Mechanical stretch induces Ca2+ influx and extracellular release of PGE2 through Piezo1 activation in trabecular meshwork cells

Abstract

The trabecular meshwork (TM) constitutes the main pathway for aqueous humor drainage and is exposed to complex intraocular pressure fluctuations. The mechanism of homeostasis in which TM senses changes in intraocular pressure and leads to normal levels of outflow resistance is not yet well understood. Previous reports have shown that Piezo1, a mechanically-activated cation channel, is expressed in TM and isolated TM cells. Therefore, we tested hypothesis that Piezo1 may function in response to membrane tension and stretch in TM. In human trabecular meshwork (hTM) cells, PIEZO1 was showed to be abundantly expressed, and Piezo1 agonist Yoda1 and mechanical stretch caused a Piezo1-dependent Ca2+ influx and release of arachidonic acid and PGE2. Treatment with Yoda1 or PGE2 significantly inhibited hTM cell contraction. These results suggest that mechanical stretch stimuli in TM activates Piezo1 and subsequently regulates TM cell contraction by triggering Ca2+ influx and release of arachidonic acid and PGE2. Thus, Piezo1 could acts as a regulator of intraocular pressure (IOP) within the conventional outflow pathway and could be a novel therapeutic strategy to modulate IOP in glaucoma patients.

TRPV4 is activated by mechanical stimulation to induce prostaglandins release in trabecular meshwork, lowering intraocular pressure

“…All experiments were approved by the University of Tokyo’s ethics committee (10984-(4)) and comply with the Declaration of Helsinki. As previously reported [ 16 ], primary human trabecular meshwork cells (HTMC) were harvested and cultured. Briefly, corneal scleral rims recovered by Rocky Mountain Lions Eye Bank from donors with written consent for use in research by next of kin and stored in Optisol were sectioned and trabecular meshwork strips were isolated and cultured in cell culture dishes coated with collagen gel (Cellmatrix Type I-A, Nitta Gelatin Inc., Osaka, Japan).…”

“…For subsequent experiments, HTMCs were used in 3 to 6 passages and grown on Trabecular Meshwork Cell Medium (ScienCell Research Laboratories, Carlsbad, CA), which contains 2% fetal bovine serum (ScienCell), 1% Trabecular Meshwork cell growth supplement (ScienCell), and 1% penicillin/streptomycin solution (ScienCell). Primary HTMC were identified and characterized by dexamethasone-induced MYOC expression as described previously [ 16 ]. Following the manufacturer’s instructions, HTMC were transfected with siRNA that specifically knockdown TRPV4 (SASI_Hs01_00013361, SASI_Hs02_00354974) and MISSION siRNA Universal Negative Control #1 siRNA (Merck, Darmstadt, Germany) with MISSION siRNA Transfection Reagent (Merck).…”

“…The TRPV4 agonist GSK1016790A (10 nM) was added into the cover glass chamber on the fluorescence microscope (Keyence, Osaka, Japan). STB-CH-24 was placed on an STB-150 attached to a fluorescence microscope and subjected to a 30% single uniaxial elongation stimulus (extend for 1 s, stop for 3 s, return in 1 s) as reported [ 16 ]. The fluorescence intensity of Fluo-8 in images taken with a fluorescence microscope was determined using ImageJ software ( https://imagej.nih.gov/ij/download.html ) as described previously [ 16 ].…”

“…Mechanical stimulation has been reported to rapidly increase cPLA2 activity leading to the synthesis and release of lipid mediators [ 14 – 16 ]. Among the lipid mediators, prostaglandins (PGs), Lysophosphatidic acid and Sphingosine-1-phosphate are involved in IOP regulation [ 17 – 21 ].…”

“…PGE 2 and some PGE 2 receptor (EP) agonists have been shown to have strong potential for IOP reduction [ 17 , 18 , 22 , 23 ]. We have previously reported that mechanical extension stimulation on HTMC increases the production of arachidonic acid and PGE 2 [ 16 ]. Therefore, we investigated the relationship between these lipid mediators released by mechanical stretch and TRPV4, and conducted a comprehensive analysis of lipid mediators generated by TRPV4 activation in TM.…”