Glaucoma Lowered Decorin With Aberrant Extracellular Matrix Remodeling in Aqueous Humor and Tenon’s Tissue From Primary Glaucoma Patients Sampath NikhalaShree,1,2 Gayathree Karthikkeyan,1 Ronnie George,3 Balekudaru Shantha,3 Lingam Vijaya,3 Vineet Ratra,3 Konerirajapuram Natarajan Sulochana,1 and Karunakaran Coral1 1R.S. Mehta Jain Department of Biochemistry and Cell Biology, KBIRVO, Vision Research Foundation, Sankara Nethralaya, Chennai, India 2School of Chemical and Biotechnology, SASTRA Deemed-to-be University, Thanjavur, India 3Smt Jadhavbai Nathmal Singhvee Glaucoma Services, Medical Research Foundation, Sankara Nethralaya, Chennai, India Correspondence: Karunakaran PURPOSE. To evaluate the inflammatory cytokine, growth factors, extracellular matrix (ECM) Coral, R. S. Mehta Jain Department of remodeling genes, profibrotic and antifibrotic molecules in patients undergoing glaucoma Biochemistry and Cell Biology, filtration surgery (GFS). Additionally, the effect of preoperative antiglaucoma medications Vision Research Foundation, Sankara (AGMs) and postoperative bleb status were related to these parameters. Nethralaya, 41, College Road, Chennai - 600 006, India; METHODS. Tenon’s tissue and aqueous humour (AH) were collected from 207 patients [email protected]. undergoing GFS with primary open-angle glaucoma (POAG) (n ¼ 77), primary angle-closure Submitted: March 14, 2019 glaucoma (PACG) (n ¼ 62), and cataract controls (n ¼ 68). Monocyte chemoattractant Accepted: September 29, 2019 protein-1 (MCP-1), connective tissue growth factor (CTGF), transforming growth factor b1/2 (TGF-b1/2), lysyl oxidase (LOX), lysyl oxidase L2 (LOXL2), elastin (ELN), collagen type 1 a 1 Citation: NikhalaShree S, Karthikkeyan G, George R, et al. Lowered decorin (COL1A1), secreted protein acidic and rich in cysteine (SPARC), a-smooth muscle actin (a- with aberrant extracellular matrix re- SMA), and decorin (DCN) were determined in tenon’s tissue by real-time PCR and in AH using modeling in aqueous humor and Ten- ELISA. on’s tissue from primary glaucoma RESULTS. A significant increase was observed in the transcripts of MCP-1, TGF-b2, and SPARC patients. Invest Ophthalmol Vis Sci. in POAG and PACG ( < 0.05); b and a in PACG 2019;60:4661–4669. https://doi.org/ P CTGF, TGF- 1, LOX, LOXL2, ELN, COL1A1, -SMA 10.1167/iovs.19-27091 (P < 0.05) compared with control. DCN transcript was significantly decreased in POAG and PACG (P < 0.05) compared with control. The protein levels of CTGF, TGF-b1/b2, ELN, SPARC, and LOXL2 was significantly elevated in POAG and PACG (P < 0.05); DCN was decreased (P < 0.05) compared with control. These parameters showed significant association with duration of preoperative AGMs and postoperative bleb status. CONCLUSIONS. This study demonstrates increased expression of growth factors and ECM molecules, both at protein and transcript levels in GFS patients. A decreased DCN in AH seems striking, and if restored might have a therapeutic role in minimizing postoperative scarring to improve GFS outcome. Keywords: glaucoma, aqueous humor, decorin, extracellular matrix laucoma is a multifactorial progressive optic neuropathy increasing its outflow rate using antiglaucoma medications G characterized by loss of visual field, degeneration of retinal (AGMs), that is b-blockers, prostaglandin analogues, a-adrener- ganglion cells (RGCs), and their axons along with damage to gic agonists, cholinergic and carbonic anhydrase inhibitors.9–11 the optic nerve head (ONH). It stands the second leading cause However, patients with medically uncontrolled IOP are of blindness after cataract and accounts for 8% of global surgically treated by trabeculectomy, a filtration surgery.12 blindness as reported by the World Health Organization.1–3 The Postoperative fibrosis and inflammation-related events are the population with glaucoma is estimated to increase to 111.8 key determinants of long-term surgical success following million by 2040 globally, particularly impactful in Asia.4–6 glaucoma filtration surgery (GFS). Antimetabolites such as Primary glaucoma is classified as primary open-angle glaucoma mitomycin-C and 5-fluorouracil are commonly used during the (POAG) and primary angle-closure glaucoma (PACG) with the initial stage of the surgery to prevent excessive postoperative latter significantly prevalent in India.5 Elevated intraocular scarring. Histopathological evidence state that failed fibrotic pressure (IOP) caused due to decreased aqueous outflow capsules reveals higher fibroblast population with increased through the trabecular meshwork (TM) is a major risk factor to production of ECM contributing to the pathophysiology.13 develop glaucoma.7 The drainage of aqueous humour (AH) is Transforming growth factor b (TGF-b)14,15 and its down- reduced due to increased accumulation of extracellular matrix stream mediator connective tissue growth factor (CTGF)16 play (ECM) in the juxtacanalicular TM.8 a crucial role in the pathogenesis of glaucoma and augment Treatment for glaucoma lies focused on reducing IOP that fibrosis. Besides, lysyl oxidase (LOX), that cross-links collagen can be controlled by either limiting the AH production or by and elastin (ELN) is reported to bind with TGF-b1 and regulate Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4661 This work is licensed under a Creative Commons Attribution 4.0 International License. Downloaded from iovs.arvojournals.org on 09/28/2021 Lowered Decorin Levels in Glaucoma IOVS j November 2019 j Vol. 60 j No. 14 j 4662 the ECM remodeling.17 There is a growing interest among 3.1 software, and was found to be minimum of 44 in each researchers to inhibit these molecules as a therapeutic strategy. group. In support to this, recombinant monoclonal neutralizing 18 antibody (mAb) to human TGF-b2 (rhAnti-TGF-b2 mAb), Collection and Storage of Samples CTGF antibody (FG-3019),19 anti-LOX (GS-639556), and anti- LOXL2 (simtuzumab-GS-607601) are being explored in animal AH samples approximately 25 to 100 lL were collected models.20 through an anterior chamber paracentesis at the beginning Decorin (DCN), a small leucine-rich proteoglycan is an of the surgical procedures, using a 30-gauge needle. The AH antagonist of TGF-b and CTGF that regulates cell proliferation, samples were transported on ice and centrifuged at 3000 survival, and differentiation.21 It has shown promising effects rpm for 10 minutes at 48C and immediately frozen in À808C by reducing subconjunctival scarring22 and IOP through deep freezer until processing. Tenon’s tissue excised from fibrinolysis of the scarred TM23 in animal models.24 There is patients undergoing trabeculectomy were transferred in evidence that tissue-specific viral gene transfer of DCN reduce RNA later and stored at À808C until processed for RNA corneal scarring significantly25 due to its inhibitory effect on extraction. TGF-b in corneal fibroblasts.26 Despite these beneficial facts, the levels of DCN in AH is still not evaluated in relevance to RNA Isolation and cDNA Conversion glaucoma in human subjects. Moreover, there is inadequate data on the transcript and Total RNA was extracted using TRIzol reagent (Life Technol- protein level of these factors in AH and Tenon’s tissue in POAG ogies Corp., Grand Island, NY, USA) as per the manufacturer’s and PACG. This study aims to address this gap by measuring the instruction and was quantified using the Nanodrop spectro- inflammatory cytokine (monocyte chemoattractant protein-1 photometer (Thermo Scientific, Waltham, MA, USA). Challeng- [MCP-1]), growth factors (TGF-b, CTGF), ECM remodeling es faced during RNA extraction was due to the fibrotic nature genes (LOX, LOXL2, SPARC, COL1A1, ELN), profibrotic (a- and small size of the specimens. One microgram of total RNA smooth muscle actin [a-SMA]) and antifibrotic (DCN) mole- was converted to cDNA using the iScript cDNA synthesis kit cules in tenon’s tissue, and their corresponding protein levels (Bio-Rad, Hercules, CA, USA). in AH of POAG, PACG, and cataract control. Additionally, we investigated the association of these specific proteins with the Real-Time PCR Analysis duration of preoperative AGMs and postoperative bleb status. Quantitative PCR (qPCR) based on SYBR green chemistry was done using the Roche Light Cycler-96 PCR system MATERIALS AND METHODS (Roche, Basel, Switzerland). The mRNA fold change expres- sion of genes namely, MCP-1, CTGF, TGF-b1/2, ELN, Patient Selection COL1A1, SPARC, LOX, LOXL2, a-SMA,andDCN in each of PACG, POAG, and cataract tenon’s tissue specimens were This was a case-control study that adhered to the guidelines of analyzed. The qPCR primers sequences are given in Table 2. the Declaration of Helsinki. The study was conducted by the Real-time PCR cycle conditions included the following steps: Medical and Vision Research Foundations, India, and was initial denaturation at 958C for 5 minutes, followed by 50 approved by the Institutional Ethics Committee (ICH-GCP): cycles of denaturation at 958C for 20 seconds, annealing at study code 289-2011-P. Informed consent was obtained from 608C for 20 seconds, and extension at 728C for 35 seconds. the patients. Subjects were (1) 18 years or older and (2) either The comparative 2(ÀDDCt) method28 was used to analyze the with POAG or PACG. The demographics and relevant clinical results of the genes of interest relative to the internal control details of glaucoma and cataract control subjects are provided gene (18S rRNA). in Table 1. Patients with other ophthalmic conditions such as history of ocular trauma or uveitis, evidence of ocular