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Altered Decorin Biology in Proliferative Vitreoretinopathy: a Mechanistic and Cohort Study

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Altered Decorin Biology in Proliferative Vitreoretinopathy: a Mechanistic and Cohort Study Retinal Cell Biology Altered Decorin Biology in Proliferative Vitreoretinopathy: A Mechanistic and Cohort Study Ghazala Begum,1,2 Jenna O’Neill,3 Rishika Chaudhary,1,2,4 Karen Blachford,5 David R. J. Snead,6 Martin Berry,1 Robert A. H. Scott,1,2,7 Ann Logan,1,2 and Richard J. Blanch1,2,5,8 1Inflammation and Ageing, University of Birmingham, Birmingham, United Kingdom 2NIHR Surgical Reconstruction and Microbiology Research Centre, University of Birmingham, Birmingham, United Kingdom 3Ridgeway Research Ltd., St. Briavels, Gloucestershire, United Kingdom 4Ophthalmology Department, University Hospitals Birmingham NHS Foundation Trust, Birmingham, United Kingdom 5Academic Unit of Ophthalmology, Birmingham and Midland Eye Centre, Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, United Kingdom 6Department of Pathology, University Hospitals Coventry and Warwickshire NHS Trust, Coventry, Warwickshire, United Kingdom 7SpaMedica, Birmingham, United Kingdom 8Academic Department of Military Surgery and Trauma, Royal Centre for Defence Medicine, Birmingham, United Kingdom Correspondence: Richard J. Blanch, PURPOSE. To determine if vitreous levels of the pro-fibrotic cytokine transforming growth Neuroscience and Ophthalmology, factor beta2 (TGF-b2) and its opposing regulator decorin predict subsequent proliferative Institute of Inflammation and Age- vitreoretinopathy (PVR) development in patients with rhegmatogenous retinal detachment ing, University of Birmingham, Edg- (RRD). baston, Birmingham B15 2TT, UK; [email protected]. METHODS. We examined the effect of TGF-b2 and decorin on epithelial-mesenchymal transition Submitted: March 8, 2018 (EMT) and collagen expression in vitro using ARPE-19 cells, and we analyzed extracellular Accepted: September 11, 2018 matrix marker expression in PVR membrane and internal limiting membrane patient samples. We performed a prospective noninterventional cohort study, recruiting 125 patients Citation: Begum G, O’Neill J, Chaudh- undergoing vitrectomy for RRD and macular hole surgery, measured vitreous levels of TGF- ary R, et al. Altered decorin biology in proliferative vitreoretinopathy: a b2 and decorin by ELISA, and followed them up for 6 months. Patients who did not develop mechanistic and cohort study. Invest PVR were compared to those who did, in order to determine whether vitreous TGF-b2 and Ophthalmol Vis Sci. 2018;59:4929– decorin levels predicted PVR development. 4936. https://doi.org/10.1167/ RESULTS. In vitro, TGF-b2 induced EMT and collagen production. Decorin strongly inhibited iovs.18-24299 EMT and collagen production at high levels. PVR membranes expressed high levels of fibrosis- associated proteins, consistent with EMT. Vitreous TGF-b2 levels were unchanged between patients with macular holes and RRD who did or did not subsequently develop PVR. Average decorin levels were higher in the vitreous of RRD patients who subsequently developed PVR compared to those who did not, but at the measured vitreous concentrations (1–2 lg/mL), decorin did not demonstrate an in vitro inhibitory effect on EMT. CONCLUSIONS. In vitro, high concentrations of decorin inhibited EMT and fibrosis. At the levels seen in human vitreous, decorin did not prevent fibrosis or EMT in vitro, and higher initial vitreous decorin levels were associated with the development of postoperative PVR after vitrectomy to treat RRD, but did not reliably predict the outcome. Keywords: proliferative vitreoretinopathy, vitreous humor, epithelial to mesenchymal transition, transforming growth factor beta 2, retinal detachment roliferative vitreoretinopathy (PVR) describes a retinal The TGF-b superfamily modulates cell growth, inflamma- P scarring process that occurs in 5% to 10% of rhegmatog- tion, matrix synthesis, and apoptosis.3 TGF-b exists in two main enous retinal detachment (RRD) patients and up to 50% of open forms (TGF-b1 and TGF-b2), with TGF-b2 being the predom- globe injury patients and is the main cause of surgical failure inant form in the posterior segment of human eyes.4,5 Both in after retinal detachment surgery.1 Clinically, PVR is character- vitro and in vivo, TGF-b isoforms regulate synthesis and ized by the growth and contraction of cellular fibrotic degradation of extracellular matrix (ECM) proteins, causing membranes within the vitreous, retina, and subretinal space, increased collagen accumulation and fibrosis,6 which makes which exert traction, open treated breaks, create new breaks, them key candidate prognostic biomarkers for PVR develop- and cause redetachment or distort the macula. Although the ment and important therapeutic targets for prophylaxis and clinical manifestations of PVR are wide, they share a common treatment of PVR. TGF-b is secreted as part of a latent sequence of underlying cellular responses that are associated complex,7 cleaved into its active form by RPE-derived with RRD and vitreous changes, and eyes with existing PVR are thrombospondin-1. Activated TGF-b causes RPE cells to at higher risk of increased retinal cell proliferation after repeat undergo epithelial-mesenchymal transition (EMT) to become vitreoretinal surgery.2 fibroblasts, to produce type 1 collagen, and to become Copyright 2018 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4929 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 09/29/2021 Decorin Biology in Proliferative Vitreoretinopathy IOVS j October 2018 j Vol. 59 j No. 12 j 4930 myofibroblast-like in the absence of normal cell-cell or cell- method based on primer efficiencies (HK-SY-hu-1200; Primer- matrix interactions in vitro.8–10 There are separate TGF-b1 and design, Camberley, UK). Data are presented normalized to TGF- TGF-b2 receptors, though many of these are cross-reactive.11,12 b2-treated groups or the Àserum group. Several studies have found elevated TGF-b2 levels in vitreous 5,13,14 samples from patients with PVR, suggesting a role in Bromodeoxyuridine (BrdU) ELISA pathogenesis and potential as a predictive biomarker for PVR development. The ELISA was performed as previously described.22 Please see Decorin is a small leucine-rich proteoglycan matrix-constit- Supplementary Methods. uent that opposes the actions of TGF-b by interacting with TGF-b and other cytokines. Decorin modulates matrix metal- Clinical Studies loproteinase (MMP) activity by increasing levels of plasmino- gen15 favoring higher ECM turnover leading to ECM This cohort study was approved by the Solihull National degradation. Decorin elevates MMP2 and MMP9 levels, reduces Research Ethics Service committee and the use of tissue for fibronectin and laminin deposition,16 and attenuates fibrosis in immunohistochemistry by Arden Tissue Bank. The study animal models of many pathological conditions, including was conducted in accordance with the Declaration of proliferative vitreoretinopathy,17 renal fibrosis,18 lung fibro- Helsinki. sis,19 juvenile communicating hydrocephalus,20 and spinal We included patients in the cohort study who were cord injury.5,21 undergoing vitrectomy for new RRD, MH, and established We hypothesized that TGF-b would induce EMT in RPE cells PVR and who were capable of consenting to participation. We in vitro and that decorin would oppose EMT. We also predicted excluded patients who had any previous vitreoretinal surgery that, in humans, high vitreous TGF-b and low vitreous decorin (except for the established PVR group) or were aged under levels would predict the subsequent development of PVR in 10. patients with RRD and that RRD patients would have higher Tissue for immunohistochemistry was collected with vitreous TGF-b levels than patients with macular holes (MH). consent from patients who were undergoing vitrectomy with We investigated the effects of TGF-b2 and decorin on ARPE- membrane peel for PVR or internal limiting membrane (ILM) 19 cell EMT in vitro, we looked for immunohistologic evidence peel for MH repair. Total number of samples collected for each of EMT in human PVR membranes, and we performed a group is represented in Supplementary Figure S1. prospective noninterventional cohort study of MH and RRD patients, following them up for 6 months to determine PVR Sample Collection development. Eyes with macula holes do not develop intraocular scarring despite having a retinal break and were Vitreous was collected into a 5-mL syringe through an used as a control for retinal detachment cases where there is a unprimed cutter at the start of surgery in the largest safe retinal break and a risk of intraocular scarring. volume possible, ranging between 0.5 and 2 mL, before the infusion was turned on. Samples were refrigerated for up to 4 hours before being centrifuged at 12,300 g for 10 minutes METHODS and the supernatant collected and frozen at À808 until analysis. Cell Culture ARPE-19 cells (CRL-2302; ATCC, Manassas, VA, USA) were 3, 3-Diaminobenzidine (DAB) Staining of Human grown in Dulbecco’s modified Eagle’s medium/F12 supple- PVR and ILM Tissues mented with 100 U/mL penicillin/streptomycin and 10% heat- inactivated fetal bovine serum. Cells were incubated at 378Cin The membranes were removed from fixative and embedded in a humidified environment containing 5% CO2. For real-time warm agar. Set agar blocks were processed into paraffin wax PCR (qPCR), 300,000 cells/well were seeded in a six-well plate, using a tissue processor (study reference: 17-0477) (Path- incubated overnight, and washed with PBS to remove FBS. Center; Shandon Scientific, Runcorn, UK). Cells were then either treated with serum plus medium, 10 ng/ Blocks were sectioned at 5 lm using a microtome (Bright mL transforming growth factor b2 (TGF-b2) in serum-free 5040; Bright Instruments, Luton, UK) and placed onto slides (Àserum) media or given Àserum medium only. After 72 hours, (Superfrost; Thermo Fisher Scientific UK Ltd, Loughborough, cells were treated with decorin concentrations from 1 ng/mL UK) and dried overnight at 508C. Slides were stained by DAB up to 100 lg/mL with and without the previous treatment of according to the manufacturer’s instructions (Leica, Wetzlar, TGF-b2inÀserum medium and left for a further 72 hours, after Germany). Please see Supplementary Methods. which they were harvested.
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