Overexpression Of Cathepsins And Growth Factors Abnormalities Identified In Diabetic Corneas By Gene Microarray Analysis

Abstract

Abstract: : Purpose: We have previously documented upregulation of matrix metalloproteinase (MMP)–10, MMP–3 and insulin–like growth factor (IGF)–I in human corneas from donors with diabetic retinopathy (DR). Our goal was to identify additional proteases and growth factors with abnormal expression in these corneas using gene microarray analysis with verification at the mRNA and protein levels. Methods: Total RNA was isolated from 20 normal, diabetic and DR corneas, as well as from 13 normal, diabetic and DR corneas organ–cultured for 10–35 days. All corneas were obtained from National Disease Research Interchange (NDRI). Amplified cRNA was generated and labeled with Cy5 (normal) or Cy3 (DR) using Low RNA Input Linear Amplification Kit (Agilent, Palo Alto, CA) and hybridized to 22K Agilent Human 1A Oligo Microarrays. Gene expression in each individual DR cornea was assessed against a pool of cDNA from 9 normal corneas (5 for organ cultures). Select genes differentially expressed in DR corneas were further analyzed by immunohistochemistry and real–time RT–PCR with SYBR green detection using Applied Biosystems machine. Various antibodies were first verified for positive reaction on cryostat sections of human brain and gliomas. Results: More than 1,500 genes were upregulated and 800 were downregulated in DR corneas with a 2–fold cutoff and p<0.01. A consistent feature of both intact and organ–cultured DR corneas was increased expression of proteases and apoptosis–related genes. Significant elevation of cathepsins B, F, L, and of hepatocyte growth factor (HGF) was seen in DR corneas, whereas FGF–3 and its receptor were downregulated. There was a good correlation between gene microarray results and gene expression analyzed by real–time RT–PCR. Moreover, increased epithelial staining was observed for HGF and cathepsins B, F, and L in intact and organ–cultured DR corneas compared to respective normal corneas. Conclusions: In accordance with our hypothesis of increased proteolysis in DR corneas, elevated expression of cathepsins was observed in these corneas both at the gene and protein expression levels. These changes may be brought about by specific growth factors that have abnormal expression in DR corneas, such as HGF, IGF–I and FGF–3. Local protease blocking could possibly reduce corneal epitheliopathy typical for diabetic patients.

Keywords: cornea: epithelium • gene microarray • diabetic retinopathy