Glaucoma Activation of FP and EP2 Receptors Differently Modulates Myofibroblast Transition in a Model of Adult Primary Human Trabecular Meshwork Cells

Georges Kalouche,1,2 Fanny Beguier,1 Michael Bakria,1 Stephane´ Melik-Parsadaniantz,1 Caroline Leriche,3 Thomas Debeir,3 William Rostene,` 1 Christophe Baudouin,1,4 and Xavier Vige´2

1Sorbonne Universites,´ Universite´ Pierre et Marie Curie, Institut National de la Sante´ et de la Recherche Medicale,´ Centre National de la Recherche Scientifique, Institut de la Vision, Paris, France 2Sanofi Research & Development, Translational Sciences Unit, Chilly-Mazarin, France 3Sanofi Research & Development, Ophthalmology, Paris, France 4Quinze-Vingts National Ophthalmology Hospital, Paris, France

Correspondence: Georges Kalouche, PURPOSE. Prostaglandin F2a analogues are the first-line medication for the treatment of ocular 1 Avenue Pierre Brossolette, 91385 hypertension (OHT), and EP2 receptor agonists are under clinical development for Chilly-Mazarin, France; this indication. The goal of this study was to investigate the effects of F prostanoid (FP) and georges.kalouche@sanofi.com. EP2 receptor activation on the myofibroblast transition of primary trabecular meshwork (TM) Submitted: July 13, 2015 cells, which could be a causal mechanism of TM dysfunction in glaucoma. Accepted: February 13, 2016 METHODS. Human primary TM cells were treated with either or butaprost and TGF- Citation: Kalouche G, Beguier F, Bakria b2. Trabecular meshwork contraction was measured in a three-dimensional (3D) TM cell– M, et al. Activation of prostaglandin FP populated collagen gel (CPCG) model. Expression of a-smooth muscle actin (a-SMA) and and EP2 receptors differently modu- phosphorylation of myosin light chain (MLC) were determined by Western blot. Assembly of lates myofibroblast transition in a model of adult primary human tra- actin stress fibers and collagen deposition were evaluated by immunocytochemistry. becular meshwork cells. Invest Oph- Involvement of p38, extracellular signal-regulated kinase (ERK), and Rho-associated kinase thalmol Vis Sci. 2016;57:1816–1825. (ROCK) pathways as well as matrix metalloproteinase activation was tested with specific DOI:10.1167/iovs.15-17693 inhibitors.

RESULTS. In one source of validated adult TM cells, latanoprost induced cell contraction as observed by CPCG surface reduction and increased actin polymerization, a-SMA expression, and MLC phosphorylation, whereas butaprost inhibited TGF-b2–induced CPCG contraction, actin polymerization, and MLC phosphorylation. Both agonists inhibited TGF-b2–dependent collagen deposition. The latanoprost effects were mediated by p38 pathway.

CONCLUSIONS. Latanoprost decreased TM collagen accumulation but promoted a contractile phenotype in a source of adult TM cells that could modulate the conventional outflow pathway. In contrast, butaprost attenuated both TM contraction and collagen deposition induced by TGF-b2, thereby inhibiting myofibroblast transition of TM cells. These results open new perspectives for the management of OHT. Keywords: FP receptor, EP2 receptor, trabecular meshwork, TGF-b2, myofibroblast transition, contraction, collagen

rimary open-angle glaucoma (POAG) is the leading cause of effect of agents disrupting the TM actomyosin system P irreversible blindness worldwide.1 Elevation of intraocular demonstrates that the TM contraction state regulates conven- pressure (IOP) is the most critical risk factor for glaucoma and tional AH outflow.14 It could therefore be hypothesized that is associated with a dysfunction of the trabecular meshwork fibrotic mechanisms may be responsible for the TM alterations (TM) responsible for an increased resistance to the aqueous observed in POAG, in which TM cells undergo a deregulated humor (AH) outflow. The TM of POAG patients is characterized myofibroblast transition promoted by fibrotic factors like TGF- by a decreased TM cell number,2,3 an accumulation of b2. Indeed, the acquisition of a myofibroblastic phenotype by extracellular matrix (ECM),4–6 and an increased rigidity7 that resident cells is a common feature of all fibrotic pathologies and may account for IOP elevation. Alterations of the TM is defined by collagen accumulation and increased contractile biophysical properties have also been extensively linked to properties.15,16 the increased concentration of the profibrotic transforming Latanoprost, a prostaglandin (PG) F2a analogue, is the first F growth factor (TGF)-b2 in the AH of glaucomatous patients.8–10 prostanoid (FP) receptor agonist approved for POAG treatment The TM tissue is mainly composed of ECM proteins11 and and is currently used as first-line therapy.17 However, agonists possesses contractile features as highlighted by the presence of of another prostanoid receptor, EP2, have also been shown to a subpopulation of resident myofibroblastic cells expressing a- promote potent hypotensive effects in several animal mod- smooth muscle actin (a-SMA).12,13 Moreover, the IOP-lowering els18,19 and are currently in clinical development for the

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treatment of ocular hypertension (OHT).20–22 The action of (DMSO). The cells were used for experiments from passages 2 both FP and EP2 agonists seems to be mediated by an increased to 5. Trabecular meshwork cells were serum starved for 24 uveoscleral outflow pathway through activation of matrix hours before pretreatment with inhibitor compounds or 0.1% metalloproteinases (MMP) and ECM remodeling.18,23–26 How- DMSO as a control for 30 minutes unless otherwise stated. ever, their effects on the TM outflow facility, the main route for Dimethyl sulfoxide (0.1%), 1 lM butaprost, or 1 lM latanoprost AH drainage, is still controversial.27,28 Nonetheless, the was added to the cell medium with or without TGF-b2. presence of both FP and EP2 receptors on TM tissues,29,30 as well as their potential effects on TM cell survival (Kalouche et Immunocytochemistry al., unpublished observations, 2015), suggests that PG ana- logues may have direct physiological functions on TM cells. The three batches of TM cells were grown on poly-L-lysine– F prostanoid and EP2 are respectively qualified as contrac- coated 96-well l-plates (Ibidi, Planegg / Martinsried, Germany). tile and relaxant receptors due to their effect on smooth For the detection of collagen deposition, L-ascorbic acid 2- muscle cell contraction.31–35 Furthermore, EP2 receptor phosphate (Sigma-Aldrich Corp.) was added to the TM cell mediates Schlemm’s canal endothelial cell relaxation36 and medium to allow collagen maturation.40 Ninety-six hours after has been shown to regulate the mechanisms of contractility cell treatment with the various compounds tested, TM cells and relaxation in the TM.35,36 F prostanoid receptor has also were fixed either with ice-cold methanol for the detection of been implicated in the development of pulmonary37 and collagen deposition or fibronectin, or with 4% paraformaldehyde myocardial fibrosis,38 while loss of EP2 receptor exacerbates for the detection of actin stress fibers, a-SMA, and vinculin. The lung fibrotic lesions.39 This dichotomy of action between FP cells were treated with 0.1% Triton X-100 and 3% normal goat and EP2 receptors may thus potentially lead to different serum (NGS) for 1 hour and then incubated overnight at þ48C outcomes on the TM alterations observed in POAG. with relevant antibodies in a 1% NGS solution. Wells were The aim of the present work was to compare the effects of washed three times and incubated with appropriate Alexa FP and EP2 agonists on the myofibroblast transition of TM cells. Fluor–conjugated secondary antibodies (Life Technologies) and 0 For that purpose, TM contraction, myofibroblast markers, and 4 ,6-diamidino-2-phenylindole (DAPI) or TO-PRO-3 for 2 hours. collagen deposition were evaluated in the presence of TGF-b2 For the detection of actin stress fibers, Alexa Fluor 546– and PG analogues. We show that the FP agonist latanoprost conjugated phalloidin (Life Technologies) was incubated with promotes TM cell contraction, increases myofibroblastic DAPI for 2 hours. Plates were then rinsed in DPBS four times and markers, and decreases TGF-b2–mediated collagen deposition. wells kept in Dulbecco’s phosphate buffered saline (DPBS) four On the other hand, the EP2 agonist butaprost inhibits TGF-b2– times and wells kept in DPBS for fluorescence detection. Total mediated myofibroblast transition of TM cells by inhibiting the fluorescence was measured by a Tecan M1000 plate reader TGF-b2–dependent contraction and collagen deposition. (M¨annedorf, Switzerland) at the appropriate wavelengths. Pictures were obtained by an inverted Olympus FV1000 laser scanning confocal microscope (Tokyo, Japan). Z-sections were METHODS processed by ImageJ software (http://imagej.nih.gov/ij/; provid- ed in the public domain by the National Institutes of Health, Reagents Bethesda, MD, USA) by maximum-intensity projection. Butaprost and latanoprost free acid forms and AL-8810 were purchased from Cayman (Ann Harbor, MI, USA). The inhibitors Immunoblotting FR180204, SB203580, Y-27632 dihydrochloride, and marima- For Western blot analysis, cell supernatants and cell lysis in stat as well as dexamethasone were from Sigma-Aldrich Corp. radioimmunoprecipitation assay (RIPA) buffer supplemented (St. Louis, MO, USA). Forskolin was obtained from EMD with proteases and phosphatase cocktail inhibitors 2 and 3 (all Millipore (Billerica, MA, USA). Recombinant human TGF-b2 from Sigma-Aldrich Corp.) were collected. After centrifugation was purchased from PeproTech (Rocky Hill, NJ, USA). The anti- for 10 minutes, equal amounts of proteins were separated by human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) SDS-polyacrylamide gel electrophoresis and transferred to (1/20000) and phospho(Thr18/Ser19)-myosin light chain nitrocellulose membranes (all from Life Technologies). Mem- (MLC) (1/1000) antibodies were from Technol- branes were blocked with Tris-buffered saline containing 0.1% ogies (Danvers, MA, USA), while anti-human a-SMA, collagen Tween-20 (TBS-T) and 5% nonfat dry milk for 1 hour. Primary type I (1/400), vinculin, fibronectin, and myocilin (1/1000) antibodies were incubated overnight at þ48C in a 5% bovine antibodies were from Sigma-Aldrich Corp. serum albumin TBS-T solution. Membranes were washed three times in TBS-T and incubated with relevant horseradish Cell Culture and Treatments peroxidase–conjugated secondary antibodies (Vector Labora- tories, Burlingame, CA, USA). After three additional washes, Three commercially available primary human TM cells were membranes were developed with enhanced chemilumines- used (ScienCell Research Laboratories, Carlsbad, CA, USA). cence reagents (Pierce, Bonn, Germany) and imaged by the Two cell batches (nos. 5987 and 7278) were obtained from Fusion FX7 acquisition system (Vilber Lourmat, Torcy, France). fetal eyes (22 and 20 weeks of age, respectively), whereas a third cell batch (no. 4973) was isolated from the juxtacana- licular region of a 25-year-old Caucasian male. The cryopre- Cell Viability served primary human TM cells were thawed and plated on Total amount of adenosine triphosphate (ATP) of metabolically poly-L-lysine–coated surfaces and cultured in Dulbecco’s active cells was assessed by CellTiter-Glo (Promega, Madison, modified Eagle’s medium (DMEM) (Life Technologies, Carlsbad, WI, USA) following manufacturer’s instructions. Luminescence CA, USA) supplemented with 10% fetal bovine serum (FBS), was measured using a Tecan M1000 plate reader. sodium pyruvate, and antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin). Cells were maintained in humidified 5% CPCG Contraction CO2 and 95% air atmosphere at 378C. The first passage allowed cryopreservation of approximately 30 vials of TM cells in Rat tail collagen I solution (Corning, Inc., Corning, NY, USA) culture medium supplemented with 10% dimethyl sulfoxide was used at a sufficient final concentration of 3 mg/mL to avoid

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spontaneous TM cell–populated cell gel (CPCG) contraction. receptors. The TM cells from fetal origin showed a significant Cold stock collagen was mixed with DPBS (13 final concen- decrease in mRNA expression of the EP2 receptor (batch no. tration), 0.1 N NaOH (2.5% final concentration), and a solution 5987) and of the FP receptor (batch nos. 5987 and 7278) in containing TM cells. Trabecular meshwork cell concentration comparison with the adult human TM cells (batch no. 4973) was adjusted to 200,000 cells/mL in DMEM and quickly added (Fig. 1c). As fetal TM cells did not express the most specific to the collagen/DBPS/NaOH mixture kept on ice. After gentle marker of TM cells and, importantly, did not allow comparison mixing, 500 lL of the solution was poured into each well of a of the effects of EP2 and FP receptor agonists, only the adult TM 24-well plate. The plate was incubated in humidified 5% CO2 cells were used for subsequent experiments and are referred as and 95% air atmosphere at 378C for 15 minutes to allow gelling. hTM cells. Furthermore, phase-contrast microscopy revealed an Then 1 mL DMEM was added on the solidified CPCG, which elongated fibroblast-like phenotype of the hTM cells in culture were freed from the well borders using thin tweezers. Twenty- (Fig. 1d) as previously observed in the literature for adult hTM four hours after seeding in collagen gels, the cells were serum cells.44,45 Finally, hTM cells exhibited significant increases in a- starved for another 24 hours and then treated in the same way SMA, vinculin, fibronectin, and collagen type I levels, as well as as the cells grown on poly-L-lysine–coated surfaces. increased actin stress fibers in response to TGF-b2 (Fig. 1e) in Cell-populated collagen gels were imaged using a Leica Z6 accordance with the TGF-b2–dependent myofibroblastic transi- APO macroscope (Wetzlar, Germany). Cell-populated collagen tion of TM cells.46,47 gel pictures were calibrated using the well diameter, and CPCG In order to assess the acquisition of a contractile phenotype surfaces were calculated by ImageJ software and expressed as a by hTM cells and the effects of PG analogues, the cells were percentage of the initial surface measured prior to treatments. embedded in a collagen type I solution. After gelling, the generated three-dimensional (3D) model of hTM cells, called Quantitative Real-Time PCR hTM CPCG, was used as a force-sensing device as previously described.48 Transforming growth factor-b2 induced a time- and Total RNA was automatically isolated by a QIACube system concentration-dependent decrease of the hTM CPCG surface using a RNeasy kit with a DNase digestion step (all from (Fig. 2a), suggesting a contraction of hTM cells. Transforming Qiagen, Valencia, CA, USA). Complementary DNA was growth factor-b2 was significantly effective in inducing hTM synthesized with a reverse transcription kit using random CPCG contraction from 0.02 ng/mL and reduced CPCG surface primers for amplification (Applied Biosystems, Foster City, CA, to approximately 20% of its initial size after 96 hours at the 2 ng/ USA) in a T100 thermal cycler (Bio-Rad Laboratories, Hercules, mL concentration. Furthermore, the EP2 agonist butaprost and CA, USA). For real-time quantitative PCR, cDNA, TaqMan the FP agonist latanoprost were incubated either alone or in the Universal PCR Master Mix, and TaqMan gene expression assays presence of 0.2 ng/mL TGF-b2, a concentration producing (PTGER2: Hs04183523_m1, PTGFR: Hs00168763-m1, CO- approximately 50% contraction at 96 hours. Latanoprost L1A1: Hs00164004_m1, GAPDH: Hs99999905_m1) were significantly induced hTM CPCG contraction from 48 hours mixed in a 96-well optical reaction plate (all from Applied and reduced CPCG surface by 22 6 3% (mean 6 SEM) at 96 Biosystems). Cycling conditions included an initial denatur- hours (Fig. 2b). Moreover, latanoprost significantly potentiated ation step at 958C for 10 minutes, followed by 40 cycles TGF-b2–mediated hTM CPCG contraction at 96 hours. Butaprost, consisting of a 15-second denaturation at 958C and a 1-minute on the other hand, did not modify hTM CPCG by itself but annealing and primer extension at 608C. GAPDH was used as significantly inhibited TGF-b2–mediated contraction (Fig. 2c). housekeeping gene, and amplification of its cDNA was done in The relaxing effect of butaprost was observed throughout the 96 parallel with the gene of interest. Threshold cycle (Ct) values hours and was most pronounced at 24 hours as observed by a 20 were determined using the Applied Biosystems software. 6 4% (mean 6 SEM) increase of CPCG surface in comparison to TGF-b2 alone. As the EP2 receptor is mainly coupled to Gas protein,31 we used forskolin, an adenylate cyclase activator, Statistical Analysis which increases cAMP levels. Forskolin (10 lM) completely All values were expressed as mean 6 SEM of at least three inhibited TGF-b2–dependent contraction over the 96-hour independent experiments. Data were analyzed using GraphPad experiment (data not shown), suggesting that the EP2 recep- Prism 6 (GraphPad Software Inc., San Diego, CA, USA). tor–dependent inhibition of contraction is mediated by cAMP. Significance was tested by a 2-way ANOVA with Sidak’s or In correlation with the results from the hTM CPCG Tukey’s correction for multiple comparisons to compare the contraction assay, latanoprost increased the formation of actin effects of TGF-b2 and PG analogues, respectively. Significance stress fibers and potentiated TGF-b2–mediated actin bundle was denoted as follows: P < 0.05 (*), P < 0.01 (**), or P < polymerization at 96 hours (Fig. 3a). Measurement of hTM cell 0.001 (***). viability showed that latanoprost, as well as TGF-b2, increased viable hTM cells at 96 hours (Fig. 3b), and these results were correlated with hTM cell nuclei counting (data not shown). RESULTS Furthermore, butaprost inhibited TGF-b2–dependent actin stress fiber formation without impacting hTM cell viability. Glucocorticoid-dependent secretion of myocilin being the most These data were not observed with the two fetal TM strains specific phenotypic TM cell marker described,41–43 the three (data not shown). We also studied the expression of the widely different batches of commercially available human primary TM used myofibroblast marker, a-SMA,49 and the phosphorylation cells were challenged with dexamethasone for 96 hours. The of the regulatory light chain of myosin II (MLC) leading to an batch from an adult donor (no. 4973) showed an increased increase in actomyosin contractility.50 Latanoprost alone secretion of myocilin in TM supernatants in response to slightly increased the expression of a-SMA at 24 and 96 hours dexamethasone, while myocilin was not detected in the although not significantly (Fig. 3c). In comparison, a-SMA supernatants of the two other strains from fetal origin (nos. expression was strongly increased at 2 ng/mL TGF-b2. 5987 and 7278) (Fig. 1a). The absence of myocilin secretion was Furthermore, MLC phosphorylation was enhanced by latano- not due to a potential toxic effect of dexamethasone, as the cell prost and TGF-b2 but following different kinetics. Latanoprost viability seemed even slightly increased with the glucocorticoid induced a weak but significant phosphorylation at 24 hours, treatment (Fig. 1b). Before comparing the effects of FP and EP2 persisting at 96 hours, while TGF-b2–induced phosphorylation receptor agonists, we evaluated the expression level of both decreased by 96 hours. In contrast, butaprost inhibited MLC

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FIGURE 1. Characterization of the human trabecular meshwork cells. Three batches of TM cells from ScienCell were grown on poly-L-lysine–coated surfaces and serum starved for 24 hours. (a) Cells were incubated in the presence of 100 nM dexamethasone (dex) for 96 hours. Cell supernatants were subjected to Western blot to detect myocilin secretion. (b) Cell viability was measured by CellTiter-Glo 96 hours after treatment with dexamethasone (100 nM) and expressed as a percentage of the control DMSO condition (mean 6 SEM, n ¼ 3). **P < 0.01, ***P < 0.001 (2-way ANOVA with Sidak’s multiple comparison test). (c) mRNA expression of the EP2 and the FP receptors was analyzed after the 24-hour serum starvation period. mRNA level was expressed as fold change over the control condition (mean 6 SEM, n ¼ 3). *P < 0.01, **P < 0.01, ***P < 0.001 (1- way ANOVA with Tukey’s multiple comparison test). (d) The adult hTM cells (batch no. 4973) were imaged using a contrast-phase microscope. (e) Representative confocal pictures of actin stress fibers (red), a-SMA, vinculin, fibronectin, and collagen type I (green), counterstained with TO-PRO-3 (gray), were obtained after treatment of adult hTM cells (no. 4973) with TGF-b2 for 96 hours.

phosphorylation at 24 hours (Fig. 3c) in correlation with the associated kinase (ROCK), an upstream effector of MLC significant relaxing effect of butaprost on TGF-b2–mediated phosphorylation, suppressed the latanoprost-dependent hTM hTM CPCG contraction (Fig. 2c). CPCG contraction (Fig. 4c) without inducing cytotoxic effect at The contracting effect of latanoprost was further studied. 96 hours (data not shown), confirming the involvement of MLC The latanoprost-dependent hTM CPCG contraction was similar phosphorylation. Moreover, as extracellular signal-regulated at 1 lM and 100 nM (Fig. 4a) and was blocked by the FP kinase (ERK) and p38 signaling pathways are known to play a receptor antagonist (Fig. 4b), AL-8810 (30 lM),51 without role in fibroblast contraction,52 their implication in the affecting hTM cell viability at 96 hours (data not shown). These results demonstrated that the contracting effect mediated by latanoprost-mediated contraction was tested by using latanoprost was specific to the FP receptor. To gain insight into FR180204 (10 lM), an ERK inhibitor, and SB203580 (10 lM), the mechanism of latanoprost-induced contraction, several a p38 inhibitor (Figs. 4d, 4e). No cytotoxic effect was observed compounds were used to assess the contribution of signaling at 96 hours (data not shown), but the p38 inhibitor abolished pathways. Firstly, Y-27632 (10 lM), an inhibitor of the Rho- latanoprost-mediated contraction while the ERK inhibitor

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FIGURE 2. Latanoprost induces contraction of hTM cells while butaprost relaxes TGF-b2–mediated contraction. hTM cells were embedded into a 3D collagen matrix to evaluate CPCG contraction. hTM CPCG were imaged before addition of compounds to define the initial area and then at 24, 48, and 96 hours using a macroscope. hTM CPCG surfaces were expressed as a percentage of the initial area. (a) hTM CPCG were treated with various concentrations of TGF-b2(left, mean 6 SEM, n ¼ 3). Illustrations of the hTM CPCG contraction were obtained at 96 hours (right). (b) hTM CPCG were treated with either 0.1% DMSO, as a control, or 1 lM latanoprost (latano) with or without 0.2 ng/mL TGF-b2 (mean 6 SEM, n ¼ 4). (c) hTM CPCG were treated with either 0.1% DMSO, as a control, or 1 lM butaprost (buta) with or without 0.2 ng/mL TGF-b2 (mean 6 SEM, n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001 when comparing DMSO with PG analogue for each time point with or without TGF-b2 (2-way ANOVA with Tukey’s multiple comparison test).

induced an additive contraction, suggesting that the FP- dependent inhibition of collagen deposition was not modified dependent contraction involved p38 but not ERK activation. by the ERK inhibitor FR180204 (10 lM) but abolished by the A rise in collagen synthesis is another characteristic of p38 inhibitor SB203580 (10 lM) (Figs. 6b, 6c), while these myofibroblast transition.16 Consequently, we evaluated by inhibitors did not affect collagen deposition in the control immunocytochemistry the end product of the complex conditions (data not shown). Neither of those inhibitors collagen production process, the deposition of the insoluble modulated butaprost effects. collagen matrix. After 96 hours of culture, hTM cells did not produce significant amounts of extracellular mature collagen (Fig. 5a). On the other hand, TGF-b2 induced a significant DISCUSSION increase in the pericellular collagen deposited in a reticular pattern. Interestingly, both PG analogues decreased the The myofibroblastic transition of TM cells, defined by the extracellular accumulation of collagen induced by TGF-b2in acquisition of a contractile phenotype and an increased collagen hTM. However, such modulation of TGF-b2–dependent colla- synthesis, could be responsible for the TM dysfunction observed gen deposition by PG analogues was not observed in fetal TM in glaucomatous patients. If PG analogues mediate potent cells (data not shown). Furthermore, the mRNA expression hypotensive effects and undoubtedly increase the uveoscleral level of the pro-a1 chain encoded by the COL1A1 gene, a major outflow pathway, their effects on the pathophysiology of the TM component of collagen type I, increased 6 hours after TGF-b2 remain largely unknown. We demonstrate, in one validated adult treatment, culminating at 24 hours and persistent to 48 hours hTM cell model, that latanoprost, the most commonly used (Fig. 5b). Butaprost significantly decreased TGF-b2–induced glaucoma medication, induces cell contraction and decreases COL1A1 upregulation at 6 and 24 hours while latanoprost did TGF-b2–mediated collagen deposition. Alternatively, the EP2 not, suggesting that only butaprost inhibited the transcription- agonist butaprost inhibits both TGF-b2–dependent contraction al activity of the TGF-b2 signaling pathway. and collagen deposition and, consequently, the myofibroblast The remodeling effect of PG analogues on the uveoscleral transition of hTM cells. outflow pathway has been extensively attributed to MMP activation.24–26 Consequently, the broad-spectrum inhibitor of To avoid the inherent bias of immortalized cell lines, MMP, marimastat (10 lM), was evaluated (Fig. 6a). Marimastat primary hTM cells were evaluated based on the dexametha- did not modify collagen deposition in the control conditions sone-dependent secretion of myocilin. One source of adult TM (data not shown) but significantly increased collagen deposi- cells was validated while two batches of fetal TM cells failed to tion induced by TGF-b2, suggesting that MMP did regulate show an increased secretion of myocilin in response to collagen turnover in TM cell cultures. However, MMP dexamethasone. Furthermore, PG receptor transcripts were activation did not seem to be involved in the inhibitory effect significantly more strongly expressed in the adult TM strain. of PG analogues on TGF-b2–induced collagen deposition, as While other markers of differentiation in TM cell lineage would marimastat did not block their effect. In contrast, latanoprost- be necessary to validate these findings, as well as extension to

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FIGURE 3. Latanoprost, but not butaprost, increases myofibroblast markers in hTM cells. hTM cells were grown on a poly-L-lysine–coated surface to assess actin fiber formation, a-SMA expression, and phosphorylation of MLC. (a) hTM cells were treated with either 0.1% DMSO, as a control, or 1 lM butaprost or latanoprost with or without 2 ng/mL TGF-b2. Representative confocal pictures of actin stress fibers (red) counterstained with DAPI (blue) were obtained at 96 hours (left). Total fluorescence was measured at 570 nm (right, mean 6 SEM, n ¼ 3). (b) hTM cells were treated with either 0.1% DMSO, as a control, or 1 lM butaprost or latanoprost with or without 2 ng/mL TGF-b2. Cell viability was measured at 96 hours by CellTiter-Glo and expressed as a percentage of the control DMSO condition (mean 6 SEM, n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001 when comparing DMSO, butaprost, and latanoprost with or without TGF-b2 (2-way ANOVA with either Tukey’s or Sidak’s multiple comparison tests for the effects of PG analogues or TGF-b2, respectively). (c) hTM cells were treated with either 0.1% DMSO, as a control, or 1 lM butaprost (buta) or latanoprost (latano) with or without TGF-b2 at the specified concentrations. Cell lysates obtained at the indicated times were analyzed by Western blot. Values were obtained from the densitometric analyses of three independent experiments and expressed as fold change over the control condition (mean 6 SEM). *P < 0.05, **P < 0.01, ***P < 0.001 when comparing the tested conditions to the DMSO control (2-way ANOVA for each TGF-b2 concentration with Sidak’s multiple comparison test).

various primary hTM cell strains, the observed differences may significantly inhibited TGF-b2–dependent contraction, which be due to the early stage of development of the fetal hTM cells. was correlated with MLC phosphorylation inhibition and a The effect of PG analogues on adult hTM cell contraction decrease in actin stress fiber formation. Interestingly, TM from was thus evaluated by embedding the cells in a 3D collagen cynomolgus monkeys treated for a year with latanoprost matrix, which allows an indirect measurement of the cellular exhibited myofibroblastic-like cells in the cribriform region,23 traction forces applied to the ECM.48 The assay parameters suggesting that our in vitro observations have physiological were defined to avoid hTM CPCG contraction in control relevancy in vivo. Other studies using CPCG as a force-sensing conditions. For that purpose, the adult hTM cells were serum device showed that latanoprost induced contraction of human starved for 24 hours and the collagen gel was used at a Tenon fibroblasts (HTF) from the conjunctiva and correlated sufficiently high concentration. In these settings, latanoprost, with the formation of actin stress fibers, consistent with our just like the profibrotic cytokine TGF-b2, induced a time- and observations.53 In contrast, endothelin-dependent short-time concentration-dependent contraction of the hTM CPCG. contraction of bovine TM strips has been reported to be Furthermore, the latanoprost-mediated contraction correlated inhibited by FP receptor agonists, suggesting that the FP with increased actin stress fiber formation, MLC phosphoryla- receptor activation could potentially lead to opposite effects at tion, and a-SMA expression, known hallmarks of myofibroblast shorter time points.54 Finally, EP2 receptor activation de- transition. In contrast, if butaprost had no effect per se, it creased CPCG contraction by dermal fibroblasts.55,56 These

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FIGURE 4. Latanoprost contraction is dependent on FP receptor and is mediated by p38 but not by ERK activation. hTM cells were embedded into a 3D collagen matrix to evaluate CPCG contraction induced by latanoprost. hTM CPCG were imaged before addition of compounds to define the initial area and then at 24, 48, and 96 hours using a macroscope. hTM CPCG surfaces were expressed as the percentage of the initial area. (a) hTM CPCG were treated with 0.1% DMSO, as a control, or various concentrations of latanoprost (mean 6 SEM, n ¼ 3). hTM CPCG were preincubated for 30 minutes with either (b) the FP antagonist AL-8810 (30 lM), (c) the ROCK inhibitor Y-27632 (10 lM), (d) the ERK inhibitor FR180204 (10 lM), or (e) the p38 inhibitor SB203580 (10 lM). Control (0.1% DSMO) or 1 lM latanoprost (latano) was then added to the cell medium (mean 6 SEM, n ¼ 3–4). *P < 0.05, **P < 0.01, ***P < 0.001 when comparing DMSO with latanoprost for each time point (2-way ANOVA with Tukey’s multiple comparison test).

authors also demonstrated that the butaprost relaxing effect tested. A p38 inhibitor suppressed latanoprost-induced con- was mediated by activation of adenylate cyclase and generation traction while an ERK inhibitor resulted in hTM CPCG of cAMP as we also observed in our hTM CPCG model by using contraction by itself. Interestingly, these results are in forskolin (data not shown). accordance with a previous report on HTF cells showing that To further explore the mechanism of action of latanoprost- latanoprost-induced HTF CPCG contraction was dependent on mediated CPCG contraction, we investigated the signaling p38 activation but also seemed mediated by ERK and c-Jun N- pathways involved on only the adult hTM cell batch. Firstly, we terminal kinase signaling.53 In another study, p38 inhibition showed that latanoprost-mediated contraction was dependent blocked TGF-b–mediated HTF CPCG contraction and myofi- on ROCK activation. Because ROCK is an upstream activator of broblast transition while ERK signaling inhibition, by U0126, MLC phosphorylation, this result is in accordance with our induced a spontaneous contraction of HTF CPCG.52 Hence, we finding of increased phosphorylation of MLC induced by extended these observations made on HTF to TM cells and latanoprost treatment. Interestingly, ROCK inhibitors have showed that latanoprost induced the generation of contractile been shown to increase the outflow pathway and to reduce forces in TM cells, a characteristic of myofibroblast phenotype, IOP,57–59 suggesting that latanoprost-mediated contraction through ROCK and p38 activation. through ROCK signaling could decrease the outflow through Collagen production is another feature of myofibroblast the TM. However, these results should be extended to other transition. Both latanoprost and butaprost inhibited TGF-b2– TM cell strains and confirmed by in vivo experiments. mediated collagen deposition. Interestingly, studies on cynomol- Furthermore, because p38 and ERK signaling have been gus monkeys treated for a year with either latanoprost23 or involved in contractile and actomyosin relaxation process- butaprost18 reported a loss of collagen materials within the TM es,46,52 their contribution to latanoprost contraction was in accordance with our present in vitro results. Furthermore, the

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FIGURE 5. Latanoprost and butaprost inhibit TGF-b2–mediated collagen deposition in hTM cells but differently affect TGF-b2–dependent collagen I transcription. hTM cells were grown on a poly-L-lysine–coated surface to assess collagen type I production. For that purpose, hTM cells were treated with either 0.1% DMSO, as a control, or 1 lM butaprost or latanoprost with or without 2 ng/mL TGF-b2. (a) Representative confocal pictures (left) were obtained at 96 hours by staining collagen deposition (green) and counterstaining with DAPI (blue). Total fluorescence was measured at 520 nm (right, mean 6 SEM, n ¼ 12). (b) mRNA expression of collagen type I gene COL1A1 was analyzed at the indicated times. mRNA level was expressed as fold change over the control condition (mean 6 SEM, n ¼ 3). **P < 0.01, ***P < 0.001 when comparing DMSO, butaprost, and latanoprost with or without TGF-b2 (2-way ANOVA with either Tukey’s or Sidak’s multiple comparison tests for the effects of PG analogues or TGF- b2, respectively).

FP agonist fluprostenol has been shown to decrease expression mediated inhibition of collagen deposition, but not ERK, while of collagen types IV and VI induced by connective tissue growth butaprost effects were not affected by inhibition of either factor (CTGF) in cultured TM cells.60 To gain insight into the pathway. These results confirm the pivotal role of p38 pathway mechanism of PG analogue–mediated inhibition of collagen in latanoprost effects and provide further evidence of the deposition, several hypotheses were tested on the only adult activation of distinct signaling pathways in the regulation of source of hTM cells used. Firstly, at the transcriptional level, the collagen deposition by FP and EP2 agonists. Finally, as the activation of EP2 receptor led to the attenuation of mRNA mechanism of action of FP agonists is known to induce MMP expression of the major component of collagen type I, the pro- activation in the ciliary body and the sclera,24–26 we tested the a1 chain, while latanoprost did not. These results are in involvement of MMP activation in our model. The broad- agreement with the suppressed mRNA level of a1-collagen spectrum MMP inhibitor, marimastat, did not block the effects of induced by an EP2 agonist in lung fibroblasts.61 Moreover, we either PG analogue, suggesting that the inhibition of collagen showed that p38 activation was involved in latanoprost- accumulation is independent of MMP activation in hTM cells.

FIGURE 6. PG analogue–mediated inhibition of TGF-b2–dependent collagen deposition is independent of MMP activation, but the latanoprost effect depends on p38 activation. hTM cells were grown on a poly-L-lysine–coated surface and preincubated for 30 minutes with (a) the broad-spectrum MMP inhibitor marimastat (10 lM), (b) the ERK inhibitor FR180204 (10 lM), or (c) the p38 inhibitor SB203580 (10 lM). hTM cells were then treated with either 0.1% DMSO, as a control, or 1 lM butaprost or latanoprost with or without 2 ng/mL TGF-b2 for 96 hours. Total fluorescence from collagen deposition stained with an anti-human collagen type I antibody and Alexa Fluor 488–conjugated secondary antibody was measured at 520 nm (mean 6 SEM, n ¼ 4–5). *P < 0.05, **P < 0.01, ***P < 0.001 when comparing DMSO, butaprost, and latanoprost with or without TGF-b2 (2-way ANOVA with Tukey’s multiple comparison test).

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These results are in accordance with the absence of MMP 7. Last JA, Pan T, Ding Y, et al. Elastic modulus determination of modulation by latanoprost in the TM, as opposed to the normal and glaucomatous human trabecular meshwork. Invest profound upregulation of MMP in the ciliary body.24 Thus, Ophthalmol Vis Sci. 2011;52:2147–2152. mechanisms other than MMP regulation can be involved in PG 8. Tripathi RC, Li J, Chan WF, Tripathi BJ. Aqueous humor in analogue–mediated inhibition of ECM accumulation to poten- glaucomatous eyes contains an increased level of TGF-beta 2. tially lead to increased AH drainage through the TM. Exp Eye Res. 1994;59:723–727. Myofibroblasts are able to develop strong contractile forces 9. Gottanka J, Chan D, Eichhorn M, Lutjen-Drecoll¨ E, Ethier CR. and synthesize ECM components crucial for normal wound Effects of TGF-b2 in perfused human eyes. Invest Ophthalmol healing processes and for endowing resistance to tissues Vis Sci. 2004;45:153–158. subjected to high pressures. However, the increased myofibro- 10. Shepard AR, Cameron Millar J, Pang IH, Jacobson N, Wang blast cell population in the TM, due to fibrotic agents such as WH, Clark AF. Adenoviral gene transfer of active human TGF-b2 or other pathologic events, could lead to the known transforming growth factor-beta 2 elevates intraocular pres- alterations observed in the TM of POAG patients. Here, we sure and reduces outflow facility in rodent eyes. Invest provide evidence, in our model of adult hTM cells, that the PG Ophthalmol Vis Sci. 2010;51:2067–2076. analogue latanoprost, used to treat OHT, promotes a contrac- 11. Keller KE, Aga M, Bradley JM, Kelley MJ, Acott TS. Extracellular tile phenotype and decreases collagen deposition induced by matrix turnover and outflow resistance. Exp Eye Res. 2009;88: TGF-b2 through activation of p38. The two mechanisms 676–682. differently regulate the AH outflow pathway, as a decrease of 12. De Kater A, Shahsafaei A, Epstein DL. Localization of smooth ECM accumulation would reduce the outflow resistance muscle and nonmuscle actin isoforms in the human aqueous whereas TM contraction would increase it. Nonetheless, the outflow pathway. Invest Ophthalmol Vis Sci. 1992;33:424– potent hypotensive effect of FP agonists should overwhelm the 429. potential adverse effect of TM cell contraction. In contrast, the 13. Gonzalez JM, Heur M, Tan JCH. Two-photon immunofluores- ability of the EP2 receptor to both relax TM cell contractility cence characterization of the trabecular meshwork in situ. and decrease collagen deposition may be potentially beneficial Invest Ophthalmol Vis Sci. 2012;53:3395–3404. for long-term treatment of glaucoma, as both mechanisms 14. Tian B, Gabelt BT, Geiger B, Kaufman PL. The role of the decrease AH outflow resistance. Such novel data warrant actomyosin system in regulating trabecular fluid outflow. Exp extension to various adult primary hTM cell strains and Eye Res. 2009;88:713–717. validation in in vivo models. 15. Kendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: novel roles and mediators. Front Pharmacol. 2014;5:1–13. 16. Baum J, Duffy HS. Fibroblasts and myofibroblasts: what are we Acknowledgments talking about? J Cardiovasc Pharmacol. 2011;57:376–379. The authors thank Charlotte Vernhes (Sanofi Pasteur, Florida, USA) 17. Zhang K, Zhang L, Weinreb RN. Ophthalmic drug discovery: for critical reading and editing of the manuscript. They thank novel targets and mechanisms for retinal diseases and Stephane´ Fouquet and David Godefroy (Imaging Core Facility, glaucoma. Nat Rev Drug Discov. 2012;11:541–559. Vision Institute, France) and Sup’Biotech school (Villejuif, France) 18. Nilsson SFE, Drecoll E, Lutjen-Drecoll¨ E, et al. The prostanoid for their collaboration. They also thank Patrick Avenet (Sanofi, EP2 receptor agonist butaprost increases uveoscleral outflow France), who made this work possible. in the cynomolgus monkey. Invest Ophthalmol Vis Sci. 2006; Supported by Sanofi Research & Development and by the Vision 47:4042–4049. Institute (Paris). 19. Prasanna G, Carreiro S, Anderson S, et al. Effect of PF- Disclosure: G. Kalouche, Sanofi (E, F); F. Beguier, None; M. 04217329 a prodrug of a selective prostaglandin EP 2 agonist Bakria, None; S. Melik-Parsadaniantz, None; C. Leriche, Sanofi on intraocular pressure in preclinical models of glaucoma. Exp (E); T. Debeir, Sanofi (E); W. Rostene` , None; C. Baudouin, None; Eye Res. 2011;93:256–264. X. Vige´, Sanofi (E) 20. Schachar RA, Raber S, Courtney R, Zhang MA. A phase 2, randomized, dose-response trial of taprenepag isopropyl (PF- 04217329) versus latanoprost 0.005% in open-angle glaucoma References and ocular hypertension. Curr Eye Res. 2011;36:809–817. 1. Tham YC, Li X, Wong TY, Quigley HA, Aung T, Cheng CY. 21. Multi-center phase II study assessing the safety and efficacy of Global prevalence of glaucoma and projections of glaucoma DE-117 ophthalmic solution in subjects with primary open- burden through 2040. A systematic review and meta-analysis. angle glaucoma or ocular hypertension (SEE-1). Available at: https://clinicaltrials.gov/ct2/show/NCT02179008. Accessed Ophthalmology. 2014;121:2081–2090. October 28, 2015. 2. Alvarado J, Murphy C, Juster R. Trabecular meshwork 22. Safety and efficacy of AGN-210961 ophthalmic solution cellularity in primary open-angle glaucoma and nonglaucom- compared with ophthalmic solution in patients atous normals. Ophthalmology. 1984;91:564–579. with glaucoma or ocular hypertension. Available at: https:// 3. Grierson I, Howes RC. Age-related depletion of the cell clinicaltrials.gov/ct2/show/NCT01110499. Accessed October population in the human trabecular meshwork. Eye (Lond). 28, 2015. 1987;1:204–210. 23. Richter M, Krauss AHP, Woodward DF, Lutjen-Drecoll¨ E. 4. Lutjen-Drecoll¨ E, Shimizu T, Rohrbach M, Rohen JW. Quanti- Morphological changes in the anterior eye segment after tative analysis of ‘‘plaque material’’ between ciliary muscle tips long-term treatment with different receptor selective prosta- in normal- and glaucomatous eyes. Exp Eye Res. 1986;42:457– glandin agonists and a prostamide. Invest Ophthalmol Vis Sci. 465. 2003;44:4419–4426. 5. Rohen JW, Lutjen-Drecoll¨ E, Flugel¨ C, Meyer M, Grierson I. 24. El-Shabrawi Y, Eckhardt M, Berghold A, et al. Synthesis pattern Ultrastructure of the trabecular meshwork in untreated cases of matrix metalloproteinases (MMPs) and inhibitors (TIMPs) in of primary open-angle glaucoma (POAG). Exp Eye Res. 1993; human explant organ cultures after treatment with latanoprost 56:683–692. and dexamethasone. Eye. 2000;14:375–383. 6. Tektas OY, Lutjen-Drecoll¨ E. Structural changes of the 25. Gaton DD, Sagara T, Lindsey JD, Gabelt BT, Kaufman PL, trabecular meshwork in different kinds of glaucoma. Exp Weinreb RN. Increased matrix metalloproteinases 1, 2, and 3 Eye Res. 2009;88:769–775. in the monkey uveoscleral outflow pathway after topical

Downloaded from iovs.arvojournals.org on 09/28/2021 FP and EP2 Receptor Effects on Trabecular Meshwork Cells IOVS j April 2016 j Vol. 57 j No. 4 j 1825

prostaglandin F(2 alpha)-isopropyl ester treatment. Arch 44. Tamm ER, Siegner A, Baur A, Lutjen-Drecoll¨ E. Transforming Ophthalmol. 2001;119:1165–1170. growth factor-beta 1 induces alpha-smooth muscle-actin 26. Weinreb RN, Lindsey JD, Marchenko G, Marchenko N, Angert expression in cultured human and monkey trabecular M, Strongin A. Prostaglandin FP agonists alter metalloprotei- meshwork. Exp Eye Res. 1996;62:389–397. nase gene expression in sclera. Invest Ophthalmol Vis Sci. 45. Yu AL, Fuchshofer R, Kampik A, Welge-Lussen¨ U. Effects of 2004;45:4368–4377. oxidative stress in trabecular meshwork cells are reduced by 27. Lim KS, Nau CB, O’Byrne MM, et al. Mechanism of action of prostaglandin analogues. Invest Ophthalmol Vis Sci. 2008;49: bimatoprost, latanoprost, and in healthy subjects. A 4872–4880. crossover study. Ophthalmology. 2008;115:790–795. 46. Pattabiraman PP, Rao PV. Mechanistic basis of Rho GTPase- 28. Toris CB, Gabelt BT, Kaufman PL. Update on the mechanism of induced extracellular matrix synthesis in trabecular meshwork action of topical for intraocular pressure cells. Am J Physiol Cell Physiol. 2010;298:C749–C763. reduction. Surv Ophthalmol. 2008;53:S107–S120. 47. Pattabiraman PP, Maddala R, Rao PV. Regulation of plasticity 29. Schl¨otzer-Schrehardt U, Zenkel M, Nusing¨ RM. Expression and and fibrogenic activity of trabecular meshwork cells by rho localization of FP and EP prostanoid receptor subtypes in GTPase signaling. J Cell Physiol. 2014;229:927–942. human ocular tissues. Invest Ophthalmol Vis Sci. 2002;43: 48. Li B, Wang JHC. Application of sensing techniques to cellular 1475–1487. force measurement. Sensors. 2010;10:9948–9962. 30. Biswas S, Bhattacherjee P, Paterson C. receptor subtypes, EP1, EP2, EP3 and EP4 in human and 49. Hinz B. Formation and function of the myofibroblast during mouse ocular tissues–a comparative immunohistochemical tissue repair. J Invest Dermatol. 2007;127:526–537. study. Prostaglandins Leukot Essent Fatty Acids. 2004;71: 50. Conti MA, Adelstein RS. Nonmuscle myosin II moves in new 277–288. directions. J Cell Sci. 2008;121:11–18. 31. Hirata T, Narumiya S. Prostanoid receptors. Chem Rev. 2011; 51. Griffin BW, Klimko P, Crider JY, Sharif NA. AL-8810: a novel 111:6209–6230. prostaglandin F2 alpha analog with selective antagonist effects 32. Kaddour-Djebbar I, Ansari HR, Akhtar RA, Abdel-Latif AA. at the prostaglandin F2 alpha (FP) receptor. J Pharmacol Exp Species differences in the effects of on MAP Ther. 1999;290:1278–1284. kinase phosphorylation, myosin light chain phosphorylation 52. Meyer-Ter-Vehn T, Gebhardt S, Sebald W, et al. p38 inhibitors and contraction in bovine and cat iris sphincter smooth prevent TGF-beta-induced myofibroblast transdifferentiation muscle. Prostaglandins Leukot Essent Fat Acids. 2005;72:49– in human tenon fibroblasts. Invest Ophthalmol Vis Sci. 2006; 57. 47:1500–1509. 33. Poyer JF, Millar C, Kaufman PL. Prostaglandin F2 alpha effects 53. Liu Y, Ko JA, Yanai R, et al. Induction by latanoprost of on isolated rhesus monkey ciliary muscle. Invest Ophthalmol collagen gel contraction mediated by human tenon fibroblasts: Vis Sci. 1995;36:2461–2465. role of intracellular signaling molecules. Invest Ophthalmol 34. Vysniauskiene I, Allemann R, Flammer J, Haefliger IO. Vis Sci. 2008;49:1429–1436. Vasoactive responses of , PGF2alpha, latanoprost, 54. Thieme H, Schimmat C, Munzer¨ G, et al. Endothelin and travoprost in isolated porcine ciliary arteries. Invest antagonism: effects of FP receptor agonists prostaglandin F Ophthalmol Vis Sci. 2006;47:295–298. 2a and fluprostenol on trabecular meshwork contractility. 35. Krauss AH, Wiederholt M, Sturm A, Woodward DF. Prostaglan- Invest Ophthalmol Vis Sci. 2006;47:938–945. din effects on the contractility of bovine trabecular meshwork 55. Parekh A, Sandulache VC, Lieb AS, Dohar JE, Hebda PA. and ciliary muscle. Exp Eye Res. 1997;64:447–453. Differential regulation of free-floating collagen gel contraction 36. Wang JW, Woodward DF, Stamer WD. Differential effects of by human fetal and adult dermal fibroblasts in response to prostaglandin E2-sensitive receptors on contractility of human prostaglandin E2 mediated by an EP2/cAMP-dependent ocular cells that regulate conventional outflow. Invest mechanism. Wound Repair Regen. 2007;15:390–398. Ophthalmol Vis Sci. 2013;54:4782–4790. 56. Parekh A, Sandulache VC, Singh T, et al. Prostaglandin E2 37. Oga T, Matsuoka T, Yao C, et al. Prostaglandin F(2alpha) differentially regulates contraction and structural reorganiza- receptor signaling facilitates bleomycin-induced pulmonary tion of anchored collagen gels by human adult and fetal dermal fibrosis independently of transforming growth factor-beta. Nat fibroblasts. Wound Repair Regen. 2009;17:88–98. Med. 2009;15:1426–1430. 38. Ding WY, Liu L, Wang ZH, et al. FP-receptor gene silencing 57. Honjo M, Tanihara H, Inatani M, et al. Effects of Rho-associated ameliorates myocardial fibrosis and protects from diabetic protein kinase inhibitor Y-27632 on intraocular pressure and cardiomyopathy. J Mol Med. 2014;92:629–640. outflow facility. Invest Ophthalmol Vis Sci. 2001;42:137–144. 39. Moore BB, Ballinger MN, White ES, et al. Bleomycin-induced E 58. Vasantha Rao P, Deng PF, Kumar J, Epstein DL. Modulation of prostanoid receptor changes alter fibroblast responses to aqueous humor outflow facility by the Rho kinase-specific prostaglandin E2. J Immunol. 2005;174:5644–5649. inhibitor Y-27632. Invest Ophthalmol Vis Sci. 2001;42:1029– 40. Chen CZ, Raghunath M. Focus on collagen: in vitro systems to 1037. study fibrogenesis and antifibrosis state of the art. Fibrogenesis 59. Inoue T, Tanihara H. Rho-associated kinase inhibitors: a novel Tissue Repair. 2009;2:7. glaucoma therapy. Prog Retin Eye Res. 2013;37:1–12. 41. Polansky JR, Fauss DJ, Zimmerman CC. Regulation of TIGR/ 60. Fuchshofer R, Kuespert S, Junglas B, Tamm ER. The MYOC gene expression in human trabecular meshwork cells. prostaglandin F2a analog fluprostenol attenuates the fibrotic Eye. 2000;14:503–514. effects of connective tissue growth factor on human 42. Wentz-Hunter K, Shen X, Okazaki K, Tanihara H, Yue BYJT. trabecular meshwork cells. J Ocul Pharmacol Ther. 2014;30: Overexpression of myocilin in cultured human trabecular 237–245. meshwork cells. Exp Cell Res. 2004;297:39–48. 61. Choung J, Taylor L, Thomas K, et al. Role of EP2 receptors and 43. O’Brien TE, Metheney CD, Polansky JR. Immunofluorescence cAMP in prostaglandin E2 regulated expression of type I method for quantifying the trabecular meshwork glucocorti- collagen a1, lysyl oxidase, and -1 genes in coid response (TIGR) protein in trabecular meshwork and human embryo lung fibroblasts. J Cell Biochem. 1998;71:254– Schlemm’s canal cells. Curr Eye Res. 1999;19:517–524. 263.

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