title: Overexpression of SPARC in human trabecular meshwork increases intraocular pressure and alters extracellular matrix.
abstract: OBJECTIVE
  Intraocular pressure (IOP) regulation is largely unknown. SPARC-null mice demonstrate a lower IOP resulting from increased outflow. SPARC is a matricellular protein often associated with fibrosis. We hypothesized that SPARC overexpression would alter IOP by affecting extracellular matrix (ECM) synthesis and/or turnover in the trabecular meshwork (TM).
  
  
  METHODS
  An adenoviral vector containing human SPARC was used to increase SPARC expression in human TM endothelial cells and perfused human anterior segments using multiplicities of infection (MOIs) 25 or 50. Total RNA from TM was used for quantitative PCR, while protein from cell lysates and conditioned media were used for immunoblot analyses and zymography. After completion of perfusion, the anterior segments were fixed, sectioned, and examined by light and confocal microscopy.
  
  
  RESULTS
  SPARC overexpression increased the IOP of perfused human anterior segments. Fibronectin and collagens IV and I protein levels were elevated in both TM cell cultures and within the juxtacanalicular (JCT) region of perfused anterior segments. Collagen VI and laminin protein levels were increased in TM cell cultures but not in perfused anterior segments. The protein levels of pro-MMP-9 decreased while the kinetic inhibitors of metalloproteinases, TIMP-1 and PAI-1 protein levels, increased at MOI 25. At MOI 50, the protein levels of pro-MMP-1, -3, and -9 also decreased while PAI-1 and TIMP-1 and -3 increased. Only MMP-9 activity was decreased on zymography. mRNA levels of the collagens, fibronectin, and laminin were not affected by SPARC overexpression.
  
  
  CONCLUSIONS
  SPARC overexpression increases IOP in perfused cadaveric human anterior segments resulting from a qualitative change the JCT ECM. Selective decrease of MMP-9 activity is likely part of the mechanism. SPARC is a regulatory node for IOP.
authors.affiliation: Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, MA, USA.
authors.forename: Dong-Jin
authors.initials: DJ
authors.lastname: Oh
authors.forename: Min Hyung
authors.initials: MH
authors.lastname: Kang
authors.forename: Yen Hoong
authors.initials: YH
authors.lastname: Ooi
authors.forename: Kyu Ryong
authors.initials: KR
authors.lastname: Choi
authors.forename: E Helene
authors.initials: EH
authors.lastname: Sage
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Rhee
mesh_terms: D000256:Adenoviridae; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007429:Intraocular Pressure / Q000502:physiology*; D008875:Middle Aged; D015676:Osteonectin; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D024042:Collagen Type I; D024141:Collagen Type IV; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D015676:Osteonectin; D012333:RNA, Messenger; C577241:SPARC protein, human; D025521:Tumor Suppressor Proteins
keywords: SPARC; adenovirus; extracellular matrix; intraocular pressure; matrix metalloproteinases; perfused anterior segment system; tissue inhibitors of matrix metalloproteinases