Comparative Proteomic Analysis of the Aqueous Humor from Patients with Pseudoexfoliation Syndrome

**Background:** Pseudoexfoliation syndrome (PEX) is an age-related systemic disorder characterized by the progressive deposition of fibrillary white flaky material in ocular tissues, and it is the most common identifiable cause of open-angle glaucoma. The etiopathology of PEX remains unknown, and previous research has focused on identifying biomarkers such as transforming growth factor β, endothelin-1, transferrin, and lysyl oxidase-like 1 (LOXL1). This study aimed to identify novel proteins in the aqueous humor (AH) of PEX patients using comparative proteomic analysis to uncover potential molecular pathways involved in PEX pathogenesis.

**Methods:** This prospective cross-sectional study included 15 patients with PEX and senile cataracts and 15 age- and sex-matched control patients with senile cataracts only. All participants had normal intraocular pressure (<21 mm Hg) and no other ocular or systemic diseases (excluding diabetes, hypertension, neurodegenerative disorders, prior ocular surgery, or laser). Aqueous humor samples (50–100 μL) were collected via paracentesis during cataract surgery. Protein concentrations were measured using the Bradford assay, and equal amounts of protein from each sample were pooled per group. Two-dimensional gel electrophoresis (2DE) was performed using 11 cm pH 3–10 nonlinear IPG strips and 12% SDS-PAGE gels. Protein spots were visualized with colloidal Coomassie blue G250, and differential expression was determined using PDQuest Advanced software, with a threshold of at least a two-fold change and p < 0.05. Differentially expressed spots were excised, trypsin-digested, and analyzed by MALDI-TOF/TOF mass spectrometry. Protein identification was performed using Mascot (Matrix Science) with a significance threshold of p < 0.05. Ingenuity Pathway Analysis (IPA) was used to identify enriched canonical pathways and protein-protein interaction networks.

**Key Results:** A total of 10 protein spots were found to be differentially expressed between the PEX and control groups. In the PEX group, eight proteins were upregulated: transthyretin (TTR; Swiss-Prot P02766, score 121, 68% sequence coverage), apolipoprotein A4 (ApoA4; P06727, score 160, 35% coverage), α-1-antitrypsin (A1AT; P01009, score 130, 29% coverage), vitamin D binding protein (VDBP; P02774, score 57, 18% coverage), serum albumin (P02768, score 286, 26% coverage), keratin type II cytoskeletal 1 (P04264, score 297, 38% coverage), apolipoprotein A1 (ApoA1; P02647, score 65, 32% coverage), and serotransferrin (P02787, score 384, 39% coverage). Two proteins were downregulated: α-1-acid glycoprotein 1 (A1AG1; P02763, score 90, 22% coverage) and α-1-acid glycoprotein 2 (A1AG2; P19652, score 77, 20% coverage). IPA analysis identified the most significant enriched canonical pathways: LXR/RXR activation (p = 3.02 × 10⁻¹⁹), FXR/RXR activation (p = 4.4 × 10⁻¹⁹), and acute phase response signaling (p = 6.54 × 10⁻¹⁰). The demographic and clinical characteristics (age, sex, axial length, BCVA, lens opacity grades) were not significantly different between groups (p > 0.05).

**Clinical Implications:** The identification of TTR and ApoA4 as significantly upregulated proteins in PEX aqueous humor suggests their potential role in PEX pathophysiology. TTR, a thyroid hormone-binding protein, may contribute to retinoic acid delivery to the anterior chamber, which could regulate LOXL1 expression via RXR. ApoA4, known to be associated with amyloid formation, may act as an initiator of PEX fibril deposition, similar to its role in Alzheimer's disease. The upregulation of ApoA1 and A1AT further supports the involvement of lipid metabolism and protease inhibition in PEX. The downregulation of A1AG1 and A1AG2, which have anti-inflammatory and antioxidant properties, may exacerbate oxidative stress and tissue damage. The IPA findings highlight LXR/RXR signaling as a potential therapeutic target, as these receptors regulate lipid metabolism and inflammation. Future studies should include PEX glaucoma patients to differentiate changes specific to glaucoma, and larger sample sizes are needed to validate these biomarkers. These results provide a foundation for developing novel diagnostic and therapeutic strategies for PEX syndrome.