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Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma

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Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma Glaucoma Connective Tissue Growth Factor Is Increased in Pseudoexfoliation Glaucoma John G. Browne,1 Su Ling Ho,2 Rosemary Kane,1 Noelynn Oliver,3 Abbot. F. Clark,4,5 Colm J. O’Brien,1,2 and John K. Crean6 PURPOSE. Pseudoexfoliation (PXF) syndrome is a generalized seudoexfoliation syndrome (PXF) is an age-related disorder disorder of the extracellular matrix (ECM) involving the trabec- Pthat manifests with abnormal fibrillar extracellular material ular meshwork (TM), associated with raised intraocular pres- (ECM) accumulation in ocular tissues.1 Fibrillar material similar sure, glaucoma, and cataract. The purposes of this study were to that in the eyes of PXF patients has more recently been to quantify aqueous humor connective tissue growth factor detected in the skin and visceral organs of patients with PXF.2 (CTGF) in PXF glaucoma, to determine the effect of CTGF on In the eye, PXF is detected by pupil dilation and subsequent slit ECM production in TM cells, and to identify intracellular CTGF lamp examination. Deposits of fibrillar material are observed in signaling pathways. the anterior segment, primarily on the pupillary border and anterior lens capsule. The disease usually has an insidious METHODS. Aqueous humor samples were obtained from pa- tients undergoing routine cataract surgery or trabeculectomy. onset, unless complications occur such as cataract and glau- CTGF levels were quantified by ELISA. The effect of CTGF on coma. PXF is reported to be responsible for more than half of the cases of open-angle glaucoma in Norway, Ireland, Greece, fibrillin-1 expression in TM cells was investigated by real-time 3 PCR. Western immunoblot analysis was used to investigate and Saudi Arabia. Patients with PXF have a 5.3% chance of developing glaucoma within 5 years, increasing to 15.4% CTGF signaling. c-Jun/AP-1 activation was measured in CHO 4 cells by ELISA after stimulation with CTGF. within 10 years. PXF-associated secondary open-angle glau- coma is a relatively severe and progressive type of glaucoma RESULTS. PXF with glaucoma had the highest aqueous humor with a worse prognosis and more extensive complications ϭ Ϯ Ͻ level of CTGF (n 18; 5.15 0.79 ng/mL [SEM]; P 0.01) with surgery.5 Many factors contribute to the pathologic fea- ϭ Ϯ compared with PXF without glaucoma (n 15; 2.76 0.64 tures of open-angle glaucoma in PXF, including obstruction of ϭ ng/mL), primary open-angle glaucoma (POAG; n 20; the trabecular meshwork (TM) area by the deposition of pseu- Ϯ ϭ Ϯ 3.05 0.40 ng/mL), and the control (n 21; 2.60 0.29 doexfoliative material, juxtacanalicular endothelial cell dys- ng/mL). In vitro exposure of TM cells to CTGF resulted in a function, and increased aqueous humor protein levels.6 50% upregulation of fibrillin-1, which was partially blocked The protein components of pseudoexfoliative material in- with the MEK (mitogen-activated protein extracellular ki- clude both noncollagenous basement membrane components nase) inhibitor PD098059. Western blot analysis demon- such as laminin, fibronectin,7 amyloid P, and vitronectin. Also strated increased phosphorylation of p42/44 MAPK, p38 contained in the pseudoexfoliative material are proteinaceous MAPK, and the JNK pathways in response to CTGF. c-Jun/ components of elastic fibers, such as elastin, tropoelastin, fibril- AP-1 activity was significantly increased in response to CTGF lin-1, microfibril-associated glycoprotein-1, and latent TGF-␤- treatment. binding proteins (LTBP-1 and -2).8,9 The elastic microfibril CONCLUSIONS. Increased levels of CTGF in the aqueous humor of theory of PXF pathogenesis hypothesizes that PXF is a type of PXF patients likely has pathologic significance through in- elastosis characterized by excessive synthesis of elastic micro- creased production of fibrillin-1 by TM cells through activation fibrillar components throughout the body. This theory was of p42/44 MAPK, p38 MAPK, and JNK pathways. (Invest Oph- supported by molecular biological studies confirming the over- thalmol Vis Sci. 2011;52:3660–3666) DOI:10.1167/iovs.10- expression of fibrillin-1, LTBP-1, and LTBP-2 mRNA in most of 5209 the affected tissues and cell types.8,10 In 1991 Bradham et al.11 discovered human connective tissue growth factor (CTGF/CCN2) as a protein secreted by human umbilical vascular endothelial cells. CTGF is a member From the Schools of 1Medicine and Medical Science and 6Biomo- of a family of proteins with a similar structure known as the lecular and Biomedical Science, The UCD Conway Institute of Biomo- CCN family.12 Members of the CCN family have four distinct lecular and Biomedical Research, University College Dublin, Dublin, modules in their structure. These modules are an insulin-like Ireland; the 2Mater Misericordiae University Hospital, Dublin, Ireland; 3Fibrogen, Inc., South San Francisco, California; and the 4Department growth factor (IGF)-binding protein–like module (IGF-BP), von of Cell Biology and Anatomy and the 5North Texas Eye Research Willebrand factor type C repeat (VWC), thrombospondin Institute, University of North Texas Health Science Center, Fort Worth, type-1 repeat (TSP1), and C-terminal module (CT). CTGF is a Texas. 36- to 38-kDa protein rich in cysteine13 and is expressed in a Supported by the Health Research Board and Science Foundation variety of cell types, including fibroblasts, vascular smooth Ireland (JGB, JKC). muscle cells, endothelial cells, neuronal cells, and epithelial Submitted for publication January 14, 2010; revised June 8, Sep- cells.14 CTGF and transforming growth factor (TGF)-␤ interact tember 22, and November 15, 2010, and January 14, 2011; accepted via a binding site located in the amino terminal von Willebrand January 19, 2011. factor domain of CTGF.15 CTGF expression is induced by Disclosure: J.G. Browne, Fibrogen (F); S.L. Ho, Fibrogen (F); R. TGF␤, and CTGF mediates some of the downstream effects of Kane, Fibrogen (F); N. Oliver, Fibrogen (F); A.F. Clark, Fibrogen (F); 16,17 C.J. O’Brien, Fibrogen (F); J.K. Crean, Fibrogen (F) TGF␤ on proliferation, migration, and ECM production. Corresponding author: John K. Crean, University College Dublin, Pathologic fibrosis is often attributed to uncontrolled matrix Conway Institute Belfield, Dublin 4, Ireland; [email protected]. deposition, perhaps mediated by CTGF.18 Investigative Ophthalmology & Visual Science, May 2011, Vol. 52, No. 6 3660 Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 09/25/2021 IOVS, May 2011, Vol. 52, No. 6 CTGF and Pseudoexfoliation Glaucoma 3661 CTGF was first identified as a profibrotic mediator that was graphic information were obtained from the case notes. The patients upregulated in both in vivo and in vitro models of diabetic were diagnosed and graded with pseudoexfoliation (PXF) syndrome nephropathy,19 with a suppression subtractive hybridization based on the biomicroscopy findings, and the PXF group was divided (SSH) screen and characterized the mechanism through which into two subcategories—PXF without glaucoma and PXF with glau- elevated CTGF expression leads to matrix accumulation20 (for coma—on the basis of case notes history. The diagnosis of glaucoma review, see Ref. 21). Current models of activity propose that included cupped optic disc, documented pattern of glaucomatous CTGF largely functions as a matricellular protein, modulating visual field loss, and a history of an intraocular pressure greater than 21 and integrating the role of other growth factor signaling path- mm Hg. The POAG patients had the same clinical features as the PXF ways, including those of the TGF␤ superfamily. Signaling and glaucoma group other than having no evidence of PXF, both groups regulatory insights have remained elusive with the observation having open drainage angles on slit lamp biomicroscopy. Patients with that CTGF can bind to multiple receptors on the cell surface glaucoma were maintained on their preoperative antiglaucoma medi- and activate divergent signaling pathways, including p42/44 cations. MAPK,20 PI3K,22 Rho GTPase,23 and p38 MAPK.24 Functional interplay of these signaling networks is proposed as the key Enzyme Immunoassay for CTGF mechanism controlling CTGF-mediated gene transcription. The three isoforms of TGF␤,-␤1, -␤2, and -␤3 are found in Levels of CTGF in the aqueous humor were determined by sandwich the eye.25,26 TGF␤ has been shown in numerous studies to play ELISAs developed by Fibrogen, Inc. (South San Francisco, CA), by using a role in ocular wound healing,27,28 and its role in the patho- pairs of CTGF-specific monoclonal antibodies that specifically bind to genesis of glaucoma is also well documented.29 Several studies distinct epitopes on the N-terminal half of the CTGF protein and thus have reported elevated aqueous humor levels of TGF␤2in allows for detection of both full-length CTGF and N-terminal half patients with POAG.30,31 On the other hand, the aqueous fragments of CTGF. CTGF is unstable in vivo and proteolytic fragments humor of patients with PXF glaucoma was found to have of full-length CTGF varying from 10 to 20 kDa have been found in ␤ 9 human biological fluids (serum, cerebrospinal, amniotic, follicular, and elevated levels of TGF 1. In their study, Schlotzer-Schrehardt 34 35 et al.9,32 showed that TGF␤1, in both the latent and intrinsi- peritoneal fluids). and in porcine uterine luminal secretory fluid by Western blot analysis. Biological activities from these low-mass frag- cally active forms, was significantly raised in PXF glaucoma 36–38 eyes compared with POAG eyes. In contrast, both the latent ments have also been reported. and active forms of TGF␤2 were elevated in POAG eyes but not A range-finding experiment was initially conducted to determine 9,32 ␤ the most suitable sample dilution, as per the method previously de- in PXF glaucomatous eyes. TGF 1 had a more pronounced 33 effect on ECM production on cultured human Tenon’s capsule scribed.
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