ABSTRACT Members of the transforming growth factor beta (TGFβ) cytokine family have long been associated with affecting several cellular functions, including cell proliferation,

differentiation and extracellular matrix (ECM) turnover. Of particular interest to this work, TGFβ2 has been linked to most types of glaucomas as a potential fibrotic agent that can cause

elevation of intraocular pressure (IOP). Given that the trabecular meshwork (TM) provides most of aqueous humor outflow resistance in the eye, an _in vitro_ bioengineered human TM (HTM)

model has been created and validated by analyzing effects of TGFβ2 on transcellular pressure changes and outflow facility. These changes were correlated with several biological alterations

notion that anti-fibrotic activities of ROCK-inhibitors could counteract the elevation of IOP and increased strain observed in glaucomatous TM. SIMILAR CONTENT BEING VIEWED BY OTHERS

GLUCOCORTICOID-INDUCED CELL-DERIVED MATRIX MODULATES TRANSFORMING GROWTH FACTOR Β2 SIGNALING IN HUMAN TRABECULAR MESHWORK CELLS Article Open access 24 September 2020 SUPPRESSION OF TGF-Β1

SIGNALING BY MATRIGEL VIA FAK SIGNALING IN CULTURED HUMAN TRABECULAR MESHWORK CELLS Article Open access 01 April 2021 ESTABLISHMENT OF APPROPRIATE GLAUCOMA MODELS USING DEXAMETHASONE OR

TGFΒ2 TREATED THREE-DIMENSION (3D) CULTURED HUMAN TRABECULAR MESHWORK (HTM) CELLS Article Open access 29 September 2021 INTRODUCTION The transforming growth factor β (TGFβ) family members

play a key role in immune responses and tissue development such as inflammation, wound healing, extracellular matrix (ECM) accumulation, bone formation, cellular differentiation, and tumor

progression1,2. The importance of TGFβ in ophthalmology is evident by its endogenous expression in the anterior segment, as well as its presence in the aqueous humor, which is responsible

for chamber-associated immune deviation, a mechanism that protects the eye from inflammation and immune-related tissue damage. In particular, TGFβ appears to be involved in the pathogenesis

of certain glaucomas3. For example, TGFβ2 is significantly elevated in the aqueous humor of patients with primary open-angle glaucoma (POAG)4 and induces pathological changes at the human

trabecular meshwork (HTM) and optic nerve. In POAG, the sheath of connecting fibrils or plaques as well as ECM components that are present in the cribriform region of the HTM increase

significantly, leading to elevated intraocular pressure (IOP)5. Treatment of cultured HTM cells with TGFβ2 could increase the expression of ECM proteins, fibronectin and tissue

transglutaminase—an enzyme known to crosslink ECM proteins into complexes that can no longer be digested by proteinases6. In human eye organ-culture perfusion studies, TGFβ2 treatment

reduced the outflow facility and increased ECM accumulation in the HTM, indicating an elevation in IOP7. Additionally, enzyme plasminogen activator inhibitor (PAI), which inhibits most

matrix metalloproteinases (MMPs), was increased as a result of TGFβ2 treatments8. The fibrogenic effect caused by TGFβ2 is believed to be mediated, in part, by activated

RhoGTPase/Rho-associated kinase (ROCK). ROCK-inhibitors are a novel potential class of glaucoma therapeutics offering distinct applications including reduced IOP, improved ocular blood flow,

inhibition of postoperative scarring, and even neuroprotection9,10. With demonstrated effectiveness in animal models, several ROCK-inhibitors are currently undergoing Phase II and III

clinical trials in glaucoma10,11. ROCK inhibitors can affect the contractile properties of HTM cells, α-smooth muscle actin (α-SMA) expression, ECM accumulation in the outflow pathway and

aqueous humor outflow9,10,11,12. Several of these factors are also affected by TGFβ2, suggesting a common pathway. However, there is a knowledge gap on how TGFβ regulates the outflow

facility of HTM mechanistically and how HTM-characteristic proteins are regulated in the presence of ROCK inhibitors. We have previously reported a 3D bioengineered HTM model that

recapitulates the ultrastructure, biological and physiological features of the _in vivo_ HTM13. Here, to address the roles of TGFβ2, we first validated effects of TGFβ2 at clinically

relevant concentrations using this model. Next, we investigated whether the effects of TGFβ2 are mediated by ROCK, analyzing actin expression pattern, ECM deposition, changes in HTM marker

expression of myocilin and αB-crystallin, modulation of MMPs and cytokines induced by TGFβ2 in the absence or presence of a representative ROCK inhibitor (Y27632) in relationship to the

regulation of outflow facility. MATERIALS AND METHODS PRIMARY HUMAN TRABECULAR MESHWORK CELL CULTURE Primary TM cells were isolated from donor tissue rings discarded after penetrating

keratoplasty and carried out in accordance with relevant guidelines and regulations. The SUNY Downstate IRB has reviewed the protocol and determined that work is IRB exempt. Isolation of the

cells was under this IRB-exempt protocol approved by the SUNY Downstate IRB. Isolation and culture conditions were as previously described13,14. Before use in experiments, all HTM cell

strains were characterized for expression of αB-crystalline and α-smooth muscle actin. HTM cells were initially plated in 75 cm2 cell culture flasks with 10% fetal bovine serum (FBS) (Atlas

Biologicals, Fort Collins, CO) in Improved MEM (IMEM) (Corning Cellgro, Manassas, VA) with 1% 10 mg/mL gentamicin. Fresh medium was supplied every 48 h. Cells were maintained at 37 °C in a

humidified atmosphere with 5% carbon dioxide until 80–90% confluence at which point cells were trypsinized using 0.25% trypsin/0.5 mM EDTA (Gibco, Grand Island, NY) and subcultured. At least

three donors’ human primary cell cultures were used during experiments. All studies were conducted using cells before the 5th passage. SCAFFOLD FABRICATION SU-8 2010 photoresist (MicroChem

Corp.) was used to develop free-standing porous microstructures that served as scaffolds on which primary HTM cells were cultured. Using photolithographic techniques and a chrome mask with

defined micrometer-scale features, we fabricated SU-8 scaffolds as previously described13. Briefly, a release layer was spin-coated on the silicon wafer (substrate) and baked at temperatures

between 120–150 °C. Photoresist was spin-coated on the substrate to a final thickness of <5 μm, then baked at 95 °C and cooled to room temperature. The photoresist was exposed to

UV-light (140 mJ/cm2) through a chrome mask containing the desired pattern, baked at 95 °C and developed in PGMEA developer (MicroChem Corp.) SU-8 scaffolds with desired features were

released from the substrate, washed with isopropyl alcohol, air dried and stored at room temperature. Scaffolds were mounted on aluminum rings, sterilized using 70% ethanol and coated with

1% gelatin before seeding HTM cells. 3D CULTURE OF HTM CELLS ON SCAFFOLDS AND TGFΒ2 +/− Y27632 TREATMENT To create 3D HTM constructs, 40,000 HTM cells were seeded on each microfabricated

SU-8 scaffold, which was surrounded by an aluminum ring. After seeding, each scaffold was placed in a well of a 24-well plate and cultured in 10% FBS-IMEM for 14 days. The aluminum ring

allowed suspension of the scaffold, preventing cell contact with the bottom of the well. Medium was changed every 2–3 days. By day 14, HTM cells-scaffold constructs formed a 3D structure13.

Samples were serum-starved in 1% FBS for 48 hrs and then treated with TGFβ2 (2.5 ng/mL in 4 mM HCl, R&D systems, Minneapolis, MN), vehicle alone, ROCK inhibitor Y27632 (10 μM, Sigma

Aldrich, ST. Louis, MO) or a combination of TGFβ2 (2.5 ng/mL) and Y27632 (10 μM) in 1% FBS-IMEM for 3, 6 and 9 days, as indicated. PERFUSION STUDIES A perfusion apparatus was used as

previously described13. After 14 days in culture, 3D HTM constructs were serum-starved in 1% FBS and treated with TGFβ2, TGFβ2/Y27632, or vehicle alone for 9 days (as described above). Then

3D HTM samples were securely placed in the perfusion chamber and perfused at various rates for 6 hrs per flow rate (2, 10, 20 and 40 μl/min or 2, 4, 6, 8, 10 μl/min). Samples were perfused

in an apical-to-basal direction with perfusion medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) (Cellgro) with 0.1% w/v gentamicin (MP Biomedicals) containing vehicle, 2.5 

ng/mL TGFβ2 or TGFβ2 with 10 μM Y27632. The temperature was maintained at 34 °C throughout the experiment. Pressure was continuously monitored and recorded. After perfusion, the outflow

facility of our bioengineered 3D HTM model was calculated from the inverse of the slope of the pressure versus flow per unit surface area. At least eight different samples per condition,

including HTM cells from three donors, were studied under perfusion. In later experiments, 10 μM Y27632 was also used as negative control. PHALLOIDIN STAINING, IMMUNOCYTOCHEMISTRY, AND

CONFOCAL MICROSCOPY 3D HTM samples were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked using 5% goat serum. To reveal cytoskeleton arrangement, samples were

stained for F-actin using phalloidin (ThermoFisher Scientific) and incubated with antibody against α-SMA (Sigma-Aldrich). To confirm HTM marker expression, samples were incubated with

antibodies against HTM markers, myocilin and αB-crystallin, washed and incubated with their appropriate secondary antibodies. To examine ECM proteins, samples were incubated with antibodies

against collagen IV, fibronectin, and laminin (Abcam), as described previously13. Samples were co-stained with DAPI to reveal cell nuclei, followed by confocal imaging. Laser scanning

confocal microscopy was performed using a Leica SP5 confocal microscope, and images were acquired at 40X and 63X magnifications using an oil-immersion objective. Confocal images were

processed using Leica LasAF software, and all confocal images within a given experiment were imaged and captured using the same laser intensity and gain settings in order to be able to

compare intensities across samples. PROTEIN EXTRACTION AND WESTERN BLOT ANALYSIS Cellular proteins were extracted with ice-cold radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCL,

pH 7.5, 150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 25 mM NaF, 0.1 mM sodium orthovanadate, 10 mM NaP4O7, 1 nM phenylmethyl sulfonyl

fluoride) containing protease inhibitors (Complete Protease Inhibitor, Roche, Manheim, Germany) on ice. Proteins were quantified by bicinchoninic acid assay (Thermo Fischer Scientific). 20 

μg of proteins from each sample were separated by SDS polyacrylamide gel electrophoresis on a 4–12% gel in MOPS running buffer (ThermoFisher Scientific), transferred onto a PVDF membrane and

probed with the following primary antibodies rabbit anti-myocilin (Sigma Aldrich), mouse anti-αB-crystallin, mouse anti-fibronectin, rabbit anti-collagen IV and mouse anti-β-actin (Abcam).

HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen) were used. Bound antibody was detected using FluorChem E (Protein Simple). Protein expression was analyzed by

densitometry using ImageJ, and normalized to the housekeeping gene β-actin. All experiments were performed in duplicate for each of three donor cells. QUANTITATIVE REAL-TIME PCR (QPCR)

ANALYSIS Total RNA was extracted from samples cultured for 3, 5, and 9 days with TGFβ2, TGFβ2/Y27632, or without treatment using an RNeasy Plus Mini kit (Qiagen Inc., Valencia, CA). RNA

concentrations were determined using a NanoDrop spectrophotometer. 20 ng of RNA per sample was used for each qPCR experiments. qPCR was carried out using TaqMan RNA-to-CT 1-Step Kit (Applied

Biosystems, Carlsbad, CA) and performed on an AB StepOnePlus Real Time PCR system (Life Technologies) using primers for MMP2, MMP3, TIMP1, IL1α, TGFβ2 and GAPDH (Table 1). The temperature

profile was as follows: 48 °C for 15 min (reverse transcription step), followed by an enzyme activation step of 95 °C for 10 min, 40 cycles of 15 s denaturation at 95 °C and 1 min

anneal/extend at 60 °C. Relative quantitation data analysis was performed using the comparative quantification method, ΔΔCt, with GAPDH as the endogenous reference. All samples were

normalized to the vehicle-treated controls. qPCR experiments were performed in triplicate (technical replicates) from duplicate biological experiments for each of the three donor cells.

Average values are presented as mean ± SD. STATISTICAL ANALYSIS Data are expressed as mean ± standard deviation. The difference between vehicle-treated (controls), TGFβ2-treated, and

TGFβ2/Y27632-treated 3D HTM samples was analyzed using two-way ANOVA followed by Bonferroni post-tests (GraphPad Prism 6.02; GraphPad Software, Inc., La Jolla, CA). P values: P < 0.05, P 

< 0.01, and P < 0.001 are considered significant, very significant, and extremely significant, respectively. RESULTS VALIDATION OF TGFΒ2-INDUCED PHYSIOLOGICAL OUTFLOW CHANGES IN THE 3D

BIOENGINEERED HTM Given that regulation of aqueous humor flow is one of the main functions performed by the HTM, we investigated the hydrodynamic behavior and transcellular pressure across

the 3D HTM after treatment with TGFβ2 over at least 9 days. We utilized a perfusion system apparatus, as previously described13, that controls perfusion flow rate while constantly monitoring

pressure changes. After treatment with 2.5 ng/mL TGFβ2 alone for 9 days, the bioengineered 3D HTM construct was place in the perfusion chamber and perfused with serum-free medium

supplemented with 2.5 ng/mL TGFβ2 at various flow rates (2, 4, 6, 8 and 10 μL/minute) in an apical-to-basal orientation. Control samples were treated with vehicle and perfused at flow rates

of 2, 5, 10, 15 and 20 μL/min in the apical-to-basal orientation. Higher flow rates were required for controls to consistently record pressure changes in the low range. For both treated and

untreated samples, the pressure was continuously recorded for 6 hours to assure equilibrium at each flow rate. Compared to controls, TGFβ2 perfusion increased transcellular pressure (Fig.

1a) and significantly decreased (0.18 ± 0.03 versus 0.05 ± 0.01) the outflow facility per unit area across the 3D HTM (N = 10, P < 0.001) (Fig. 1b). These results show that our 3D HTM

model is responsive to TGFβ-2 by drastically changing the transcellular pressure and outflow facility as expected. VALIDATION OF TGFΒ2-INDUCED F-ACTIN REARRANGEMENTS AND CO-LOCALIZATION OF

Α-SMOOTH MUSCLE ACTIN TO STRESS FIBERS The cytoskeleton provides the framework for multiple cell functions including resisting deformation, coordination of forces that enable cell movement

and control of shape15. Given the dynamic nature of the HTM, it is believed that HTM cells’ cytoskeletal alterations may allow the effective control of aqueous humor outflow resistance. We

applied our previously established, 3D-bioengineered HTM model to study changes in actin arrangement upon TGFβ2 exposure. After primary HTM cells were cultured on gelatin-coated SU-8

scaffolds for 14 days, as described previously13, these 3D HTM constructs were conditioned overnight in 1% serum media and subsequently incubated for 9 days in the presence or absence of

TGFβ2 (2.5 ng/mL) While cells in vehicle-treated controls exhibited aligned actin fibers, TGFβ2 treated samples showed increased staining of F-actin fibers that exhibited actin

rearrangements and appeared more disorganized, despite seemingly aligned nuclei (Fig. 2). The actin expression was intensified in 3D HTM after treatment with TGFβ2. No significant change in

nuclei size between vehicle-treated controls (35 ± 3 μm2, N = 20) and TGFβ-2 treated samples (38 ± 4 μm2, N = 15) was observed. Given previous reports of the induction of α-SMA by this

cytokine _ex vivo_ and _in vitro_16,17, we further studied the expression of α-SMA in our model. Our studies show an increase of α-SMA in 3D HTM after exposure to TGFβ2 and interestingly, a

colocalization with F-actin fibers. While our controls expressed diffuse α-SMA in the HTM cytosol, after TGFβ2 treatment, this protein appears to be upregulated and arranged into fibers

(Fig. 2g). These changes in actin arrangements could have a direct impact on cell behavior and cell interactions with the microenvironment, including the ECM and neighboring cells.

VALIDATION OF TGFΒ2-INDUCED ECM DEPOSITION IN BIOENGINEERED 3D HTM Increased interest in the pathogenic, fibrotic expression of several ECM proteins in glaucomatous HTM encouraged us to

evaluate the effect of TGFβ2 on ECM protein expression in our bioengineered 3D HTM model. Fibrotic glaucomatous HTM is believed to result from the elevated amount of TGFβ2 found in the

aqueous humor3,18. Exogenous treatment with TGFβ2 increased the expression of ECM proteins, collagen IV, fibronectin, and laminin, as shown by immunocytochemistry analysis (Fig. 3a–j). After

treatment with TGFβ2, collagen IV fibers appeared highly disorganized and arranged into highly crosslinked bundles compared to vehicle-treated controls (compare Fig. 3b,g). Denser and

thicker fibrous arrangements of fibronectin and laminin were seen after treatment with TGFβ2, in which multiple fibers joined together (fibrils) longitudinally, making fibers appear thicker

and denser compared to those seen in controls (compare Fig. 3c,h, Fig. 3d,i). western blot analysis corroborated the induction of collagen IV (Fig. 3.k) and fibronectin (Fig. 3l) in 3D HTM

cultures after TGFβ2 treatment and confirmed the significant increase in collagen IV (N = 4, P < 0.01) and fibronectin (N = 4, P < 0.05) proteins (Fig. 3m). All together, these results

not only support the involvement of TGFβ2 in fibrotic HTM, as seen in several types of glaucomas, but also suggest the role of TGFβ2 in tissue hardening that may take place as a result of

crosslinked, dense arrangements of collagen IV, fibronectin and laminin. INDUCED EXPRESSION OF HTM-MARKER PROTEIN MYOCILIN BUT NOT ΑB-CRYSTALLIN IN 3D HTM AFTER EXTENDED EXPOSURE TO TGFΒ2

Despite decades of research in the field, myocilin and αB-crystallin are two characteristic, but poorly understood proteins that have been found in the HTM. Myocilin, a secreted protein

known to interact with the ECM, apparently causes disease only in the eye19. αB-crystallin is a small heat shock protein with chaperone activity found mostly in the cribriform region of the

HTM, the area adjacent to the inner wall of the human Schlemm’s canal (HSC), which is believed to exert the most pressure regulation20. αB-crystallin expression increases after a few days of

exposure to TGFβ221. However, the effects of extended exposure to TGFβ2, e.g., a week or more, are unclear. We studied the effects of prolonged (9 days or more) TGFβ2 exposure on the

expression of myocilin and αB-crystallin using immunocytochemistry (Fig. 4a–h) and western blot analysis (Fig. 4I,j). Confocal imaging showed an increase in myocilin protein expression in 3D

HTM cultures after TGFβ2 perfusion (Fig. 4f) compared to vehicle-treated controls (Fig. 4b). The myocilin accumulation, induced by TGFβ2, appeared punctate and occasionally plaque-like in

areas between cells (Fig. 4F). On the other hand, 3D HTM cultures treated with TGFβ2 showed no change in αB-crystallin expression (Fig. 4g) compared to control (Fig. 4c). These results were

confirmed by western blot analysis (Fig. 4I) in which the densitometry analysis showed that expression of myocilin after TGFβ2 treatment was significantly higher than control (N = 4, P < 

0.01) while there was no significant difference in expression of αB-crystallin (N = 4, P > 0.05) (Fig. 4j). SUPPRESSION OF TGFΒ2-INDUCED OVEREXPRESSION OF ECM PROTEINS AND MYOCILIN IN 3D

HTM BY A ROCK INHIBITOR ROCK inhibitors are currently being considered as a potential new class of glaucoma drugs. Despite extensive studies on their effects on the HTM, it is not clear

whether ROCK inhibitors can correct TGFβ2-induced fibrotic ECM build-up and myocilin over-expression, which have been linked to glaucoma. Therefore, we assessed the effect of

co-administering TGFβ2 along with a ROCK inhibitor, Y27632, on ECM protein secretion and myocilin expression. Immunocytochemistry analysis of our bioengineered 3D HTM model co-treated with

TGFβ2/Y27632 for 9 days showed substantially decreased collagen IV and fibronectin expression compared to samples treated with TGFβ2 alone (Fig. 5a–l). In the Y27632 co-treated samples,

shorter collagen IV fibers and more aligned fibronectin fibers (Fig. 5i–l) were observed compared to samples treated with TGFβ2 alone (Fig. 5e–h). Similarly, treatment with TGFβ2/Y27632

combined, significantly reduced the expression of myocilin while slightly increasing the expression of αB-crystallin (Fig. 5u–x) compared to treatment with TGFβ2 alone (Fig. 5q–t). western

blot analysis further confirmed these immunocytochemistry results, showing that TGFβ2/Y27632 treatment caused a significant decrease in ECM proteins, fibronectin (N = 5, P < 0.05) and

collagen IV (N = 5, P < 0.01 for all) in comparison to TGFβ2 alone, to the level of vehicle-treated controls or Y27632-treated samples (Fig. 5y), demonstrating that the presence of ROCK

inhibitor could prevent the ECM accumulation caused by TGFβ2. Interestingly, treatment with either TGFβ2 or Y27632 alone induced overexpression of myocilin (Fig. 5z). This increase in

myocilin expression was significantly suppressed by combined TGFβ2/Y27632 treatment when compared to vehicle-treated controls (N = 6, P < 0.001) and TGFβ2 samples (N = 6, P < 0.001)

(Fig. 5z.). As mentioned previously, TGFβ2 treatment slightly, but not significantly, decreased αB-crystallin expression (Fig. 4j). The presence of Y27632 during TGFβ2 treatment

significantly increased αB-crystallin expression (N = 6, P < 0.05), bringing it back to the level of vehicle-treated control or Y27632-treated samples. Compared to vehicle treated

controls, TGFβ2/Y27632 combined treatments decreased myocilin (N = 6, P < 0.01) and increased fibronectin (N = 5, P < 0.05) expression (Fig. 5y,z). These results demonstrate that TGFβ2

combined with the ROCK inhibitor counteracts the otherwise fibrotic effect of TGFβ2, effectively lowering ECM protein accumulation and myocilin expression. ECM REMODELING THROUGH MMPS AND

CYTOKINES IN 3D HTM TREATED WITH TGFΒ2 IN THE ABSENCE OR PRESENCE OF ROCK INHIBITOR To explore the factors that may be responsible for modulation of the TGFβ induction of ECM and other

secreted proteins by ROCK inhibitor, the transcriptional expression of several MMPs and cytokines, including tissue inhibitor of metalloproteinase-1 (TIMP1), interleukin 1-alpha (IL1α) and

TGFβ2, were evaluated through a 9-day time course study. MMP2 gene expression increased ~2.7-fold after a 3-day TGFβ2 treatment compared to vehicle-treated controls (P < 0.001) (Fig. 6a).

This increase was further enhanced to ~7.1-fold in TGFβ2/Y27632 co-treatment while Y27632 treatment alone did not change MMP2 gene expression. After 5 days in culture, TGFβ2 treatment

showed no significant difference in MMP2 gene expression from vehicle-treated control while in TGFβ2/Y27632 co-treated samples, MMP2 gene expression remained high (~5.1-fold increase

compared to control, P < 0.001). Interestingly, MMP2 expression decreased after 9 days treatment with Y27632, TGFβ2, or TGFβ2/Y27632 (P < 0.001 for all treatments compared to control)

(Fig. 6a). MMP3 expression showed no significant difference between control and TGFβ2/Y27632 treated cultures at day 3, while down-regulation in TGFβ2-treated samples (~9.4-fold decrease, P 

< 0.001) was observed. By day 5, MMP3 expression was down-regulated ~15.3-fold (P < 0.001) for TGFβ2 treatment compared to control; the TGFβ2 downregulation was suppressed by

co-treatment with Y27632 (Fig. 6b). By day 9 of treatment, TGFβ2 and TGFβ2/Y27632 significantly decreased MMP3 mRNA expression by ~52- (P < 0.001) and ~13-fold (P < 0.05),

respectively. HTM cells secrete several cytokines that modulate cell behavior and ECM turnover in the conventional outflow pathway. Increased cytokine expression has been observed in the

aqueous humor of glaucoma patients, including TIMP122 and TGFβ23,24. In addition, IL1α is an inflammatory cytokine that has been shown to be involved in regulating outflow facility25.

Therefore, studying cytokine expression could provide insight into the pathology of glaucoma as pertaining to TGFβ2. TIMP1 mRNA expression was up-regulated ~7- fold after 3 days of TGFβ2 (P 

< 0.001) or TGFβ2/Y27632 (P < 0.001) treatment. On day 5, TIMP1 gene expression levels were still higher than controls, ~4-fold increased by TGFβ2 (P < 0.001) or TGFβ2/Y27632

combined (P < 0.001). By day 9, TIMP1 was down-regulated by ~2.5-fold (P < 0.001) after TGFβ2 and by ~1.5 (P < 0.001) in TGFβ2/Y27632 treatments (Fig. 6c). Y27632 treatment alone

did not affect expression of TIMP1, nor did the presence of Y27632 change TGFβ2-induced gene expression of TIMP1. On the other hand, IL1α gene expression exhibited no significant change

after treatment with Y27632, TGFβ2, or Y27632/TGFβ2 for 3 and 5 days (Fig. 6d). On day 9, IL1α mRNA expression decreased by ~7.7-fold for TGFβ2 treated samples (P < 0.001) while

co-treatment with Y27632 during TGFβ2 treatment further down-regulated this gene by ~23-fold (P < 0.001) (Fig. 6d). Both treatments exhibited a time-dependent effect on IL1α mRNA

expression. As TGFβ2 can also be produced and secreted by HTM cells, we evaluated its expression in the presence of exogenous TGFβ2. TGFβ2 gene expression was down-regulated throughout days

3, 5 and 9 of exogenous TGFβ2 treatment by 7.4-, 4.4- and 3.2-fold, respectively. TGFβ2/Y27632 co-treatment initially (day-3) up-regulated TGFβ2 gene expression by 1.3-fold and later, during

5 and 9 days of treatment, down-regulated TGFβ2 mRNA expression by 1.4- and 1.6-fold, respectively, compared to control (Fig. 6e); however, in comparison with TGFβ2 treatment alone, the

expression in the co-treated cultures was much closer to control levels. Overall, these data demonstrate a complex ECM regulation that takes place as a result of elevated exogenous TGFβ2 or

TGFβ2/Y27632 exposure at the TM. ROCK INHIBITOR PREVENTS TGFΒ2-INDUCED ELEVATED FLOW RESISTANCE Given the dramatic changes in protein and cytokine expression resulting from the combined

treatment with TGFβ2 and ROCK inhibitor, we examined the effect of combined treatment on outflow facility through perfusion studies. As Y27632 alone increases outflow facility, we compared

vehicle-treated controls, TGFβ2 treatment, Y27632 alone, and Y27632 in conjunction with TGFβ2 in 3D HTM cultures. Compared to vehicle-treated controls, TGFβ2/Y27632 perfusion maintained a

similar outflow facility as shown in Fig. 7 (N = 6, ns). Vehicle- and Y27632-treated cultures showed an outflow facility of 0.184 ± 0.003 μL/min/mmHg/mm2 and 0.23 ± 0.04 μL/min/mmHg/mm2,

respectively. On the other hand, TGFβ2 treatment significantly lowered the outflow facility of the bioengineered 3D HTM cultures to 0.049 ± 0.004 μL/min/mmHg/mm2 (P < 0.001) and

TGFβ2/Y27632 treatment returned the outflow facility to 0.176 ± 0.008 μL/min/mmHg/mm2 (Fig. 7). These data demonstrate that the bioengineered 3D HTM regulates flow in a similar fashion as

aqueous humor outflow regulation seen in _in vivo_ and _ex vivo_ HTM after TGFβ2 treatment, by increasing pressure and decreasing the outflow facility across this tissue. In addition,

combined TGFβ2/Y27632 treatment maintained “normal” outflow regulation comparable to the vehicle-treated bioengineered HTM. Altogether, these data demonstrate that TGFβ2 is capable of

modulating outflow of the bioengineered 3D HTM comparably to _in vivo_ and _ex vivo_ HTM, and the presence of ROCK inhibitor Y27632 during TGFβ2 treatment can prevent TGFβ-2-induced

elevation of transcellular pressure and maintain the outflow facility close to control levels. These data provide insight into the use of ROCK inhibitors to treat elevated IOP in glaucoma

patients who have elevated TGFβ2 in their aqueous humor. DISCUSSION In this study, we demonstrated that was our previously established bioengineered 3D HTM model13 responds to TGFβ2

treatment similarly to _in vivo_ or _ex vivo_ HTM, including elevated transcellular pressure, decreased outflow facility, and increased ECM accumulation. These data further validate the

utility of the 3D HTM model for perfusion studies of biological agents believed to affect the TM, in a physiologically relevant setting, which are otherwise difficult to conduct using _ex

vivo_ organ cultures. Using a bioengineered 3D HTM model system, we further revealed that TGFβ2 affects a range of proteins that can elicit tissue stiffening and outflow facility changes,

including ECM proteins, F-actin, and α-SMA. Lastly, we explored the potential of ROCK inhibitor in conjunction with TGFβ2 to rescue the homeostatic behavior of HTM through MMPs and

inflammatory cytokines. The actin cell machinery polymerizes to form different network organizations that provide cells with mechanical elements and properties26. Filamentous actin such as

stress fibers also connects the cytoskeleton to the ECM via focal adhesion sites. Treatment with TGFβ2 caused cytoskeletal actin changes including polymerization of F-actin fibers and

increased expression of α-SMA. After treatment, a majority of these F-actin fibers appeared to be organized into bundles, F-actin fibers in close proximity to other fibers. Actin bundles

have innate compressive mechanical properties that can be measured by their buckling force, which is directly proportional to the number of actin filaments that make up a bundle26. In this

way, tissues can increase their compressive force/strength through their actin arrangement. Based on our results, the induction of actin bundles suggests a more rigid cytoskeleton

arrangement that can provide greater compressive forces to their microenvironment. Treatment of our 3D HTM model with TGFβ2 for 9 days also induced α-SMA overexpression and its

rearrangement. α-SMA is a mechanosensitive protein, reported to be induced in response to critically stiffened cell mircroenvironments27 and by exogenous TGFβs. Furthermore, α-SMA is

co-localized or recruited to stress actin fibers under high tension, and localization of this protein from the cytosol to the stress fibers can be mediated under specific topographic cues

that affect the structure of focal adhesions28. In this study, we observed increased expression of ECM proteins collagen IV, fibronectin and laminin, along with elevated expression of α-SMA

after TGFβ2 exposure. These induced ECM proteins together could substantially increase the stiffness of the immediate cell substrate, inducing the co-localization of α-SMA to the stress

actin fibers as observed and previously discussed29,30. Furthermore, fibronectin, via feedback activation, induces α-SMA expression16. Therefore, the increased expression of fibronectin seen

in this work could also be involved in the increased α-SMA expression. Collectively, these observations further support the interplay between HTM cell contractile hardware and ECM

synthesis/deposition in the aqueous humor outflow pathway. In addition, the elicited expression of α-SMA and ECM proteins by TGFβ2 suggests transdifferentiation of HTM cells into

myofibroblast-like cells expressing fibrogenic and fibroblast-like markers, as recently reported by others31,32. Increased ECM deposition in the TM is associated with POAG. Cell-ECM

interactions play a key role in regulating outflow in the eye33,34. The overproduction of ECM components such as fibronectin and collagen IV by HTM cells under pathological conditions has

been described previously. For instance, we have shown that steroid treatment upregulates secreted and cell-associated ECM proteins, including fibronectin, collagen type IV, and laminin in a

3D bioengineered HTM model. Exposure to high glucose concentrations also induces fibronectin production by HTM cells35. In this work, ECM overproduction, including collagen IV, fibronectin,

and laminin, was induced by TGFβ2. These proteins are the major components of the HTM basement membrane and are found throughout the conventional outflow tract. ECM proteins are dynamically

remodeled by several enzymes including MMPs. HTM cells constitutively express significant levels of MMPs, such as stromelysin-1 (MMP3) and gelatinase A (MMP2); expression can be further

elevated in response to a variety of stimuli such as mechanical stretching and biologically active cytokines36,37. Exposure to TGFβ2 enhances accumulation of the pro form of MMP-3 and

decreases active MMP3 in the TM38. The unbalanced expression of MMP3 may result in decreased proteolytic degradation and increased accumulation of ECM components such as fibronectin, a known

MMP3 substrate39. Indeed, in this study, exposure to TGFβ2 led to a significant increase in fibronectin expression and decreased gene expression of MMP3. In addition, MMP2 expression

increased after 3 and 5 days of TGFβ2 exposure, which is consistent with previous short-term studies40. It is important to highlight that over an extended 9 days of treatment, MMP2 mRNA

expression decreased, dropping well below the control levels. The initial increase in MMP2 was accompanied by TIMP1 induction. A similar relationship between MMP2 and TIMP1 has been shown

after cyclic mechanical stretching of bovine TM cells41. In our model, TGFβ2 induces actin and ECM changes that can exert biomechanical stress on HTM cells leading to ECM turnover,

accompanied by modulating MMP and cytokine expression, which is attenuated during longer term exposure to TGFβ2. The initial (day 3) up-regulation of TIMP1, along with the sustained

down-regulation of MMP3 during TGFβ2 treatment could account for the accumulation of ECM proteins. The early upregulation of MMP2 suggests an active ECM remodeling during early TGFβ2

exposure, perhaps due to TM cells attempting to counteract ECM protein overproduction. With extended exposure to TGFβ2, MMPs and TIMPs are down-regulated, potentially leaving considerable

paracellular accumulation of ECM proteins and myocilin at the HTM basement membrane. A similar trend in MMP gene expression was observed after extended TGFβ2/Y27632 co-treatment, but towards

the beginning (day 3–5) MMP2 mRNA-level was enhanced to a greater extent (over 2-fold) compared to TGFβ2 treatment alone. This potent induction of MMP2 gene expression could be sufficient

to prevent or even disrupt proper assembly of proteins including myocilin and/or ECM proteins that require focal-adhesions. By the same token, given that IL1α has been shown to regulate MMPs

in the TM or organ cultures25, down-regulation of this gene could induce the down-regulation of MMPs seen towards later days in this study. RhoA/Rho kinase signaling activity affects the

expression of ECM proteins and αSMA16, in addition to its well-recognized role in actin cytoskeletal organization, and several ROCK inhibitors are currently undergoing Phase II and III

clinical trials in glaucoma9,10. Using ROCK inhibitor, Y27632, in combination with TGFβ2, we assessed its effects on the expression of collagen IV, fibronectin, laminin, myocilin and

αB-crystallin. This combination significantly decreased expression of ECM proteins as well as myocilin back to vehicle-treated controls levels, but failed to reduce αB-crystallin expression.

Interestingly, treatment with Y27632 alone for 9 days upregulated myocilin and maintained unchanged levels of αB-crystallin compared to controls (Fig. 5). These results reinforce the

anti-fibrotic nature, as related to ECM proteins, of ROCK inhibitors. On the other hand, these findings raise questions as to the relationship between myocilin, the ROCK pathway, and TGFβ2

and elucidate their probable interplay. Based on this work and current literature, there are several changes caused by TGFβ2 that may induce TM stiffening, eliciting elevated IOP (Fig. 8).

Overall, this work supports the idea that misregulated accumulation of ECM proteins, and perhaps myocilin, may influence the biomechanical properties and stiffness of TM cells, and

therefore, aqueous humor outflow facility. ADDITIONAL INFORMATION HOW TO CITE THIS ARTICLE: Torrejon, K. Y. _et al_. TGFβ2-induced outflow alterations in a bioengineered trabecular meshwork

are offset by a Rho-associated kinase inhibitor. _Sci. Rep._ 6, 38319; doi: 10.1038/srep38319 (2016). PUBLISHER'S NOTE: Springer Nature remains neutral with regard to jurisdictional

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MMP-2 in response to mechanical stretching. Jpn. J. Ophthalmol. 42, 90–94 (1998). Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work is partially supported

by the College of Nanoscale Science and Engineering Startup Funds (Sharfstein), NIH Grant R01 EY20670 and R01 EY025543 (Danias), unrestricted challenge grant from Research to Prevent

Blindness Inc (Department of Ophthalmology), American Glaucoma Society Mid-Career award (Danias), SUNY Health Network of Excellence (Xie), SUNY TAF, and NSF STTR 1448900. Torrejon was

supported by the NSF Graduate Research Fellowship Program. The authors thank Dr. Sandeep Kumar for technical support, and Cula Dautriche for experimental assistance. AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 fuller road, Albany, 12203, New York, USA Karen Y. Torrejon, Ellen L. Papke, Justin

R. Halman, Magnus Bergkvist, Susan T. Sharfstein & Yubing Xie * Department of Ophthalmology, SUNY Downstate Medical Center, Brooklyn, 11203, New York, USA John Danias Authors * Karen Y.

Torrejon View author publications You can also search for this author inPubMed Google Scholar * Ellen L. Papke View author publications You can also search for this author inPubMed Google

Scholar * Justin R. Halman View author publications You can also search for this author inPubMed Google Scholar * Magnus Bergkvist View author publications You can also search for this

author inPubMed Google Scholar * John Danias View author publications You can also search for this author inPubMed Google Scholar * Susan T. Sharfstein View author publications You can also

search for this author inPubMed Google Scholar * Yubing Xie View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.Y.T., J.D. and Y.X. designed

research; K.Y.T., E.L.P. and J.R.H. performed research; K.Y.T., M.B., S.T.S. and Y.X. analyzed data; and K.Y.T., S.T.S. and Y.X. wrote the paper; All authors reviewed the manuscript. ETHICS

DECLARATIONS COMPETING INTERESTS Karen Torrejon is the founder of Glauconix. This work is related to a pending patent (US20140038221 A1). ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY

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CITE THIS ARTICLE Torrejon, K., Papke, E., Halman, J. _et al._ TGFβ2-induced outflow alterations in a bioengineered trabecular meshwork are offset by a rho-associated kinase inhibitor. _Sci

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