aldosterone-induced pathological features of CSC; (3) KW6002 treatment suppressed aldosterone-induced activation of retinal glial cells and retinal inflammatory responses; (4) A\u003csub\u003e2A\u003c/sub\u003eR deletion preserves BRB integrity by preventing downregulation of TJs proteins; (5) Mechanistically, A\u003csub\u003e2A\u003c/sub\u003eR contributes to the disruption of both choroidal vascular endothelial and RPE barriers in CSC via the TNF-α/NF-κB-MMP2/9 signaling axis. Collectively, these results highlight the critical role of the A\u003csub\u003e2A\u003c/sub\u003eR in CSC pathogenesis and provide experimental support for its potential as a therapeutic target. All experimental results are schematically summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eA\u003csub\u003e2A\u003c/sub\u003eR signaling as a critical mediator of barrier integrity in central serous chorioretinopathy\u003c/h2\u003e \u003cp\u003eThe integrity of both the choroidal vascular system and RPE serves as a critical defensive mechanism against the pathological progression of CSC(Kaye et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the present study, we observed a significant increase in adenosine A\u003csub\u003e2A\u003c/sub\u003eR expression within the RPE-choroid-sclera complex under CSC pathological conditions, indicating that the aberrant adenosine A\u003csub\u003e2A\u003c/sub\u003eR signaling may be a valid therapeutic target. Indeed, we found that A\u003csub\u003e2A\u003c/sub\u003eR-KO attenuated aldosterone-induced choroidal thickening and vasodilation, and increased the choroidal-retinal barrier integrity by preventing the reduction of TJs protein levels. This finding aligns with our finding that knockout of A\u003csub\u003e2A\u003c/sub\u003eR in the choroid plexus epithelium delayed experimental autoimmune encephalomyelitis onset and alleviated pathological damages by preventing T-cell infiltration into the choroid plexus region(Zheng et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The protection against aldosterone-induced CSC pathology was also consistent with previously reported protective effects of A\u003csub\u003e2A\u003c/sub\u003eR antagonists on the blood-brain barrier(Fernandez et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kim and Bynoe \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yamamoto et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Based on these collective findings, we propose that A\u003csub\u003e2A\u003c/sub\u003eR plays a crucial role in CSC pathogenesis, with its involvement closely related to choroidal-retinal barrier dysfunction and CSC pathological development.\u003c/p\u003e \u003cp\u003eHow does the A\u003csub\u003e2A\u003c/sub\u003eR exert its control over CSC pathology? The pathogenesis of CSC with pathological accumulation of subretinal fluid involves the well-established core pathological components: enhanced permeability of larger choroidal vessels and disruption of the RPE barrier(Lee et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). One of the key mechanisms is the ability of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist to protect against disruption of the RPE barrier, the core of CSC pathogenesis. Increased permeability of choroidal capillaries leads to elevated hydrostatic pressure within the choroid, which subsequently results in RPE dysfunction(Gass \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). Specifically, cadherin-5, a key regulator of cell-cell adhesion, is essential for maintaining vascular integrity in the choroid. Dysfunction of cadherin-5 compromises vascular permeability and weakens intercellular connections in large choroidal vessels, potentially underlying the vascular abnormalities observed in CSC(Schubert et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Indeed, our analysis reveals that 24hours aldosterone treatment significantly disrupted the continuous distribution of RPE65 and ZO-1 signals, with evident TJs structural damage. Importantly, we found that KW6002 treatment markedly attenuated these pathological changes. Similarly, genetic deletion of A\u003csub\u003e2A\u003c/sub\u003eR-KO increased protein expression of ZO-1, Claudin-1, and claudin-5 in the RPE-choroid-sclera complex in aldosterone-treated mice. Thus, KW6002 protects BRB structural and functional integrity through A\u003csub\u003e2A\u003c/sub\u003eR signaling inhibition, primarily by maintaining TJs protein stability, providing compelling evidence for A\u003csub\u003e2A\u003c/sub\u003eR inhibition as a therapeutic strategy for BRB protection.\u003c/p\u003e \u003cp\u003eAnother pathological component that A\u003csub\u003e2A\u003c/sub\u003eR antagonists target to control CSC pathology is inflammation, an established as a critical pathogenic factor in CSC to disrupt the integrity of both the choroidal vascular system and RPE(Shen et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The characteristic choroidal vascular hyperpermeability in CSC appears to result from the synergistic effects of inflammatory mediators and oxidative stress(Nicol\u0026ograve; et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, breakdown of the BRB may trigger a cascade of secondary inflammatory responses, leading to prolonged disease duration and neurosensory retinal detachment. Recent studies have investigated serum-based inflammatory biomarkers as potential predictors of CSC progression or severity. Alterations in plasma cytokine levels have been observed in CSC patients, characterized by elevated expression of VEGF, IL-6, IL-10, and IL-12(Karska-Basta et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Matet et al(Matet et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported decreased lipocalin-2 levels in acute CSC patients compared to healthy controls. Lipocalin-2, an acute-phase reactant, exhibits both anti-inflammatory and pro-inflammatory properties. Recent investigations have demonstrated that systemic inflammatory markers, including the neutrophil-to-lymphocyte ratio and monocyte-to-high-density lipoprotein ratio, are significantly elevated in patients with acute CSC(Erol et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sirakaya et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several studies have demonstrated the efficacy of topical NSAIDs in acute CSC. For example, Alkin Z et al(Alkin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that 0.1% nepafenac significantly enhanced subretinal fluid absorption and improved best-corrected visual acuity (BCVA) in acute CSC patients. Subsequent investigations by Bahadorani S et al. (Bahadorani et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Consistent with this, we demonstrated that aldosterone administration induced intraocular inflammation accompanied by concurrent upregulation of adenosine A\u003csub\u003e2A\u003c/sub\u003eR expression. Importantly, the pharmacological blockade of A\u003csub\u003e2A\u003c/sub\u003eR with KW6002 effectively mitigated retinal inflammation and attenuated both choroidal and retinal edema, consistent with previous reports. This also collaborates with the previous report that intravitreal administration of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist ZM 241385 in ocular hypertensive models effectively suppresses microglial activation and downregulates pro-inflammatory cytokine expression(Liu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, during ischemic injury, KW6002 treatment attenuates microglial reactivity and reduces IL-1β levels, conferring neuroprotective effects on retinal tissue(Boia et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, the A\u003csub\u003e2A\u003c/sub\u003eR antagonist SCH 58261 has been shown to inhibit pathological overactivation of retinal microglia while promoting retinal cell survival(Aires et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEarly studies with transcriptomic analyses reveal that excessive aldosterone significantly upregulates genes encoding inflammatory response proteins, neutrophil chemotaxis, responsiveness to IL-1, IL-6, IL-12, and TNF-α, as well as other inflammatory genes involved in the NF-κB and TNF-α pathways, CCL3, CXCL1, and the CCL2/CCR2 axis(Canonica et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sanz-Rosa et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Activation of NF-κB has also been shown to trigger inflammation in age-related macular degeneration(Ghosh et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our analysis confirmed that the TNF-α/NF-κB signaling pathway was activated in the CSC model. Furthermore, inhibition of the NF-κB pathway can effectively prevent vascular leakage and inflammatory cell infiltration, reduce the expression of pro-inflammatory mediators and leukocyte adhesion molecules, and regulate vascular tension and permeability(Reddy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sanz-Rosa et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Thus, aldosterone may induce CSC pathology via the TNF-α/NF-κB signaling pathway.\u003c/p\u003e \u003cp\u003eIndeed, our results revealed that A\u003csub\u003e2A\u003c/sub\u003eR-KO significantly inhibited TNF-α, p65, and p-p65 levels, indicating that A\u003csub\u003e2A\u003c/sub\u003eR antagonism can block the expression of these inflammatory mediators. This finding further collaborates with our early findings that A\u003csub\u003e2A\u003c/sub\u003eR antagonists or A\u003csub\u003e2A\u003c/sub\u003eR-KO can modulate microglial reactivity, inhibit the expression of inflammatory factors TNF-α and IL-1β, and protect the retina from transient ischemic injury(Boia et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 is a potential therapeutic option for CSC. While half-dose PDT remains the first-line option for chronic CSC, its broad clinical application faces significant limitations(Radke et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The treatment's dependence on the costly photosensitizer verteporfin, the requirement for specialized laser equipment and trained personnel, and its relatively complex procedure compared to intravitreal injections all hinder its routine use. Currently, the most used drugs in clinical treatment are anti-VEGF agents (such as ranibizumab and aflibercept). These anti-VEGF drugs need to be administered through intravitreal injection (due to poor permeability of the blood-ocular barrier), which requires strict aseptic conditions and high professional requirements for medical resources. Although intravitreal injections have the advantages of high local drug concentration, wide applicability, and small incision, this operation also has multiple potential risks of complications(Patel et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Clinical research data show that after the operation, various potential complications may occur, such as ocular pain, subconjunctival hemorrhage (occurrence rate 23%-74%)(Fasih et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), temporary high intraocular pressure(de Vries et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gismondi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), retinal detachment (0.013%-0.067%)(Meyer et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Storey et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tolentino \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), endophthalmitis (0.019%-1.6%)(McCannel \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Scott and Flynn \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and lens damage. Moreover, repeated intravitreal injections may accelerate lens opacification and increase the incidence of complications in cataract surgery(HahnJiramongkolchai et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; HahnYashkin et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Compared with the currently widely used anti-VEGF drugs, KW6002 has unique advantages in drug administration: KW6002 can better cross the blood-ocular barrier. Our research group previously demonstrated in the study of retinopathy of prematurity (ROP) that intraperitoneal injection of KW6002 could effectively protect retinal vascular regeneration: this drug selectively inhibit pathological cell proliferation, effectively reduce the area without blood vessels and the formation of new blood vessels, while not affecting the normal physiological development of blood vessels(Zhong et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), showing similar therapeutic effects to anti-VEGF drugs. Additionally, as an FDA-approved drug, the safety of KW6002 has been fully verified by a large amount of preclinical and clinical data(Chen and Cunha \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These studies provide important theoretical support for the use of KW6002 in the treatment of ocular diseases.\u003c/p\u003e \u003cp\u003eIn summary, our findings indicate that adenosine A\u003csub\u003e2A\u003c/sub\u003eR antagonists can significantly reduce aldosterone-induced choroidal thickening and vasodilation, maintain BRB integrity by preventing the reduction of TJs protein levels, and decrease the levels of inflammatory cytokines, chemokines, and MMPs. These effects are likely achieved through inhibition of the TNF-α/NF-κB signaling pathway. This study provided a directly evidence that the A\u003csub\u003e2A\u003c/sub\u003eR is an effective therapeutic target to prevent CSC development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLimitations of the study\u003c/h2\u003e \u003cp\u003eThis study mainly used the aldosterone-induced model, which was generally accepted as the model representing the acute CSC, but cannot mimic the progressive forms of CSC, such as subretinal fluid accumulation or serous retinal detachment. Thus, further investigations based on improved CSC animal models for elucidating the specific mechanisms underlying CSC are essential. Additionally, our investigation focused mainly on acute CSC models. Further studies are needed to evaluate the efficacy of A\u003csub\u003e2A\u003c/sub\u003eR antagonists in chronic and recurrent forms of CSC.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of this study preliminarily confirm the protective effect of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 on acute CSC, which is achieved by reducing inflammation, maintaining BRB integrity, and simultaneously inhibiting the TNF-α/NF-κB pathway. This study provides a new insight into the treatment of CSC and lays a foundation for future clinical strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCSC: Central serous chorioretinopathy, A\u003csub\u003e2A\u003c/sub\u003eR: A\u003csub\u003e2A\u003c/sub\u003e receptor, AMD: age-related macular degeneration, RPE: retinal pigment epithelium, BRB: blood-retinal barrier, AQP4: aquaporin-4, BBB: blood-brain barrier, CP: choroid plexus, MMP2: Matrix metallopeptidase 2, MMP3: Matrix metallopeptidase 3, MMP9: Matrix metallopeptidase 9, A\u003csub\u003e2A\u003c/sub\u003eR -KO: A\u003csub\u003e2A\u003c/sub\u003eR knockout, WT: wild-type, OCT: Optical coherence tomography, FFA: Fluorescein fundus angiography, H\u0026amp;E: Hematoxylin-eosin, qPCR: Quantitative real-time PCR, TJs: tight junction, BCVA: best-corrected visual acuity, ROP: retinopathy of prematurity\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuarantor of integrity of the entire study: Jiang-fan Chen Study concepts; Qiaoli Liu, Jia Qu, Jiang-fan Chen, Wu Zheng Study design: Qiaoli Liu, Zijun Yu, Wei-hang Tang, Jia-nan Que, Jiaqi Li Data acquisition; Qiaoli Liu, Jiasheng Yang, Kailang Xu Data analysis; Qiaoli Liu Manuscript preparation; Jiang-fan Chen, Wu Zheng Manuscript editing; Jiang-fan Chen Manuscript review. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (Grant No. 31800903 and No. 82150710558) and the Natural Science Foundation of Zhejiang province, China (Grant No. LMS25H090008 and No. LQ18H090007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments conducted in this study received approval from the Institutional Ethics Committee for Animal Use in Research and Education at Wenzhou University, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdair TH, Cotten R, Gu JW, Pryor JS, Bennett KR, McMullan MR. 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Mol Med. 2018;24:41.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Adenosine A2A receptor, blood-retinal barrier, central serous chorioretinopathy, TNF-α/NF-κB signaling pathway, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-8526598/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8526598/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eCentral serous chorioretinopathy (CSC), a prevalent disease characterized by choroidal vascular abnormalities, has extremely limited treatment options. This study investigates the effects of the selective adenosine A\u003csub\u003e2A\u003c/sub\u003e receptor (A\u003csub\u003e2A\u003c/sub\u003eR) antagonist KW6002 on choroidal vascular hyperpermeability and the blood-retinal barrier (BRB), and explores its therapeutic potential in experimental CSC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe examined the expression of A\u003csub\u003e2A\u003c/sub\u003eR in the retinal pigment epithelium (RPE)-choroid-sclera complex using quantitative real-time PCR (qPCR) and Western blotting (WB) in mice with an established aldosterone-induced acute CSC model. Before modeling, mice were administered 5 mg/kg KW6002 or a vehicle control via intraperitoneal injection. The retinal and choroidal thickness was assessed by optical coherence tomography (OCT) and hematoxylin-eosin(H\u0026amp;E) staining. We observed M\u0026uuml;ller cells activation, retinal microglia infiltration, and proinflammatory cytokine expression via immunofluorescence and qPCR. Next, we employed the effects of the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 and genetic A\u003csub\u003e2A\u003c/sub\u003eR knockout on BRB integrity using immunofluorescence and WB. Finally, to clarify how A\u003csub\u003e2A\u003c/sub\u003eR knockout confers therapeutic benefits in CSC, we assessed activation of the TNF-α/NF-κB-MMP2/9 signaling pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that A\u003csub\u003e2A\u003c/sub\u003eR signaling was significantly upregulated in the RPE-choroid-sclera complex in CSC models, and both A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002 and A\u003csub\u003e2A\u003c/sub\u003eR knockout significantly inhibited the aldosterone-induced central retinal and choroidal pathologic thickening. Moreover, KW6002 treatment decreased the activation of M\u0026uuml;ller cells and the proliferation of microglia, inhibited the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and ameliorated the retinal damage caused by aldosterone; in contrast, A\u003csub\u003e2A\u003c/sub\u003eR knockout resulted in significant upregulation of key tight junction proteins (ZO-1, Claudin-1, and Claudin-5). In summary, these results suggest that the protective effects are likely due to A\u003csub\u003e2A\u003c/sub\u003eR inhibiting TNF-α/NF-κB\u0026ndash;MMP2/9 signaling axis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings show that the A\u003csub\u003e2A\u003c/sub\u003eR antagonist KW6002, or A\u003csub\u003e2A\u003c/sub\u003eR knockout, offers a protective effect in experimental CSC, reduces inflammation and maintains the integrity of the BRB, and mediates such protection through inhibiting TNF-α/NF-κB pathway. These findings present a new method for treating CSC, which will guide our future clinical strategy development.\u003c/p\u003e","manuscriptTitle":"The Adenosine A2A Receptor Antagonist KW6002 Mitigates Aldosterone-induced Central Serous Chorioretinopathy in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 13:03:01","doi":"10.21203/rs.3.rs-8526598/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d8be053d-3a17-4560-9617-1cf572935360","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:41:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 13:03:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8526598","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8526598","identity":"rs-8526598","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}