Vascular Cell Adhesion Molecule-1 (VCAM-1) contributes to macular fibrosis in neovascular age-related macular degeneration through modulating macrophage functions

**Background:** Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in the elderly, characterized by abnormal choroidal neovascularization (CNV). Intravitreal anti-VEGF therapy is standard, but up to 50% of patients respond poorly, and 40–70% develop macular fibrosis within 10 years, leading to irreversible vision loss. Inflammation, particularly macrophage involvement, is implicated in fibrosis, but the role of adhesion molecules like VCAM-1 in this process is unclear. This study investigated intraocular levels of adhesion molecules (ICAM-1, VCAM-1, CD44, CD62L, CD62P) in nAMD patients with and without macular fibrosis, and explored VCAM-1's role in macrophage function and subretinal fibrosis in a mouse model.

**Methods:** Aqueous humor was collected from 48 nAMD patients (24 with macular fibrosis, 24 without) and 24 senile cataract controls during intravitreal injection or cataract surgery. Adhesion molecule levels were measured using a magnetic bead-based multiplex assay. Clinical parameters included BCVA, central retinal thickness (CRT), and fibrosis presence assessed by fundus photography, FFA, ICGA, and OCTA. In vivo, subretinal fibrosis was induced in C57BL/6J mice using a two-stage laser protocol. Mice received intravitreal injections of anti-VCAM-1 antibody (6.85 µg), VLA-4 inhibitor (8.17 µg), or vehicle immediately after the second laser. Eyes were collected 10 days later for immunofluorescence staining of collagen-1 and F4/80+ macrophages. In vitro, bone marrow-derived macrophages (BMDMs) were treated with recombinant VCAM-1 (100, 500, 1000 ng/ml) for 24 h, and gene expression (iNOS, Il1b, Il6, Arg1, Mmp12, etc.) was analyzed by RT-qPCR. Macrophage migration was assessed using a Boyden chamber with VCAM-1 (25 µg/ml) in the bottom chamber, with or without anti-VCAM-1 antibody (20 µg/ml) or VLA-4 inhibitor (2, 10 µM).

**Key Results:** Aqueous levels of ICAM-1, VCAM-1, CD44, and CD62L were significantly higher in nAMD patients vs controls (all p<0.001). VCAM-1 was significantly higher in nAMD patients with macular fibrosis vs those without (p=0.002) and correlated positively with CRT (r=0.313, p=0.031). ICAM-1 negatively correlated with CRT change (r=-0.316, p=0.029), and CD62L negatively correlated with BCVA improvement (r=-0.316, p=0.029). In the mouse model, Vcam1 and Itga4 mRNA were significantly upregulated in RPE/choroid with fibrosis (p<0.01). VCAM-1 was expressed on CD31+ endothelial cells and F4/80+ macrophages in fibrotic lesions. Anti-VCAM-1 antibody and VLA-4 inhibitor significantly reduced collagen-1+ fibrotic lesion area (p<0.01) and F4/80+ macrophage infiltration (p<0.01). In vitro, recombinant VCAM-1 significantly increased macrophage migration (p<0.05), which was blocked by anti-VCAM-1 antibody or VLA-4 inhibitor. VCAM-1 treatment (1000 ng/ml) significantly downregulated iNOS (p<0.05) and Il1b (p<0.01), and upregulated Il6 (p<0.05), Arg1 (p<0.05), and Mmp12 (p<0.001) in BMDMs, but did not affect Col1a1, Fn1, or Acta2 expression.

**Clinical Implications:** This study identifies VCAM-1 as a key contributor to macular fibrosis in nAMD, with higher intraocular levels associated with fibrosis and macular thickness. Blocking VCAM-1/VLA-4 reduces fibrosis and macrophage infiltration in a mouse model, suggesting a potential therapeutic target for preventing or treating macular fibrosis in nAMD patients, especially those with poor anti-VEGF response. The findings highlight the role of VCAM-1 in modulating macrophage migration and profibrotic polarization, offering a new avenue for adjunctive therapy to improve long-term visual outcomes.