Abstract
Purpose :
Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, a leading cause of irreversible blindness consequent to retinal ganglion cell (RGC) degeneration. Elevated IOP induces biomechanical aberrations within ocular tissues – including the transmission of stretch through the lamina cribrosa (LC) region of the optic nerve head (ONH), the initial site of RGC damage. ONH astrocytes (ONHA), a primary cell type of the LC, respond to stretch in a manner that promotes pathological extracellular matrix (ECM) remodeling (fibrosis) and mechanical damage of RGC axons within the ONH. A complex set of molecular mechanisms regulate ECM remodeling. Part of this regulation may involve microRNAs (miRNAs), small molecules that inhibit protein expression by binding to and silencing mRNA. In this study, we examined miRNA expression profiles of ONHA exposed to cyclic stretch. We hypothesized that cyclic stretch would increase expression of miRNAs that silence anti-fibrotic protein translation and decrease expression of miRNAs that silence pro-fibrotic protein translation, promoting a net-fibrotic molecular signaling environment.
Methods :
Primary human normal ONHA cell strains (n=3) were exposed to 0-12% cyclic stretch for 24 hours; controls were exposed to 0% stretch. RNA samples were collected from stretched and control cells. miRNA PCR arrays were used to determine expression changes for miRNAs associated with fibrosis. Expression fold changes were normalized to SNORD68. The bioinformatics tool TargetScan was used to predict mRNA targets for dysregulated miRNAs. Western blotting of conditioned medium was used to compare TGFβ2, fibronectin, and transglutaminase 2 expression between control and stretched ONHA.
Results :
miR-146b-5p was upregulated by +5.97-fold (P = 0.029) in stretched ONHA. Predicted mRNA targets for miR-146b-5p are involved in fibrosis and cell survival, among other functions. Preliminary data indicates the upregulation of secreted proteins associated with fibrosis (TGFβ2, fibronectin, transglutaminase 2) by stretched ONHA.
Conclusions :
Stretch modulates miRNA expression in cultured human ONHA, miR-146b may mediate ECM alterations and other pathological changes at the LC. Future experimental directions will include assessing co-expression of other miR-146 family miRNAs, validating putative mRNA targets and elucidating the mechanisms by which specific miRNAs and their targets modulate ECM remodeling.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.