Abstract
Purpose :
Actomyosin regulated contraction and cell adhesion within the trabecular meshwork (TM) modulate aqueous humor outflow and intraocular pressure (IOP). The role played by various cytoskeletal components in influencing these actin-based cellular characteristics, however, is poorly understood. This study investigates the role of septin-9 (SEPT9) which is induced by glucocorticoids and TGF-β2 in regulation of TM cell actin cytoskeletal organization and cell adhesive interactions, and IOP in mice and humans.
Methods :
The effects of SEPT9 on actin stress fibers, cell adhesion and myosin II activity were investigated by suppressing or enhancing expression of SEPT9 in human TM cells using SEPT9 siRNA and lentiviral vector, respectively, with immunofluorescence and biochemical analyses. Since SEPT9 has been identified as a risk loci gene for IOP elevation in a UK Biobank cohort, the replication of association between common variant rs9038 at SEPT9 and IOP was assessed using data from a previous genome-wide association study (GWAS) conducted in the Kaiser Permanente Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (N= 69,756 individuals). The effects of expressing human SEPT9 on IOP were tested using lentiviral vectors administered into the anterior chamber of live mice and a rebound tonometer.
Results :
Increased expression of SEPT9 in human TM cells induced actin stress fibers, focal adhesions, cell-cell junctions, and myosin II activity by interacting with actin stress fibers. In contrast, most of these characteristics were suppressed under SEPT9 deficiency. Importantly, a significant association between SEPT9 rs9038 and IOP was observed in the GERA cohort (P<5.3x10-7). Moreover, expression of recombinant SEPT9 in the anterior chamber of live mice was associated with a consistent increase in IOP (by 2-5 mmHg; n=6) over a period of three weeks compared to control mice injected with control vector.
Conclusions :
Septin-9, a known component of the septin cytoskeleton which is expressed abundantly in TM cells and whose gene variant (rs9038) is associated with IOP induces actomyosin contraction and cell adhesion in human TM cells, and IOP when overexpressed in mice. In addition, the GWAS findings offer further strength to our hypothesis on the role of SEPT9 in regulation of IOP. Collectively, these studies indicate a promising role for the septin cytoskeleton in TM biology and homeostasis of IOP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.