title: Cathepsin S expression is up-regulated following balloon angioplasty in the hypercholesterolemic rabbit.
abstract: OBJECTIVE
  Neointimal development following balloon angioplasty involves many factors including smooth muscle cell (SMC) migration and proliferation and extracellular matrix (ECM) remodeling. Further, in hypercholesterolemic (HC) conditions, there is an influx of macrophage foam cells (FCs) into the restenotic lesion, which also involves degradation of the basement membrane and surrounding ECM. The ECM remodeling that occurs during restenosis has been shown to be mediated by various proteases. Here we have investigated the role of cathepsin S (CatS), a cysteine protease, in this process.
  
  
  RESULTS
  We have demonstrated by Taqman quantitative PCR, Western blot, and immunohistochemistry that CatS is up-regulated in restenotic lesions of HC rabbits following balloon injury of the iliofemoral artery. CatS mRNA expression was elevated 28-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.05). CatS protein expression was detected within 1 day post-injury, persisted throughout the entire time course evaluated (60 days post-injury), and was co-localized with SMCs, macrophages, and FCs. In contrast, cystatin C (CysC), the endogenous inhibitor of cathepsins, was only minimally up-regulated following injury. CysC mRNA expression was elevated 3.5-fold in balloon-injured vessels relative to uninjured contralateral vessels in HC rabbits 8 weeks post-angioplasty (p<0.005), and up-regulation of protein expression was not detected until days 28 and 60 post-injury. Additional biochemical studies using recombinant rabbit CatS revealed that rabbit CatS digests laminin, fibronectin, and type I collagen. Further, CatS expression was evaluated in SMCs that were induced to migrate through a matrix-coated Boyden chamber upon platelet-derived growth factor (PDGF) stimulation. The addition of a selective CatS inhibitor reduced SMC migration dose-dependently with an 80% reduction in migration at 30 nM (p<0.005). Additionally, we have shown that CatS protein expression by human macrophages was increased upon stimulation with oxidized low density lipoprotein (ox-LDL), implying augmentation of CatS production during foam cell formation.
  
  
  CONCLUSIONS
  Taken together, our results indicate an enhanced expression of CatS during neointima formation and it is associated with invading SMCs, macrophages, and FCs, highlighting the importance of CatS in the pathogenesis of restenosis.
authors.affiliation: Department of Vascular Inflammatory Diseases, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA. cynthia_l_kurtis@gsk.com
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Burns-Kurtis
authors.forename: Alan R
authors.initials: AR
authors.lastname: Olzinski
authors.forename: Saul
authors.initials: S
authors.lastname: Needle
authors.forename: Josephine H
authors.initials: JH
authors.lastname: Fox
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Capper
authors.forename: Fiona M
authors.initials: FM
authors.lastname: Kelly
authors.forename: Michael S
authors.initials: MS
authors.lastname: McQueney
authors.forename: Anne M
authors.initials: AM
authors.lastname: Romanic
mesh_terms: D000800:Angioplasty, Balloon / Q000009:adverse effects*; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D002403:Cathepsins / Q000378:metabolism*; D002465:Cell Movement; D024042:Collagen Type I / Q000378:metabolism; D003251:Constriction, Pathologic; D055316:Cystatin C; D015891:Cystatins / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005263:Femoral Artery / Q000473:pathology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D006937:Hypercholesterolemia / Q000378:metabolism* / Q000473:pathology; D007150:Immunohistochemistry / Q000379:methods; D007797:Laminin / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism; D008297:Male; D009000:Monocytes / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits
chemical_list: C525030:CST3 protein, human; D024042:Collagen Type I; D055316:Cystatin C; D015891:Cystatins; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D007797:Laminin; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D002403:Cathepsins; C028217:cathepsin S