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

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.


Mechanical stretch induces Ca
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 Ca 2+ influx and release of arachidonic acid and PGE 2 . Treatment with Yoda1 or PGE 2 significantly inhibited hTM cell contraction. These results suggest that mechanical stretch stimuli in TM activates Piezo1 and subsequently regulates TM cell contraction by triggering Ca 2+ influx and release of arachidonic acid and PGE 2 . 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.
Intraocular pressure (IOP) is regulated by the production and outflow of aqueous humor and has been associated with increased risk of glaucoma development. The main route of aqueous humor drainage is the conventional outflow pathway, which involves the flow of aqueous humor to the Schlemm's canal through the juxtacanalicular trabecular meshwork (TM) 1,2 . Conventional outflow tissues are constantly subjected to fluctuations in IOP, and cells in these tissues sense alterations in IOP as mechanical stretch or strain 2,3 . Changes in pressure of 8-30 mmHg can cause length extension by 50% in cells in the outflow pathway 4,5 . In response to mechanical stretch or strain, TM cells activate intracellular signaling pathways leading to changes in gene expression, extracellular matrix (ECM) turnover, contractile properties 2,6 .
The non-selective cation channel TRPV4 and the two-pore domain potassium channel TREK1 are expressed in TM cells, sense mechanical stretch generated by fluctuations in IOP, and play an essential role in IOP adjustment [7][8][9][10] . Following membrane stretch, TM-resident TRPV4 channels initiate calcium (Ca 2+ ) signals and cytoskeletal reorganization, and TRPV4 antagonists increased the outflow facility and lowered IOP in glaucomatous mouse eyes 10 . Recent studies showed that TREK-1 collaborates with TRPV4, regulating TM tensile homeostasis 8 . Although numerous studies highlighted the importance of mechanosensation in modulating TM outflow, the molecular links between mechanotransduction, Ca 2+ homeostasis, and reorganization of the conventional outflow pathway remain unclear. Hence, the mechanosensory mechanisms mediating force-coupling and IOP regulation should be further explored.
Piezo1 and Piezo2 were first described in 2010 as components of a mechanically activated cation channel expressed in neurons and neuronal cell lines 11 . Piezo1 has been implicated in mechanical stretch response and extracellular Ca 2+ influx regulation in the bladder urothelium 12 , red blood cell 13 , and neural stem cells 14 . Nevertheless, its role in TM remains poorly understood. We hypothesized that in TM, Piezo1 might respond to mechanical stress and regulate IOP by locally releasing lipid mediators. Lipid mediators are well known to have  [17][18][19][20] . Thus, we investigated the involvement of Piezo1 on the mechanical stress and the release of lipid mediators in primary cultured human TM cells.

Results
Expression of Piezo and TRPV family members in primary hTM cells. First, we examined the expression of human Piezo and TRPV family members in primary hTM cells by qPCR. Piezo1 mRNA levels were profoundly higher than Piezo2 and TRPV family mRNA levels in hTM cells (Fig. 1).
Knockdown of Piezo1 using siRNA in hTM cells. To Fig. 3A,B). These findings suggest that mechanical stretch stimulation in primary hTM cells induces Ca 2+ influx in a Piezo1-dependent manner.

Mechanical stretch stimulation in hTM cells promotes PGE 2 release.
We then performed lipid analysis to investigate which lipid mediators are produced by hTM cells following mechanical stretch stimulation. We found that mechanical stretch stimulation significantly increased the amounts of arachidonic acid and PGE 2 released from hTM cells (Fig. 4A (Fig. 4C), and other lipid mediators such as PGF 2α and TXB 2 were not detected. Moreover, PIEZO1 silencing in hTM cells significantly abrogated the mechanical stretch stimulation-induced release of arachidonic acid and PGE 2 (Fig. 5A  www.nature.com/scientificreports/ These results suggesting that Piezo1 plays an essential role in the release of arachidonic acid and PGE 2 following stretch stimulation.

Yoda1 promotes PGE 2 release in hTM cells.
To assess the direct effect of Piezo1 activation, we measured lipid mediators in hTM cell supernatants after Yoda1 treatment. This analysis revealed that Yoda1 treatment significantly increased the levels of arachidonic acid and PGE 2 released from hTM cells, but not PGD 2 (Fig. 6).

Effects of Yoda1 and PGE 2 on collagen gel contraction.
To investigate the effect of Yoda1 and the arachidonic acid metabolite PGE 2 on hTM cells contraction, we performed an in vitro collagen gel contraction assay. This assay correlates that cells have contraction ability, which is supported by the fact that collagen alone does not cause contraction 21,22 . Compared to controls, Yoda1 significantly suppressed collagen gel contraction after 48 h and later, and PGE 2 at 10 and 100 nM PGE 2 after 24 h and later (Fig. 7A,B).

Discussion
In this study, we demonstrated that Piezo1 was abundantly expressed in primary hTM cells, mediating mechanical stretch-induced Ca 2+ influx and PGE 2 release. Additionally, the Piezo1 agonist Yoda1 triggered Ca 2+ influx and PGE 2 release in hTM cells. Piezo1 activation in response to mechanical stimuli and subsequent PGE 2 release www.nature.com/scientificreports/ suppressed hTM cell contraction. These results indicate that Piezo1 plays a crucial role in intracellular signaling induced by mechanical stretch and regulating TM contraction. First, we analyzed the expression of TRPV1-6 and PIEZO1-2 in hTM cells and found that Piezo1 was most abundantly expressed at the mRNA level. Consistently, previous studies suggested that Piezo1 was one of 11 mechanotransduction channels identified in TM tissue and isolated TM cells 23 . Therefore, we hypothesized that Piezo1 might play a crucial role in TM physiology.
Next, we explored the role of Piezo1 signaling pathway in regulating TM contraction. As shown in Fig. 3, mechanical stretch-induced Ca 2+ influx was inhibited when PIEZO1 was silenced. This finding is consistent with previous findings in urothelial cells 12 , red blood cell 13 , and neural stem cells 14 , suggesting that Piezo1 constitutes Ca 2+ influx pathway in mechanical stretch of hTM cells. Arachidonic acid and its metabolites have been reported to play important roles in mechanical signaling 15,16 . Additionally, cyclic mechanical stress induced the release of arachidonic acid and its metabolic product PGE 2 in porcine TM cells 24 . Therefore, we next investigated the profile of lipid mediators released after mechanical stretch and found a significant increase in the levels of arachidonic acid and PGE 2 secreted from primary hTM cells following mechanical stretch (Fig. 4). We further found that the stretch-induced secretion of arachidonic acid and PGE 2 was abrogated by PIEZO1 silencing (Fig. 5), and pharmacological activation of Piezo1 with Yoda1 triggered the release of arachidonic acid and PGE 2 in hTM (Fig. 6), suggesting an essential role of Piezo1 in the mechanical stretch-induced secretion of these lipid mediators.
Moreover, we found that PGE 2 produced by Piezo1 activation inhibited hTM contraction. The contraction of TM cells decreases the size of intercellular spaces and permeability, resulting in reduces aqueous humor outflow 25 . Agents that induce contraction of TM cells have been shown to increase IOP by reducing the outflow rate of aqueous humor, whereas relaxation of TM cells increases outflow and lowers IOP [26][27][28][29][30] . Consistent with these, EP2 agonists inhibited gel contraction 29 and have an IOP lowering effect in vivo 18,30,31 . The response of TM cells by mechanical stimulation leading to the release of PGE 2 may corroborate that recently introduced EP2 agonist, omidenepag, and previously reported EP2 agonists can significantly reduce IOP 30,31 . Our result suggests new homeostatic mechanism in the eye that elevated IOP stretches TM and subsequently TM cells respond to protect eyes through enhancing aqueous outflow via endogenously produced PGE 2 . The proposed mechanism www.nature.com/scientificreports/ underlying Stretch-mediated Piezo1 activation in human trabecular meshwork cells is diagrammatically summarized in Fig. 8.
To assess the effect of direct activation of Piezo1 in hTM cells, we used the Piezo1-selective activator Yoda1. Yoda1 was identified among approximately 3.25 million compounds as a synthetic small molecule that acts as an agonist of both human and mouse Piezo1 32 . We investigated the effect of Yoda1 on Piezo1 activation in hTM cells and confirmed that it can be reliably activated at 10 μM (Fig. 2). Previous studies showed that high concentration (10-100 μM) of Yoda1 simultaneously caused substantial cytotoxicity and compromise of the HUVEC monolayer after 24 h 33 . In this study, we found that 10 μM Yoda1 reduced survival by approximately 10% after 24 h (See Supplementary Fig. S1 online). Although 10 μM Yoda1 significantly inhibited hTM cell contraction after 48-72 h in the gel contraction assay (Fig. 7A), we also showed the effect of PGE 2 in gel contraction assay www.nature.com/scientificreports/ because of prolonged 10 μM Yoda1exposure (72 h) not only activates Piezo1 but may also have toxic effects. As shown in Fig. 7B, PGE 2 significantly inhibited hTM collagen gel contraction in a dose-dependent manner, suggesting PGE 2 production after Piezo1 activation contributes to IOP regulation. On the other hand, activation of EP 2 (and EP 1 , EP 4 ) receptors have been demonstrated to increase TM cell contractility 34 . However, this result is a response within 1 h of agonist exposure. PGE 2 lowers IOP, and also temporarily increases IOP in the early stages after instillation 18 . The increase in TM cell contractility due to activation of EP 2 receptor may reflect this transient increase in IOP and change at later time. EP 2 receptor was also expressed in Schlemm's canal (SC) and its activation was shown to reduce SC cell stiffness 34 . Accordingly, PGE 2 released from hTM cells may target SC in addition to TM. While our findings highlighted the critical role of Piezo1 in IOP regulations upon TM stretch, there were several limitations in the present study. Piezo1 and TRPV4 were compared only for mRNA expression in this study. There are several reports comparing the role of Piezo1 and TRPV4 in response to stretch stimulation. In bladder urthelium, the sensitivity of Piezo1 to stretch stimuli was higher than that of TRPV4 12 . In chondrocyte, Piezo1 was largely responsible for the stretch-activated current, whilst TRPV4 had no involvement in the specific mechanoelectrical transduction pathway 35 . However, there are no reports comparing the importance of Piezo1 and TRPV4 in TM. Thus, future studies are required to compare the sensitivity of Piezo1 and TRPV4 to stretch stimuli, as well as the relationship between Piezo1 and TRPV4 mRNA levels and function. We focused on lipid mediator and its involvement in the TM contraction, and didn't further explore whether Piezo1 are involved in the stretch-induced cytoskeletal remodeling in hTM cells, which should be determined in the future study. In addition to lipid mediators, nitric oxide (NO) is a candidate that link intracellular Ca 2+ elevation and regulation of TM outflow. NO is formed by the enzyme called nitric oxide synthase (NOS) and Three different isoforms have been identified including neuronal NOS or brain NOS (nNOS, bNOS or NOS1), endothelial NOS (eNOS or NOS3), and inducible NOS (iNOS or NOS2). nNOS1 and eNOS are constitutively expressed in cells and are activated by an increase in intracellular Ca 2+36 . iNOS is induced by a variety of stimuli, such as endotoxins and inflammatory cytokines, regardless of Ca 2+ level. Three isoforms of NOS has been reported to be expressed in TM 37 . Several studies in vitro and in vivo have demonstrated IOP-lowering effects of NO, and treatment with www.nature.com/scientificreports/ the ROCK inhibitor Y-26732 increased the expression of eNOS and NO production more significantly 38,39 .
Increased visual field damage results in decreased eNOS expression and Ca 2+ -dependent NOS activity in TM of POAG patients 40 . Thus, elevated intracellular Ca 2+ in TM may increase eNOS expression and NO production, leading to decreased IOP. In this study, we applied a uniaxial stretch stimuli to Piezo1 knockdown cells, but by applying a multiaxial stretch stimuli to knockout cells, the exact contribution of Piezo1 in the cellular response to stretch stimuli could be obtained under more physiological conditions. Moreover, since our conclusions are based solely on evidence from in vitro experiments, we couldn't determine the pathophysiological relevance of our in vitro observations in regulation of IOP or outflow facility. Further study using animal models or perfusion experiment will be needed to determine this issue.  The present data has shown that Piezo1 regulates Ca 2+ influx in response to mechanical stretching and that PGE 2 is essential for Piezo1-mediated hTM cell contraction inhibition. Our study revealed a novel role for Piezo1 in hTM cells and proposed a new therapeutic strategy for the conventional outflow pathway.

Methods
Cell culture. Primary human trabecular meshwork (hTM) cells were isolated from human corneal scleral rims obtained from the Rocky Mountain Lions Eye Bank from 8 donors (both male and female, the mean age ± SD was 47.3 ± 25.9), as described previously 41 . All experiments were conducted according to the principles of the Declaration of Helsinki and approved by the ethics committee of the University of Tokyo. Written informed consent was obtained from donors. Briefly, corneal scleral rims maintained in Optisol were cut and divided into 6 to 8 sections. The trabecular meshwork strip was picked and isolated with forceps from sections. TM strips were cultured in a dish coated with collagen gel (Cellmatrix Type I-A, Nitta Gelatin Inc., Osaka, Japan). After the TM cells migrated from the tissue, the gel was digested with collagenase, and the collected TM cells were grown on dishes coated with 1 μg/mL fibronectin. hTM cells from passages 3 to 6 were used in subsequent experiments and maintained in Trabecular Meshwork Cell Medium (ScienCell Research Laboratories, Carlsbad, CA) ) containing 2% fetal bovine serum (ScienCell), 1% Trabecular Meshwork cell growth supplement (ScienCell) and 1% penicillin/streptomycin solution (ScienCell). The identity of the isolated cells was confirmed by the dexamethasone-induced upregulation of MYOC. After 7 days of exposure to vehicle (DMSO), 100 or 500 nM dexamethasone, dexamethasone enhanced MYOC expression (see Supplementary Fig. S2  Quantitative real-time polymerase chain reaction (qPCR) analysis. Total RNA was extracted from hTM cells at 24 h after siRNA transfection using ISOGEN (Nippon Gene, Tokyo, Japan). The concentration of Total RNA was measured by the absorbance at a wavelength of 260 nm using a spectrophotometer NanoDrop 2000 (Thermofisher, Waltham, MA). Complementary DNA (cDNA) was prepared using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan). Quantitative real-time PCR was run on a Thermal Cycler Dice Realtime System (Takara Bio Inc., Shiga, Japan) using SYBR Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc.). Standard curves were used to determine mRNA transcript copy number in individual reactions. Primers specific for TRPV1-6 and PIEZO1-2 were designed by us or purchased from Takara Bio Inc., as detailed in Table 1.
Calcium imaging and mechanical cell stretch stimulation. hTM cells or those 24 h after siRNA transfection were seeded on cover glass chambers (Iwaki, Shizuoka, Japan) or stretch chamber STB-CH-24 (STREX Inc., Osaka, Japan) coated with 1 μg/mL fibronectin. After washing with PBS, hTM were loaded with 0.1% BSA (Sigma) and 5 μM fluorescent Ca 2+ indicator Fluo-8 AM (AAT Bioquest, Inc., Sunnyvale, CA) for  AAG TTC CTG CTG CAG AAC TC  130  Reverse  TGC TCG TCA CAA ACT TCG TG   TRPV2  Forward AAA CTG CTG CAG GCG AAA TG  95  Reverse  GCA ACA GCG GTG AAG ATG AAC   TRPV3  Forward GTT AGC TAC CCG CAT TAA GCC TGA  107  Reverse  AGC AAT TCT GGA ATT CCC AGCTC   TRPV4  Forward TGC ATG CGC CAC CAT TTT TG  114  Reverse  TAT TGA GCA CCG GCA AAT 32 was applied directly to the cover glass chambers. STB-CH-24 was attached to a stretch device STB-150 (STB-150 in this study was modified to stretch up to 40% using STB-CH-24; STREX Inc.) on the stage of a fluorescence microscope and subjected to a single uniaxial stretch with a nominal 30% extension (1 way/s, 3.0 s pause) at room temperature. As evaluated from images of the chamber at 30% extension, the actual extension of the cell culture surface was about 20%. Fluorescent images were acquired under a fluorescence microscope (Keyence, Osaka, Japan). The fluorescence intensity of Fluo-8 was quantified using ImageJ software (https ://image j.nih.gov/ij/downl oad.html). First, the captured images were stacked and the misalignment was corrected with the StackReg plugin. Next, the image with the highest fluorescence intensity from the cells was binarized by setting a threshold value by the triangle method, and the cells were selected with Analyze particles. Finally, the fluorescence intensity of the selected cells was measured on all images. The change ratio (F T /F 0 , F max /F 0 ) was calculated using peak and basal values. F T , F max , and F 0 represent the fluorescence intensity at that time, the maximum fluorescence intensity, and the fluorescence intensity before stimulation, respectively.
Gel contraction assay. Gel contraction assay was performed using the Collagen Gel Culturing Kit (Nitta Gelatin, Inc., Osaka, Japan) as per the manufacturer's instructions. Collagen type I, 10 × MEM, and reconstitution buffer (pH 7.3) were mixed at a ratio of 7:1:1 at 4 °C. The final hTM cell concentration was adjusted to 1 × 10 6 cells/mL. The resultant mixture and cell suspension were mixed at a 9:1 ratio and 500 μL of the mixture was added to each well of 24-well plates and incubated at 37 °C to allow for collagen gel formation. After 60 min, gels were placed in 6-cm dishes containing 5 mL Trabecular Meshwork Cell Medium with DMSO, 10 μM Yoda1, or PGE 2 (1, 10, or 100 nM). After 0, 12, 24, 48, and 72 h, gels were imaged with a gel imaging device, and the area was quantified with ImageJ software (https ://image j.nih.gov/ij/downl oad.html).