Treatment of glaucoma by prostaglandin agonists and beta‐blockers in combination directly reduces pro‐fibrotic gene expression in trabecular meshwork

Abstract Prostaglandin analogues (PG), beta‐blockers (BB) or their combination (PG+BB) are used primarily to reduce the intraocular pressure (IOP) pathologically associated with glaucoma. Since, fibrosis of the trabecular meshwork (TM) is a major aetiological factor in glaucoma, we studied the effect of these drugs on fibrosis‐associated gene expression in TM of primary glaucoma patients. In the present study, TM and iris of primary open‐angle (n = 32) and angle‐closure (n = 37) glaucoma patients were obtained surgically during trabeculectomy and categorized based on the type of IOP‐lowering medications use as PG, BB or PG+BB. mRNA expression of pro‐fibrotic and anti‐fibrotic genes was quantified using qPCR in these tissues. The gene expression levels of pro‐fibrotic genes were significantly lower in PG+BB as compared to other groups. These observations and underlying signalling validated in vitro in human TM cells also showed reduced fibrotic gene and protein expression levels following PG+BB treatment. In conclusion, it is observed that PG+BB combination rather than their lone use renders a reduced fibrotic status in TM. This further suggests that IOP‐lowering medications, in combination, would also modulate fibrosis‐associated molecular changes in the TM, which may be beneficial for maintaining aqueous out‐flow mechanisms over the clinical treatment duration.

loss of visual field. Trabecular meshwork (TM) contributes to 90% of the aqueous humour out-flow via the conventional drainage pathway 2 and hence is important for maintaining the IOP. The increase in resistance to conventional out-flow due to changes in TM structure, that is pore closure resulting from aberrant extracellular matrix (ECM) deposition, plays a major role in glaucoma. [3][4][5] The TM is composed of collagens, laminins, fibronectin, proteoglycans and matricellular proteins. 2 Physiological TM ECM remodelling is orchestrated by a balanced regulation between pro-fibrotic and anti-fibrotic factors such as TGFβ, CTGF, fibronectin, collagens, hevin and decorin. 6 Pro-fibrotic events contribute to pore closure in TM, 7,8 and TGFβ has been shown to be key inducer of pore closure proteins in humans, animal models and in vitro studies. 9,10 The current management of glaucoma focuses on controlling increase in IOP by the use IOP-lowering medications such as prostaglandin analogues (PG), beta-blockers (BB), carbonic anhydrase inhibitors (CAI) and alpha agonists (AA). These medications function through decreasing production of aqueous humour and/or increasing out-flow by alternative pathways. 11,12 Prostaglandin analogues and beta-blockers reduce IOP by increasing uveo-scleral out-flow and by reducing the production of aqueous humour, respectively.
Prostaglandin analogues and beta-blockers are the most potent drugs in terms of IOP-lowering capabilities and tolerability, [13][14][15] hence, are the most commonly used drugs in glaucoma patients. It has been also shown that PG when used in combination with BB led to better outcomes due to additional reduction in IOP. 16 Sustaining a functional trabecular meshwork (TM) during the management of glaucoma could improve prognosis. Hence, it would be relevant to choose IOPlowering medication that, in addition to reducing IOP, can also reduce fibrotic changes in the TM. Currently, there is no evidence regarding the effect of IOP-lowering medications directly on the fibrotic status of TM in human patients. Exogenous PGs have been reported to have a role in initiating as well as modulating fibrosis in different organs such as lung, renal and cardiac tissues, [17][18][19] but their exact role on human TM fibrosis remains unknown. Similarly, reports on the effect of BB on ECM remodelling in TM are rather sparse. 20,21 However, BB has been reported to have anti-inflammatory effects and favourable ECM remodelling with reduced fibrosis in vascular tissues. 22,23 Considering that both PG and BB exert differential outcomes in different systems in terms of ECM regulation and remodelling, [24][25][26][27][28] it is imperative to study the effect of primary IOP-lowering medications used in glaucoma on the TM. The current study investigated the effect of prostaglandin analogues (PG), beta-blockers (BB) or their combination (PG+BB) on the expression of fibrosis-associated factors (TGFβ1, transforming growth factor beta 1; TGFβ2, transforming growth factor beta 2; TGFβR2, transforming growth factor beta receptor 2; CTGF, connective tissue growth factor; FN, fibronectin; LOXL2, lysyl oxidase-like 2; WNT3A, wingless-type family member 3A; decorin, dermatan sulphate proteoglycan II-DSPG2 and hevin, secreted protein acidic and rich in cysteine-like 1-SPARCL1) in TM of primary glaucoma patients and validated the observations in vitro.
Our findings suggest the beneficial effects of combination of prostaglandin analogue and beta-blockers on TM.

| ME THODS
The cross-sectional study was approved by the institutional ethics committee. The study was conducted as per the guidelines stated by Indian Council of Medical Research and the Declaration of Helsinki.
Patient information and samples were obtained following informed written consent.

| Study population
Patients with a diagnosis of POAG or PACG that underwent trabeculectomy in a tertiary eye care centre were included in the study. All these patients underwent a complete clinical evaluation, that is visual acuity, refraction, Goldmann applanation tonometry to determine IOP, gonioscopy to determine whether angles are open or closed, optic nerve head evaluation, perimetry with Humphrey Field Analyser (HFA) and/or imaging of the optic nerve head with optical coherence tomography (OCT) to establish the glaucomatous damage. These patients were diagnosed as POAG when the IOP was high with open angles on gonioscopy and glaucomatous changes on clinical optic nerve evaluation. The changes in the nerve were substantiated with corresponding perimetric changes and/or RNFL thinning on OCT. Primary angle-closure glaucoma (PACG) diagnosis was considered for patients that had narrow angles with signs of occlusion such as patchy pigmentation of trabecular meshwork, pigments on anterior lenticular surface, sphincter changes or synechial angle closure. These angle changes along with optic nerve damage substantiated by corresponding changes in perimetry and/or OCT were considered as PACG. All PACG patients underwent laser peripheral iridotomy followed by the medical treatment. These glaucoma patients whether POAG or PACG were subjected to glaucoma filtration surgery, that is trabeculectomy, when (a) the IOP could not be controlled with medical treatment, (b) intolerant to medical treatment or (c) the cataract surgery was indicated and the patient was already on more than one IOP-lowering medications for that eye.

| Inclusion criteria
Patients with POAG or PACG undergoing trabeculectomy with or without cataract surgery. Further, the patients that were on IOPlowering medications that includes PG and/or BB for more than a week.

| Exclusion criteria
(a) Patients with secondary glaucoma or type of glaucoma other than POAG and PACG, (b) TM tissue with poor RNA yield, (c) patients with age less than 18 years and more than 85 years, (d) patients with serology positive for HIV, HBS and HCV, (e) patients who were operated without any IOP-lowering medications or its use for less than a week and (f) patients using IOP-lowering medications other than PG or BB, for example alpha agonist, carbonic anhydrase inhibitors or cholinergic agonists only.

| Sample collection and storage
Trabecular meshwork was obtained while performing trabecular meshwork block excision, and iris was collected while performing iridectomy during trabeculectomy procedure. The tissue samples were collected in sterile micro-centrifuge tubes containing Ringer Lactate solution and stored in a bio-repository at −80°C until further use.

| Classification of groups
The samples were divided into subgroups based on the types of primary glaucoma or types of IOP-lowering medications. Based on primary glaucoma, the samples were divided into those from POAG (n = 32) or PACG (n = 37) patients. Based on the type of IOP-lowering medications, the samples were divided into those on either prostaglandin analogues (PG, n = 14), beta-blockers (BB, n = 28) or a combination of prostaglandin analogues and beta-blockers (PG+BB, n = 27). Prostaglandin analogues used by the study cohort include bimatoprost, travoprost and latanoprost. All these three PGs are structural analogues of prostaglandin F 2α and they bring about their action by binding to prostaglandin F receptor. The vast majority of the study subjects on PG were using bimatoprost in the study cohort. Further, study subjects using beta-blockers were all on timolol.

| RNA isolation and quantitative real-time PCR
Total RNA was isolated from trabecular meshwork tissue and iris tissue using TRIzol method according to manufacturer's protocol (Invitrogen). The concentration and purity of the extracted mRNA was assessed, and samples that had at least 1000 ng of RNA with a purity by optical density 260/280 ratio of >1.6 were selected for further analysis. RNA was converted into cDNA using Bio-Rad iSCRIPT cDNA conversion kit (Bio-Rad). Real-time PCR was performed using SYBR green reagent (Kapa Biosystems Inc). The quantitative real-time PCR cycle includes pre-incubation at 95°C for 3 minutes, 40 amplification cycles at 95°C for 10 seconds, 58°C for 30 seconds using a CFX Connect™ real-time PCR detection system (Bio-Rad). Primer sequence details are provided in Table S1.
The expression levels of human TGFβ1 (transforming growth factor beta 1), TGFβ2 (transforming growth factor beta 2), TGFβR2   (Abcam plc) and phosphorylated SMAD3 (Abgenex Pvt. Ltd) were used at a dilution of 1:1000 except for GAPDH (Abcam plc) which was used at 1:5000 in 5% fat free milk in TBST. The membranes were incubated with respective primary antibodies overnight at 4°C. The membranes were washed using TBST and incubated with the relevant secondary antibodies (antimouse and anti-rabbit, BosterBio) at 1:5000 dilutions for an hour at room temperature.

| Immunoblotting
The membranes were then washed and incubated with Clarity ECL Western blotting substrate (Bio-Rad) and resulting chemiluminescence was imaged using Image quant (GE Image Quant LAS 500, GE Healthcare). Densitometry analysis was done using ImageJ software (Version 6).

| Cytometric bead array
The levels of TGFβ1 in hTM cell culture supernatants were meas-

| Statistical analysis
Statistical significance between the groups was determined by using unpaired t test or Mann-Whitney test based on the distribution of the data. Shapiro-Wilk normality test was used to determine the distribution type of the data. P < .05 was considered to be statistically significant. GraphPad prism version 6 (GraphPad Software, Inc) was used to perform statistical analysis.

| RE SULTS
A total of 32 POAG and 37 PACG patients were included in the study. The clinical characteristics as described in Table 1 show no significant differences in the parameters such as age, sex, IOP, MD, PSD and VFI between the two groups. The expression of fibrosisassociated genes measured in the TM tissues exhibited no significant differences between POAG and PACG patients ( Table 2). The overall cohort of primary glaucoma patients (POAG and PACG) was further divided based on the type of IOP-lowering medication usage prior to surgery, and their clinical characteristics are listed in Table 3. All parameters among the groups based on IOP-lowering medication type used were similar except for the age and duration of drug usage.   Figure 1B).
Nevertheless, the TM tissues from PACG patients exhibited reduced expression pattern of pro-fibrotic genes in those using PG+BB as compared those using PG or BB ( Figure 1B) alone, a pattern similar to that observed in TM tissue of primary glaucoma patients ( Figure 1A).
The expression of genes in TM of PACG patients normalized to the expression of respective iris tissue (TM/iris ratio) is shown in Figure   S2. The expression of TGFβ1, CTGF, FN, LOXL2 and WNT3a in TM tissues of POAG patients was observed to be lower in patients on PG+BB, rather than those on either PG or BB ( Figure 1C). Similar trend in the expression pattern of the genes was observed when the expression of these genes in TM was normalized to the expression of matched iris-TM/iris ratio in POAG patients ( Figure S3).  Figure 3 indicates that the expression of FN was lower in hTM cells treated with PG+BB compared to either PG or BB and controls. The level of CTGF was observed to be higher following PG or BB treatment,

| D ISCUSS I ON
Impediment to the aqueous humour out-flow mechanisms in primary glaucoma, either in POAG or chronic PACG, is related to obstruction of the conventional out-flow pathways. 33 Aberrant ECM deposition in TM serves as a primary aetiopathological factor in POAG, while in PACG, it occurs secondary to physical apposition of cornea and iris angle. This eventually leads to pore closure, increased resistance to aqueous drainage and raised IOP. 33  Aberrant TGFβ response is known to contribute to fibrosis in general. 38 Dysregulation in the factors regulating ECM remodelling has been implicated in TM fibrosis. 10 TGFβ produced by hTM cells is also reported to regulate the pore closure mechanisms in human TM. 10

F I G U R E 2
The effect of prostaglandin analogues or beta-blockers alone and in combination on the differential gene expression of fibrosis-associated genes in human trabecular meshwork cells in vitro. Graphs indicate mean mRNA expression of TGFβ1, TGFβ2, TGFβR2, CTGF, FN, LOXL2, WNT3A, DECORIN and HEVIN normalized to expression of β-ACTIN (housekeeping gene) in human TM cells in vitro following IOP-lowering medication for 24 h. The categories include untreated controls (Ctrl), prostaglandin analogue (PG), beta-blocker (BB) or combination of prostaglandin analogue and beta-blocker (PG+BB) treatments. Bar graphs represent the mean ± SEM of six independent experiments. *P < .05, **P < .01, ***P < .001, Mann-Whitney test remodelling. 24,43,44 In addition, possibly due to reduction in pro-fibrotic factors, an increase in the expression of decorin by PG+BB was also observed, in vitro. Decorin has been reported to have anti-fibrotic functions and have been shown to resolve fibrosis in various systems including TM and cornea. [45][46][47] The role of beta-adrenergic signalling in driving TGFβ expression and fibrosis-associated changes in cardiac tissues has been reported. 48,49 Beta-blockers have been shown to reduce pro-fibrotic mediators and fibrotic changes in liver and cardiac tissues in animal models. [50][51][52] TM tissues have been shown to express beta-adrenergic receptors 53,54 and was observed to be present in hTM cells and TM tissues from the current study cohort ( Figure S6). This suggests the plausibility of beta-blockers regulating fibrosis-asso- and/or ultrastructural studies using electron microscopy would F I G U R E 3 Protein validation of prostaglandin analogues or beta-blockers alone and in combination on the differential gene expression of fibrosis-associated genes in human trabecular meshwork cells in vitro. Panel A shows protein expression validation by immunoblotting for FN, CTGF, pSMAD3 (phosphorylated SMAD3), tSMAD3 (total SMAD3) and decorin following treatment of TM cells with IOP-lowering medications for 24 h in vitro. The categories include untreated controls (Ctrl), prostaglandin analogue (PG), beta-blocker (BB) or combination of prostaglandin analogue and beta-blocker (PG+BB) treatments. GAPDH was used as protein loading controls. The blots shown are representative images of three independent experiments. Panel B exhibits quantification of protein expression of the immunoblot by densitometry analysis. The expression of the protein is indicated as ratio of respective protein to GAPDH (indicated in the y-axis). The expression of pSMAD3 was quantified by normalizing its level to total SMAD3 expression (indicated in the y-axis). Bar graphs represent the mean ± SEM of three independent experiments provide structural evidence to the observations made. In addition, since our data provide evidence of modulation of gene expression patterns at the TM, future studies using RNA-seq or proteomic methods are warranted. In addition to the known improved IOPlowering efficiency by the combination of PG and BB, our data provide additional molecular evidence that they also render fibrosis-reducing effects in TM.

ACK N OWLED G EM ENTS
This work was supported by Narayana Nethralaya Foundation, Bangalore, India. The funding organization had no role in the design or conduct of this research.

CO N FLI C T O F I NTE R E S T
The authors have no financial disclosures or conflicts of interest to declare pertaining to the study.

AUTH O R CO NTR I B UTI O N S
ST contributed towards data acquisition, data interpretation and manuscript preparation. PM contributed towards data acquisition, data interpretation and manuscript preparation. APN contributed towards research design, data acquisition and analysis, and AG contributed towards research design and manuscript preparation.
RKD contributed to data analysis and manuscript preparation. ASG contributed to research design, data interpretation and manuscript preparation. SS contributed towards research design, data analysis, data interpretation and manuscript preparation. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.