title: Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.
abstract: OBJECTIVE
  To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.
  
  
  METHODS
  RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA). Gene expression in each diabetic corneal cRNA was assessed against pooled cRNA from 7 to 9 normal corneas. Select differentially expressed genes were validated by quantitative real-time RT-PCR (QPCR) and immunohistochemistry. Organ cultures were treated with a cathepsin inhibitor, cystatin C, or MMP-10.
  
  
  RESULTS
  More than 100 genes were upregulated and 2200 were downregulated in DR corneas. Expression of cathepsin F and hepatocyte growth factor (HGF) genes was increased in ex vivo and organ-cultured DR corneas compared with normal corneas. HGF receptor c-met, fibroblast growth factor (FGF)-3, its receptor FGFR3, tissue inhibitor of metalloproteinase (TIMP)-4, laminin alpha4 chain, and thymosin beta(4) genes were downregulated. The data were corroborated by QPCR and immunohistochemistry analyses; main changes of these components occurred in corneal epithelium. In organ-cultured DR corneas, cystatin C increased laminin-10 and integrin alpha(3)beta(1), whereas in normal corneas MMP-10 decreased laminin-10 and integrin alpha(3)beta(1) expression.
  
  
  CONCLUSIONS
  Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.
authors.affiliation: Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
authors.forename: Mehrnoosh
authors.initials: M
authors.lastname: Saghizadeh
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Kramerov
authors.forename: Jian
authors.initials: J
authors.lastname: Tajbakhsh
authors.forename: Annette M
authors.initials: AM
authors.lastname: Aoki
authors.forename: Charles
authors.initials: C
authors.lastname: Wang
authors.forename: Ning-Ning
authors.initials: NN
authors.lastname: Chai
authors.forename: Julia Y
authors.initials: JY
authors.lastname: Ljubimova
authors.forename: Takako
authors.initials: T
authors.lastname: Sasaki
authors.forename: Gabriel
authors.initials: G
authors.lastname: Sosne
authors.forename: Marc R J
authors.initials: MR
authors.lastname: Carlson
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ljubimov
mesh_terms: D000368:Aged; D003315:Cornea / Q000378:metabolism*; D003316:Corneal Diseases / Q000201:enzymology / Q000235:genetics*; D003930:Diabetic Retinopathy / Q000201:enzymology / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006133:Growth Substances / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D015854:Up-Regulation
chemical_list: D006133:Growth Substances; D012333:RNA, Messenger; D010447:Peptide Hydrolases