Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis

**Background:** Glaucoma is a leading cause of irreversible blindness, with primary open-angle glaucoma (POAG) being the most common type. Elevated intraocular pressure (IOP) is a major risk factor, often resulting from increased resistance to aqueous humor outflow through the trabecular meshwork (TM). The renin-angiotensin-aldosterone system (RAAS) is known to regulate blood pressure, and its components are expressed in ocular tissues. Angiotensin II (ANGII), the main effector of RAAS, has profibrotic effects in various organs, but its role in POAG pathogenesis was unclear. This study investigated whether ANGII levels are altered in POAG patients and whether ANGII contributes to TM fibrosis and IOP elevation.

**Methods:** Aqueous humor samples were collected from 20 POAG patients and 28 cataract controls. ANGII concentrations were measured by ELISA. Human trabecular meshwork cells (HTMCs), both immortalized and primary, were treated with 100 nM ANGII for 48 hours, with or without inhibitors: NOX4 inhibitor GLX351322 (10 μM), ROS scavenger NAC (40 μM), or Smad3 inhibitor SIS3 (100 nM). NOX4 knockdown was achieved using siRNA. Fibrosis-related gene expression (Col1, FN, αSMA) was assessed by qPCR, Western blotting, and immunofluorescence. ROS levels were measured using DCFH-DA probe. In vivo, female C57BL/6J mice received periocular conjunctival fornix injections of ANGII (0.5 μM) alone or with GLX351322 (1 μM) or SIS3 (1 μM) once weekly for 4 weeks. IOP was measured weekly using a rebound tonometer. TM fibrosis was evaluated by immunofluorescence staining of eye sections.

**Key Results:** ANGII concentration was significantly higher in POAG patients (44.35 ± 60.77 pg/ml) compared to controls (27.67 ± 50.63 pg/ml, P < 0.001). ANGII levels positively correlated with IOP (R = 0.422, P < 0.05). In vitro, ANGII treatment upregulated Col1, FN, and αSMA mRNA and protein in both immortalized and primary HTMCs. ANGII increased intracellular ROS levels and selectively upregulated NOX4 expression. NOX4 knockdown or pharmacological inhibition with GLX351322 or NAC reduced ROS levels and reversed ANGII-induced fibrosis-related gene expression. ANGII increased Smad3 phosphorylation, which was attenuated by GLX351322. The Smad3 inhibitor SIS3 also blocked ANGII-induced fibrosis-related gene expression. In vivo, ANGII injections significantly increased IOP compared to controls, and this effect was partially rescued by co-treatment with GLX351322 or SIS3. Immunostaining showed increased Col1 and FN in the TM of ANGII-treated mice, which was reduced by both inhibitors.

**Clinical Implications:** This study identifies elevated ANGII in the aqueous humor as a potential contributor to POAG pathogenesis through a NOX4/ROS/Smad3-mediated fibrotic pathway in TM cells. The findings suggest that targeting ANGII signaling, particularly NOX4 or Smad3, could represent novel therapeutic strategies for lowering IOP and preventing TM fibrosis in glaucoma. The positive correlation between ANGII levels and IOP also suggests ANGII as a potential biomarker for POAG. However, further studies are needed to validate these findings in larger patient cohorts and to assess the clinical utility of NOX4 or Smad3 inhibitors in glaucoma treatment.