Triad of homocysteine, ROCK2 and vimentin as a convergent biomarker signature in pseudoexfoliation

Pseudoexfoliation (PEX) is a complex age-related disorder traditionally considered ocular in origin but increasingly recognized as a systemic condition involving extracellular matrix (ECM) dysregulation, oxidative stress, and cytoskeletal alterations. The disease typically presents as pseudoexfoliation syndrome (PEXS), with a subset of patients progressing to pseudoexfoliation glaucoma (PEXG), characterized by elevated intraocular pressure and optic nerve damage. While genetic susceptibility has been explored, dynamic molecular markers that reflect disease activity and progression remain underdeveloped. This study investigates the plasma levels of three candidate biomolecules, homocysteine (Hcy), Rho-associated coiled-coil containing protein kinase 2 (ROCK2), and vimentin (VIM), in individuals with PEXS, PEXG, and age-matched controls, to evaluate their potential as systemic indicators of disease presence and progression. A total of 72 participants (24 controls, 24 PEXS, 24 PEXG) were recruited from a single Indian cohort. Plasma Hcy was measured via fluorometric assay, and ROCK2/VIM by ELISA. Analyses included ANOVA, effect size estimation, logistic regression, LASSO modelling, ROC analysis, and unsupervised clustering. Marker interactions and stage associations were assessed through correlation and distribution analyses. Functional validation involved HLE-B3 cells treated with Hcy, followed by immunoblotting for ROCK2 and VIM. Bioinformatics (pathway enrichment, STITCH, TF prediction) identified upstream regulators and pathways. All three biomarkers were significantly elevated in PEXS and PEXG compared to controls (p < 0.001), with large effect sizes. Hcy and VIM levels progressively increased from PEXS to PEXG, while ROCK2 peaked in PEXS. The three-marker model combining Hcy, ROCK2, and VIM demonstrated a robust diagnostic profile, achieving a high sensitivity of 0.94 and specificity of 0.88, indicating a more balanced classification performance compared to individual or two-marker combinations. Clustering analyses revealed three molecular subgroups with moderate alignment to clinical staging (ARI = 0.32). Correlation and distribution analyses suggested stage-specific marker interactions. Bioinformatic findings present SP1 as a shared upstream regulator linking Hcy to ROCK2 and VIM expression, supported by in vitro findings. Homocysteine, ROCK2, and vimentin form a clinically relevant biomarker panel with strong diagnostic and staging utility. The findings suggest a mechanistic axis driven by Hcy, possibly through SP1-mediated regulation, contributing to cytoskeletal and fibrotic alterations. This integrated pathway highlights potential molecular targets for pharmacological intervention and offers a foundation for future risk-based screening for pseudoexfoliation.