DTCAMD, a disease-associated truncated form of CRYAB in neovascular age-related macular degeneration: a potential driver of subretinal fibrosis

Abstract

Abstract Purpose: Subretinal fibrosis causes irreversible vision loss in neovascular age-related macular degeneration (nAMD) and remains poorly controlled by anti-vascular endothelial growth factor (VEGF) therapy. We investigated the role of B-crystallin (CRYAB) in angiogenic and fibrotic pathology and evaluated disease-associated CRYAB truncation as a therapeutic target. Methods: Aqueous humor from patients with nAMD was analyzed by quantitative proteomics and receiver operating characteristic (ROC) analysis. CRYAB function was assessed using knockout and knockdown approaches in a choroidal neovascularization (CNV) mouse model. CRYAB truncation and associated proteins were examined by immunoblotting, co-immunoprecipitation, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). C-terminal truncation was further examined in mouse retinal tissue using antibodies generated against full-length CRYAB and its C-terminal region. Primary human retinal pigment epithelial (hRPE)-THP-1 co-cultures were used to examine CRYAB truncation under fibrosis-associated conditions. Pharmacological effects were tested using ({+/-})-threo-3-methylglutamic acid (T3MG) and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). Results: CRYAB was upregulated in the aqueous humor of patients with nAMD. Higher CRYAB levels were associated with persistent lesions after initial anti-VEGF treatment. CRYAB knockout or knockdown reduced CNV pathology and decreased VEGFA and -smooth muscle actin (SMA) expression, without a significant substrain effect between C57BL/6N and C57BL/6J mice. A Ser45-phosphorylated truncated CRYAB species was detected in mouse laser-induced CNV model. We termed this species disease-associated truncated CRYAB in AMD (DTCAMD). Peptide mapping showed reproducible loss of distal C-terminal peptide coverage in truncated CRYAB. Consistent with this finding, C-terminal antibody recognition was reduced in CNV lesions. In hRPE-THP-1 co-cultures, DTCAMD accumulated with increased SMA expression. Proteomic analysis identified Prss1 as a candidate CRYAB-associated serine protease linked to truncation under CNV conditions. T3MG and AEBSF reduced DTCAMD-associated signals and CNV pathology. Conclusions: CRYAB contributes to angiogenic and fibrotic pathology in CNV. Disease-associated CRYAB truncation, particularly Ser45-phosphorylated DTCAMD, represents a potential therapeutic target for fibrotic remodeling in nAMD.