Abstract
Purpose :
Changes in intraocular pressure (IOP) are “sensed” by stretching of trabecular meshwork (TM) cells and shear stress on Schlemm’s canal (SC) cells. We previously reported stretch-induced primary cilia (PC)-mediated activation of autophagy as a crucial physiological response for regulating IOP homeostasis in TM cells. Here we investigate the role of autophagy upon shear stress in SC cells.
Methods :
Primary human SC cells (6 strains) and human umbilical vein endothelial cells (HUVECs) were subjected to shear stress (1~10 dyne/cm2) for up to 24 h. Autophagy activation was monitored by measuring LC3-II and SQSTM1 levels, tandem fluorescent-tagged LC3 (tfLC3) or SQSTM1-RFP using western blot or live cell imaging. PC were detected by PC markers (acetylated-TUBA4A, IFT88 and 5HT6-mcherry) and disrupted by chloral hydrate (2 mM for 3 day). The expression and activity of eNOS were measured by GFP under the control of eNOS promoter (eNOSpd-GFP) and phosphorylated eNOS (S1177). Autophagy was inhibited by bafilomycin A (50~100 nM) or siRNA targeting LC3B.
Results :
Autophagy marker proteins (LC3 II and SQSTM1) significantly increase by approximately 2 fold (p<0.05, n=6) upon flow application (10 dyne/cm2 for 24 h) in the SC cells. Time-lapse live imaging of tfLC3 and SQSTM1-RFP showed that autophagic activity increases and SQSTM1 is aggregated and continuously removed by autophagic vacuole-like structures upon shear stress. Immunochemical and live cell imaging analyses revealed the presence of PC in SC cells. Deciliation significantly prevented the increase in LC3-II levels by shear stress in SC cells (CNT vs deciliated: 2.36±0.58 vs 0.78 ±0.62, n=4, p<0.05). Time-lapse live imaging of eNOSpd-GFP and western blot showed that the transcription and phosphorylation of eNOS increase by shear stress, and removal of PC decreased eNOS phosphorylation in SC cells. Intriguingly, bafilomycin treatment decreased eNOS protein levels in HUVECs in a concentration-dependent manner, and LC3 knockdown decreased eNOS protein level of HUVECs in static and shear stress conditions (10 dyne/cm2 for 24 h).
Conclusions :
We demonstrate here for the first time the PC-mediated autophagy activation in response to physiological levels of shear stress in SC cells. Moreover, our data strongly suggest that the shear stress-induced autophagy regulates eNOS/NO production in SC cells.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.