title: Lentiviral Delivery of Small Hairpin RNA Targeting Connective Tissue Growth Factor Blocks Profibrotic Signaling in Tenon's Capsule Fibroblasts.
abstract: OBJECTIVE
  Trabeculectomy is a surgical procedure for lowering intraocular pressure in glaucoma patients, in which excessive scarring leading to failure of the filtering bleb adversely affects the surgical outcome. Heightened Tenon's capsule fibroblast (TCF) proliferation and extracellular matrix (ECM) deposition are implicated in this process but endogenous factors that regulate TCF functions remain largely elusive. This study sought to elucidate the role of connective tissue growth factor (CTGF) in the regulation of TCF phenotypes and signaling.
  
  
  METHODS
  Expression of CTGF in scarring and nonscarring Tenon's capsules was measured by real-time PCR and immunofluorescence. Knockdown of CTGC was achieved by lentivirus delivery of small-hairpin RNA. Cell proliferation was measured by CCK8, cell cycle progression, and apoptosis by flow cytometry, adhesion, migration, and invasion of TCF by functional assays in vitro. Proteins and cytokines related to fibrosis were measured by Western blot and ELISA, respectively.
  
  
  RESULTS
  Expression of CTGF was significantly upregulated in scarring Tenon's capsules and their isolated fibroblasts when compared with the nonfibrotic counterparts. Functionally, targeting CTGF with lentivirus-delivered small-hairpin RNA inhibited the proliferation, adhesion, migration, and invasion of TCFs, accompanied by downregulation of p38 and nuclear factor-κB as well as matrix metalloproteinase-2, cyclin D1, and collagen I. In addition, lentiviral targeting of CTGF reduced the release of fibrosis-related cytokines from TCFs and inhibited TCF-conditioned, medium-induced macrophage chemotaxis.
  
  
  CONCLUSIONS
  Our study supports a crucial role of CTGF in the regulation of TCF proliferation and ECM deposition. Targeting CTGF using lentiviral vector may be a promising approach for preventing excessive scarring after trabeculectomy.
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China 2The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Lei
authors.affiliation: Department of Biology, University of Science and Technology of China, Hefei, China.
authors.forename: Changgui
authors.initials: C
authors.lastname: Dong
authors.affiliation: Department of Anesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, China 5State Key Laboratory of Digestive Disease, LKS Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: William Ka Kei
authors.initials: WK
authors.lastname: Wu
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China 2The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province, China.
authors.forename: Aimeng
authors.initials: A
authors.lastname: Dong
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China 2The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Anesthesia and Intensive Care, The Chinese University of Hong Kong, Hong Kong, China.
authors.forename: Matthew T V
authors.initials: MT
authors.lastname: Chan
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China 2The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province, China.
authors.forename: Xinrong
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology, the Second Affiliated Hospital of Harbin Medical University, Harbin, China.
authors.forename: Huiping
authors.initials: H
authors.lastname: Yuan
mesh_terms: D015153:Blotting, Western; D002455:Cell Division; D049109:Cell Proliferation; D002478:Cells, Cultured; D002921:Cicatrix / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D011183:Postoperative Complications / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control; D012333:RNA, Messenger / Q000235:genetics*; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D058475:Tenon Capsule / Q000378:metabolism* / Q000473:pathology; D014130:Trabeculectomy / Q000009:adverse effects
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D055513:Connective Tissue Growth Factor