title: PTX3 controls activation of matrix metalloproteinase 1 and apoptosis in conjunctivochalasis fibroblasts.
abstract: OBJECTIVE
  Conjunctivochalasis (CCh) is an age-related inflammatory ocular surface disease manifesting redundant, loose conjunctiva folds. The pathogenic role of Pentraxin 3 (PTX3) in controlling upregulation of matrix metalloproteinase 1 (MMP-1) and MMP-3 in CCh remains undefined.
  
  
  METHODS
  Cytolocation of PTX3 and apoptosis were compared by immunostaining and terminal deoxyribonucleotidyl transferase-mediated FITC-linked dUTP nick-end DNA labeling (TUNEL) assay between normal and CCh specimens containing the conjunctiva and the Tenon. Second to third cultures of normal and CCh fibroblasts were treated with or without Aprotinin, Batimastat, or N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), followed by transfection with or without PTX3 siRNA, and TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis measured by the Cell Death Detection ELISA and expression of PTX3, MMP-1, and MMP-3 transcripts and proteins by quantitative RT-PCR and Western blot, respectively.
  
  
  RESULTS
  PTX3 immunostaining was negative in normal specimens, but strongly positive in the subconjunctival stroma of CCh specimens. More apoptotic cells were found in CCh samples than in normal specimens. Expression of PTX3 transcripts and protein was not constitutive in resting normal fibroblasts but was in resting CCh fibroblasts and was upregulated by IL-1β in both cell lysates and culture media of both fibroblasts. PTX3 siRNA further upregulated MMP-1 and MMP-3 transcripts in resting normal fibroblasts, but synergistically with IL-1β upregulated the expression of MMP-1 and MMP-3 transcripts only in CCh fibroblasts, with activation of MMP-1 more so than MMP-3. PTX3 siRNA knockdown also promoted cell death characterized by apoptosis and necrosis, and such cell death could be rescued by inhibitors against serine proteinase, MMP1, or MMP3.
  
  
  CONCLUSIONS
  Perturbation of PTX3 expression might partake in apoptosis and pathogenesis of CCh by upregulating expression of MMP-1 and MMP-3, and activation of MMP-1 and MMP-3.
authors.affiliation: Shenzhen Eye Hospital of Jinan University, Shenzhen, Guangdong, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Guo
authors.forename: Su-Zhen
authors.initials: SZ
authors.lastname: Zhang
authors.forename: Hua
authors.initials: H
authors.lastname: He
authors.forename: Ying-Ting
authors.initials: YT
authors.lastname: Zhu
authors.forename: Scheffer C G
authors.initials: SC
authors.lastname: Tseng
mesh_terms: D000209:Acute-Phase Proteins / Q000502:physiology*; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis; D007611:Aprotinin / Q000494:pharmacology; D015153:Blotting, Western; D002097:C-Reactive Protein / Q000502:physiology*; D002478:Cells, Cultured; D003229:Conjunctival Diseases / Q000201:enzymology* / Q000473:pathology*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism*; D008875:Middle Aged; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D000683:Serum Amyloid P-Component / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D058475:Tenon Capsule / Q000201:enzymology / Q000473:pathology; D013876:Thiophenes / Q000494:pharmacology; D014162:Transfection; D000099153:Pentraxins
chemical_list: D000209:Acute-Phase Proteins; D007611:Aprotinin; D002097:C-Reactive Protein; D006877:Hydroxamic Acids; D020781:Matrix Metalloproteinase 1; D010649:Phenylalanine; D000683:Serum Amyloid P-Component; D013449:Sulfonamides; D013876:Thiophenes; D000099153:Pentraxins; C500716:N-isobutyl-N-(4-methoxyphenylsulfonyl)glycylhydroxamic acid; C080985:batimastat