Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.
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.
Saghizadeh
Mehrnoosh
M
Ophthalmology Research Laboratories, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.
Kramerov
Andrei A
AA
Tajbakhsh
Jian
J
Aoki
Annette M
AM
Wang
Charles
C
Chai
Ning-Ning
NN
Ljubimova
Julia Y
JY
Sasaki
Takako
T
Sosne
Gabriel
G
Carlson
Marc R J
MR
Nelson
Stanley F
SF
Ljubimov
Alexander V
AV
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
D006133:Growth Substances; D012333:RNA, Messenger; D010447:Peptide Hydrolases