title: Overexpression of cystatin C in synovium does not reduce synovitis or cartilage degradation in established osteoarthritis.
abstract: BACKGROUND
  Cathepsin K (catK) expression is increased in cartilage, bone and synovium during osteoarthritis (OA). To study the role of catK expression and elevated cathepsin activity in the synovium on cartilage destruction in established OA, we overexpressed cystatin C (cysC), a natural cysteine protease inhibitor, in the synovium of rabbit OA joints.
  
  
  METHODS
  The ability of cysC to inhibit activity of cathepsins in rabbit OA synovium lysates was tested in vitro using protease activity assay. In vivo, the tissue localization of recombinant adeno-associated virus (rAAV) with LacZ gene after intra-articular injection was determined by β-galactosidase staining of rabbit joints 4 weeks later. To inhibit cathepsin activity in the synovium, a rAAV2-encoding cysC was delivered intra-articularly into rabbit joints 4 weeks after OA was induced by anterior cruciate ligament transection (ACLT). Seven weeks postinjection, endogenous catK and cysC levels as well as the vector-derived cysC expression in the synovium of normal and OA joints were examined by RNA quantification. Synovial cathepsin activity and catK, catB and catL protein levels were determined by activity and Western blot analyses, respectively. Synovitis and cartilage degradation were evaluated by histopathological scoring.
  
  
  RESULTS
  In vitro, the ability of cysC to efficiently inhibit activity of purified catK and OA-induced cathepsins in rabbit synovial lysates was demonstrated. In vivo, the intra-articular delivery of rAAV2/LacZ showed transduction of mostly synovium. Induction of OA in rabbit joints resulted in fourfold increase in catK mRNA compared to sham controls while no change was detected in endogenous cysC mRNA levels in the synovium. Protein levels for catK, catB and catL were also increased in the synovium with a concomitant fourfold increase in cathepsin activity. Joints treated with rAAV2/cysC showed both detection of vector genomes and vector-derived cysC transcripts in the synovium. Production of functional cysC by the vector was demonstrated by complete block of cathepsin activity in the synovium. However, this did not decrease synovitis, bone sclerosis or progression of cartilage degradation.
  
  
  CONCLUSIONS
  Increased production of natural cathepsin inhibitor, cysC, in OA synovium does not alleviate synovitis or cartilage pathology during a preexisting OA.
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. sirkka.kyostio-moore@genzyme.com.
authors.forename: Sirkka
authors.initials: S
authors.lastname: Kyostio-Moore
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. susan.piraino@genzyme.com.
authors.forename: Susan
authors.initials: S
authors.lastname: Piraino
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. patricia.berthelette@genzyme.com.
authors.forename: Patricia
authors.initials: P
authors.lastname: Berthelette
authors.affiliation: Orthopaedic Research, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. nance.moran@genzyme.com.
authors.forename: Nance
authors.initials: N
authors.lastname: Moran
authors.affiliation: Orthopaedic Research, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. joseph.serriello@genzyme.com.
authors.forename: Joseph
authors.initials: J
authors.lastname: Serriello
authors.affiliation: Bolder BioPATH, 5541 Central Avenue, Boulder, CO, USA. Alison@bolderbiopath.com.
authors.forename: Alison
authors.initials: A
authors.lastname: Bendele
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. Cathleen.sookdeo@genzyme.com.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Sookdeo
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. Bindu.nambiar@genzyme.com.
authors.forename: Bindu
authors.initials: B
authors.lastname: Nambiar
authors.affiliation: Pathology, Genzyme, a Sanofi Company, 5 Mountain Road, Framingham, MA, USA. lessleyb@aol.com.
authors.forename: Patty
authors.initials: P
authors.lastname: Ewing
authors.affiliation: Gene Therapy, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. Donna.armentano@genzyme.com.
authors.forename: Donna
authors.initials: D
authors.lastname: Armentano
authors.affiliation: Orthopaedic Research, Genzyme, a Sanofi Company, 49 New York Avenue, Framingham, MA, USA. Gloria.matthews@genzyme.com.
authors.forename: Gloria L
authors.initials: GL
authors.lastname: Matthews
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D055316:Cystatin C / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D011817:Rabbits; D013583:Synovial Membrane / Q000378:metabolism* / Q000473:pathology; D013585:Synovitis / Q000378:metabolism* / Q000473:pathology
chemical_list: D055316:Cystatin C