Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Abstract

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p<0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.