Abstract
Purpose :
Studies by our laboratory and others have shown a role of primary cilium (PC) in regulating IOP homeostasis. The PC is a non-motile hair-like structure protruding from the cellular membrane, which is involved in mechanosensation and mechanotransduction. The PC is composed of a microtubule-based structure, the ciliary axoneme. Microtubule acetylation of α-tubulin is thought to play an important role in the mechanoproperties of the PC. Here, we investigated the effect of aging, mechanical forces and TGFβ2 treatment on tubulin acetylation and PC prevalence and length in human trabecular meshwork (TM) and Schlemm’s canal (SC) cells.
Methods :
Human TM (n=6) and SC cells (n=2) isolated from donor tissues were used for these studies. The effect of aging was evaluated in cells subjected to the hyperoxic in vitro model of aging. Mechanical stretch was applied to TM cells using the computer-controlled and vacuum-operated Flexcell FX-5000 Tension System (cyclic stretch, 15% elongation, 24h). SC cells were subjected to shear stress (1~10 dyn/cm2) for up to 24 h, using the Ibidi system. TGFβ2 (10 ng/mL) was added to the culture media prior to application of mechanical forces. Immunocytochemistry and WB analysis were used to detect and quantify PC and acetylated tubulin, respectively, using specific antibodies (acetylated-TUBA4A, IFT88).
Results :
Aged TM cells show a decrease in PC prevalence (39.7±5 vs 57±3, p=0.01). No significant difference in PC length with aging were observed. In contrast, decreased PC prevalence (49.80±7.59% vs 13.67±6.37%, p<0.001) and PC length (4.18±1.10μm vs 2.02±0.70μm, p<0.001) were observed in TM cells treated with TGFβ2. Interestingly, mechanical stretch and fluid flow increased the levels of acetylated tubulin in TM cells (3.04±1.7 fold, n=4) and SC cells (~ 2 fold, n=2), respectively. Moreover, the increased in tubulin acetylation induced by mechanical stretch completely blocked upon TGFβ2 treatment (3.04±1.7 fold vs 1.22±0.85 fold, n=4)
Conclusions :
Our data results show a role of aging, mechanical forces and TGFβ2 treatment on tubulin acetylation and ciliogenesis. Given the role of tubulin acetylation on microtubule stabilization in response to mechanical forces and contractility, we hypothesize that the TGFβ2 reduction in acetylated alpha tubulin could represent an important molecular mechanism underlying resistance to AH outflow and elevated IOP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.