Abstract
Purpose :
Septins, integral components of the cytoskeleton, are abundantly expressed in trabecular meshwork (TM) cells, and their gene variants are linked to glaucoma and intraocular pressure (IOP) regulation. However, the specific role of septins in TM biology and IOP homeostasis remains poorly understood. This study investigates the effects of septin-9 (SEPT9) overexpression on human TM cells and IOP in live mice.
Methods :
SEPT9 overexpression’s impact on human TM cell phagocytosis, permeability, cell-cell junctions, cell-ECM adhesion, and fibrogenic and contractile activities was assessed using lentiviral vectors (LV) expressing human SEPT9, along with pHrodo fluorescence based flow cytometry, fluorescein dextran, immunofluorescence, and immunoblot analyses. Additionally, the effects of SEPT9 overexpression on IOP were evaluated in C57BL/6J mice via intracameral injections using a rebound tonometer.
Results :
Human TM cells (from at least two strains, each with duplicates) infected with hSEPT9 LV exhibited significantly elevated levels of phospho-paxillin, p-MYPT1 (a Rho kinase substrate), p-myosin light chain (marker of cell contraction), and markers of fibrogenic activity such as α-smooth muscle actin, fibronectin, and collagen-1α1. This overexpression also showed increased staining for F-actin, vinculin, and cell-cell junctional proteins compared to control cells expressing GFP. Under these conditions, SEPT9 overexpression led to a significant decrease in TM phagocytosis and permeability compared to control cells. Moreover, increased hSEPT9 expression in the tissues of the aqueous humor (AH) outflow pathway of mouse resulted in a significant rise in IOP, accompanied by increased levels of αSMA, p-MLC, F-actin, fibronectin, and collagen. Additionally, transmission electron microscopy-based histological evaluation of the mouse AH outflow pathway revealed subtle differences, including, reduced number of giant vacuoles in Schlemm's canal (SC) and a reduction in SC lumen in specimens expressing hSEPT9 compared to controls.
Conclusions :
Collectively, this study demonstrates that dysregulated SEPT9 expression induces actin stress fibers, enhances contractile activity, cell adhesion, and fibrogenic processes, leading to decreased phagocytic activity and permeability in TM cells, ultimately resulting in ocular hypertension.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.