Study participants

The study protocol was approved by the Research Ethics Committee of Changsha Aier Eye Hospital (Ethical batch number: AIER2019IRB12) and was in accordance with the tenets of the Declaration of Helsinki on research into humans. Informed consent was obtained from all participants. Forty-eight nAMD and 24 senile cataract patients were recruited from Changsha Aier Eye Hospital during 2020.12.01 and 2022.03.22 to this study.

The inclusion criteria were (1) older than 55 years of age; (2) diagnosed with nAMD (including CNV and PCV) by medical retina specialists following fundus photography, optical coherence tomography angiography (OCTA), fluorescein fundus angiography (FFA), and indocyanine green angiography (ICGA). Exclusion criteria were (1) history of ocular trauma; (2) presence of other eye diseases such as pathological myopia, diabetic retinopathy, and glaucoma; (3) history of intraocular surgery or laser therapy within 6 months; (4) presence of intraocular or systemic inflammatory or autoimmune diseases; (5) the use of immunosuppressive medications such as steroids. The inclusion criteria of the control group were elderly cataract patients who needed phacoemulsification and the exclusion criteria were the same as above.

Clinical examination

Comprehensive ophthalmic examinations were performed at baseline, including best corrected visual acuity (BCVA), intraocular pressure (IOP), axial length, slit lamp examination, fundus stereoscopic biomicroscopy, colour fundus photography, FFA, ICGA and OCTA. Data on axial length were collected for both the study group and control group to rule out myopia-associated CNV. All patients received intravitreal anti-VEGF (ranibizumab or conbercept) treatment at their first visit, followed by 3 × monthly injections and then subjected to a ‘injections as pro re nata’ (PRN) regime during monthly follow-up visits. Monthly follow-up examinations included BCVA, slit lamp-assisted fundus biomicroscopy and OCTA. The improvement of BCVA and the changes in central retinal thickness (CRT) were calculated by subtracting the values (LogMar score for BCVA, retinal thickness of CRT) collected from the time of the intravitreal anti-VEGF injection from the values collected at the next fellow up visit. The presence or absence of subretinal macular fibrosis was assessed by fundus examinations (colour photography, FFA and OCTA) at the first hospital visit.

Aqueous sample collection

Aqueous humour from nAMD patients was collected during intravitreal injection of anti-VEGF. Briefly, anterior chamber paracentesis was performed to control the intraocular pressure, and ~ 60μL of aqueous humor was collected aseptically using a 1 ml disposable sterile syringe in an ophthalmology surgical operating room. Aqueous humor from cataract patients was collected during phacoemulsification surgery. The samples were placed into a sterile 1.5 mL Eppendorf tube and stored at − 80 °C freezer until analysis.

Measurement of adhesion molecules

The aqueous humor samples were thawed on ice and centrifuged at 3000 rpm for 5 min. The levels of ICAM-1, VCAM-1, CD44, CD62L and CD62P were determined through a magnetic bead-based multiplex assay (Human Magnetic Luminex® Assay, R&D Systems Inc.) following the manufacturer's specifications. For each measurement of ICAM-1, VCAM-1, CD44, and CD62L, 50μL of supernatants (25μL for CD62P) were diluted to 100μL using sample diluent buffer immediately prior to the assay. In brief, the samples were incubated for 2 h with a specific cocktail of antibodies pre-coated onto magnetic microparticles, followed by 1 h incubation with biotinylated antibodies cocktail and additional 30 min incubation with streptavidin–phycoerythrin conjugate (Streptavidin-PE), which binds to the biotinylated antibody. Finally, the microparticles were resuspended in a buffer and read using a MagPix™ System (Bio-Rad Laboratories, Inc.). The data were calculated using the spline curve-fitting method (Milliplex Analyte, Application Version: 5.1.0.0).

Animals

C57BL/6 J mice were purchased from the SJA Laboratory Animal Co., Ltd (Changsha, China).

Mice lived under a 12-h light/dark cycle in pathogenic-free conditions with open access to dry feed and water in the Department of Laboratory Animals of Hunan Normal University, China. All animal-related procedures were conducted following the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the protocols were approved by the Animal Welfare Ethics Committee of Hunan Normal University.

Induction of subretinal fibrosis

Subretinal fibrosis was induced using a two-stage laser protocol detailed previously by us [31]. Briefly, mice were anaesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg, Sigma Aldrich), and pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine (Santen Pharmaceutical, JPN). Each eye received four laser burns (Laser settings: 200 mW power, 100 ms duration and 60 μm spot size, Topcon, 532 nm Laser, JPN). Seven days later, a second laser burn was applied to each lesion using the same setting.

Intravitreal injection

We conducted two treatments in mice with subretinal fibrosis: (1) anti-VCAM-1 antibody treatment and vehicle control (PBS), (2) VLA-4 inhibitor treatment and vehicle control (dimethyl sulfoxide, DMSO). 6–8 mice were included in each group. One microliter (1 µL) of anti-VCAM-1 antibody (6.85 µg, BE0027, BioXcell, endotoxin < 0.002EU/µg, Lebanon, USA) or VLA-4 inhibitor (8.17 µg, BIO5192, MedChemExpress LLC, USA) or relevant vehicle was administered via intravitreal injection immediately after the second laser. Mice were sacrificed 10 days after the second laser and eyes were collected and processed for further investigation.

Immunofluorescence staining

Mouse eyes were fixed in 4% paraformaldehyde (PFA) for 4 h and processed for either cryosections or RPE/choroid flatmounts preparation. For cryosections, mouse eyes were embedded in optimal cutting temperature (OCT) and cryosectioned with 10 µm thickness. The sections were blocked with 10% goat serum and 2% BSA, permeabilized with 0.1% Triton X-100 for 1 h, followed by incubation with the cocktails of primary antibodies at 4 °C overnight. After thorough washes, samples were incubated with Alexa Fluor 594 or Alexa Fluor 488 conjugated secondary antibodies for 1.5 h at room temperature. The detailed information of the antibodies, including the sources, catalog numbers and dilutions are listed in Supplementary Table S1.

For RPE/choroid flatmount staining, after blocking and permeabilization, the samples were incubated with rabbit anti-collagen-1 and overnight, followed by incubation with Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated donkey anti-rat IgG. The samples were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI, Cat: D8200, Solar-bio) to illustrate cell nuclei and imaged by using the Zeiss LSM 880 Confocal Microscope (Zeiss, Braunschweig, Germany).

Quantitative real‑time PCR

Total RNAs were extracted from RPE-choroid or bone marrow-derived macrophages (BMDMs) using the miRNeasy Micro Kit (Cat: 1071023, Qiagen, Dusseldorf, Germany) or total RNA Kit II (Cat: R6934-01, Omega, Norcross, GA) respectively following manufacturer’s instructions. Then the extracted RNA was used to synthesize cDNA via the PrimeScript RT Reagent Kit (Cat: 6110A, Vazyme Biotech, Nanjing, China). Quantification of gene expression was performed by real-time PCR using SYBR green fluorescence (Cat: Q711-02, Vazyme Biotech) on a LightCycler 96 (Roche, Basel, Switzerland) (Primers are listed in Supplementary Table S2). Target gene expression was calculated using the ΔΔCt method and was normalized to Gapdh expression levels.

Isolation and culture of bone marrow-derived macrophage

Murine bone marrow cells were isolated and cultured as previously described [32]. In brief, femurs and tibiae were isolated from mice, ensuring that joints were kept intact, and placed in cold DMEM (Cat: 11,995, Gibco) containing 1% penicillin/streptomycin (P/S). The muscle was removed using a sterile scalpel and scissors, the ends of the bones were cut in a ventilated hood, and bone marrow cells were flushed out with cold DMEM. Cells were filtered through a 40-μm cell strainer, centrifuged at 1100 rpm for 5 min, and the supernatant was then removed. Subsequently, the red blood cells were removed with lysis buffer and followed by centrifugation to elute the lysate. Cells were resuspended in DMEM medium containing 20% L929-conditioned medium, 15% FBS (Cat:BS-1102, OPCEL, Inner Mongolia Opcel Biotechnology Co., Ltd), 1% P/S. and seeded in 10 cm nontreated petri dish for 6–7 days to generate bone marrow derived-macrophages (BMDM). Differentiation was assessed using the flow cytometry and the associated antibodies anti-mouse F4/80-FITC (1:100, Cat: 11–4801-82, eBioscience) and anti-mouse CD11b-PerCP/Cy5.5 (1:100, Cat:101227, Biolegend) were stained for mature BMDM. Then the cells were collected for further study (see below).

In vitro treatment of macrophages with VCAM-1

The differentiated mature BMDM were seeded in 6 well plates and treated with different concentrations (100, 500 and 1000 ng/ml) of recombinant murine VCAM-1 (Cat: 643-VM, R&D) for 24 h in DMEM without FBS. Then cells were thoroughly washed with PBS and harvested for RT-qPCR.

Macrophage migration assay

Cell migration assays were performed in a 24-well plate Boyden chamber (8-µm pore size, Cat: 3422, Corning). Briefly, BMDMs were starved overnight before seeding into the insert (105 cells/well). The recombinant murine VCAM-1 (25 µg/ml) in serum-free DMEM was added to the bottom chambers. After incubating at 37◦C and 5% CO2 for 10 h with or without anti-VCAM-1 neutralizing antibody (20 µg/ml) or VLA-4 inhibitor (2, 10 µM) in the inserts. The transwell inserts were removed and the cells were fixed with 4% paraformaldehyde in PBS for 10 min and processed for DAPI staining. The cells remaining on the inner side of the insert were removed with a cotton swab. An inverted fluorescence microscope (10 × objectives, Zeiss) was used to obtain images of the cells attached to the outer side of the insert membrane. Images from five random visual fields from each sample were used to count the cell numbers using Image J software (Version 2.9.0; Java 1.8.0).

Statistical analysis

The clinical data were analysed using the Statistical Package for the Social Sciences (version 25.0; IBM SPSS Statistics, Armonk, NY, USA). Categorical demographic and clinical data were compared using Pearson’s chi-square test. The distribution of continuous variables was assessed for normality using the Shapiro–Wilk test and histogram test with a normal distribution curve. Logarithmic transformation was performed in data with non-normal distribution to achieve normal distribution. Normally distributed continuous samples were then compared using the independent sample t-test or one-way ANOVA followed by Tukey’s multiple comparisons test. For the associations that were significant in the univariate analysis, multivariable linear regression analysis was performed to adjust for age and gender etc. Pearson’s correlation was used to assess the correlation between the clinical ophthalmic parameters and the adhesion molecules. For the data from in vitro and animal studies, GraphPad Prism (Version 9.4.1, GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Unpaired Student’s t-test was used for two group data and one or two-way ANOVA with Dunnett's multiple-comparisons test or Turkey’s multiple comparisons test was used for three and more group data analysis, respectively. All data were presented as mean ± standard deviation (SD). P values < 0.05 were considered statistically significant.