Gene microarray analysis is a powerful tool for identifying gene expression profiles of healthy and diseased tissue. In the cornea, this technology has been successfully used to identify gene expression in specific cell types48
and its changes after growth factor, cytokine, drug, or hypoxia treatment,53-55
during wound healing,56,57
and in keratoconus and bullous keratopathy.47,58
Although gene microarray results are generally accurate and reproducible,57
many authors agree that these data must be verified by subsequent mRNA and protein analysis.52,57
The present microarray results were validated by QPCR and immunostaining. However, we did not detect significant changes in the expression of MMP-3 and MMP-10 that were previously shown to be increased in DR corneas compared with normal corneas using various methods.21,25
The expression of these genes was only increased in a minority of diabetic and DR samples. Previously noted discrepancies between gene array and QPCR or protein data were ascribed to errors in conventional array analysis software, errors in cRNA amplification, lower array sensitivity compared with QPCR, or individual subject variability by age and sex.59-61
For these reasons, validation of microarray results by QPCR and immunohistochemistry or Western blotting remains a necessary step in identifying gene expression changes in disease.
Here we have attempted to identify gene and protein expression abnormalities in diabetic and DR human corneas compared with normal ones using Agilent 22,000-gene microarrays. Because diabetes affects all corneal cells, whole corneal gene expression was analyzed. We hypothesized21,28
that molecular alterations in diabetic corneas could have been caused by increased proteolysis, possibly triggered by altered growth factor/cytokine expression. Consequently, we focused on gene microarray analysis of proteinases and growth factors/cytokines comparing the expression of each gene in individual diabetic and DR corneas with the expression of the same gene in a pool of normal corneas. This approach21
allowed us to minimize individual variations between normal corneas, compare individual diseased corneas to reveal most common changes, and reduce the cost of analysis. Results were verified by QPCR (mRNA expression) and immunohistochemistry (protein expression) for select differentially expressed genes. None of these genes were altered in the same way in keratoconus or bullous keratopathy (Refs. 47, 58
and Spirin KS, Brown DJ, Ljubimov AV, Kenney MC, unpublished data, 2000) as they were in diabetic corneas, suggesting that their changes were specific for diabetes.
Previously, cathepsins A, B, D, G, K, L, and V/L2 were found in corneal cells.62-66
We provide here the first account of cathepsins F and H in human cornea. They were mostly localized in the epithelium, and cathepsin F expression was significantly increased in DR corneas (; ). Two cathepsin inhibitors, cystatins F and S, were significantly decreased in diabetic and DR corneas (), which could lead to a net increase in cathepsin activity. Cathepsin F was described in macrophages and in atherosclerotic lesions, where it participates in major histocompatibility complex class 2 processing and can modify low-density lipoprotein particles by cleaving apolipoprotein B-100.67-69
Cathepsins are a family of proteolytic enzymes, many of which, including cathepsin F, are cysteine proteinases; some cathepsins are serine or aspartic proteinases.38
Cathepsins are mostly lysosomal proteinases that are increased in tumors and may degrade extracellular substrata such as laminin. The half-life of a cathepsin at neutral pH increases when it uses extracellular matrix proteins as substrata.70,71
Therefore, an increase in cathepsin F could play a role in the degradation of diabetic corneal epithelial BM and integrins.20
In fact, our data show that treating organ-cultured DR corneas with a potent cathepsin F inhibitor, cystatin C, restored normal laminin-10 and integrin α3
staining patterns (, left). In experimental nephrosis, the activation of cathepsin B is accompanied by a decrease of α3
;a similar situation may exist in diabetic corneas.
We previously observed an increased expression and activity of stromelysins MMP-3 and, especially, MMP-10 in diabetic and DR corneas.21
Therefore, it seemed important to examine a direct effect of MMP-10 (contrary to MMP-3, it was primarily upregulated in the epithelium in DR corneas) on diabetic markers and epithelial wound healing by treating normal organ-cultured corneas with MMP-10. The treatment delayed epithelial wound healing and led to diabetic-like alterations of laminin-10 α5 chain and integrin α3
(, right). Therefore, the upregulation of BM-degrading MMP-10 in DR corneas may play a functional role in diabetic corneal epithelial abnormalities. Overall, the data on cathepsin inhibitor and MMP-10 treatment of organ-cultured corneas provide additional support to our hypothesis of the importance of increased proteolysis in corneal diabetic alterations.
Stromelysin activity may be increased in diabetes by the downregulation of TIMPs. Previously, no increase of TIMP-1, TIMP-2, or TIMP-3 was found in diabetic corneas.21
However, the present analysis revealed the downregulation of TIMP-4 in such corneas (Figs. , , ), suggesting an increase in net proteolytic activity.72
TIMP-4 can inhibit MMP-2 and MMP-9. To date, its interactions with MMP-3 and MMP-10 have not been studied and may be of interest for future investigations. TIMP-4 is able to induce cell apoptosis.73
Therefore, its decrease in DR corneas may also be related to a possible attempt of the corneal tissue to reduce cell death in high glucose conditions.
The HGF/c-met system is important for wound healing, cell migration, epithelial branching morphogenesis, and angiogenesis.41,42,75
In DR corneas, epithelial HGF increased, whereas c-met expression decreased (Tables , ). The downregulation of c-met might impair HGF signaling, which could lead to reduced migration of DR corneal epithelium and delayed wound healing. A somewhat similar situation exists in fulminant hepatic failure, with abundant circulating HGF but lack of c-met in the liver, which results in massive cell death and liver failure.42
It would be interesting to identify the effects of increasing c-met in the cornea by gene therapy on wound healing in DR corneas.
Another indirect indication for decreased HGF activity in DR corneas may be the downregulation of Tβ4
. This 5-kDa polypeptide is an actin-sequestering protein and a mediator of cell proliferation, differentiation, angiogenesis, and metastasis.45
It also promotes cell migration and wound healing.43-45
In DR corneas, Tβ4
expression is decreased in the epithelium (Figs. , ; Tables , ). Because HGF can enhance Tβ4
its reduction in DR corneas may result from possible downregulation of HGF signaling because of decreased c-met. In turn, reduced expression of laminin expression-stimulating77
may contribute to BM degradation and delayed wound healing in DR corneas.
FGF-3 plays a role in corneal development and epithelial differentiation.46
In DR corneas, it is decreased coordinately with one of its receptors, FGFR3 (). FGF-3 is identical to the int-2 proto-oncogene upregulated in different tumors.78
Certain data suggest that the FGF-3 locus on chromosome 11q may be implicated in genetic susceptibility to IDDM.79
FGFR3 is upregulated in skin wounds and in migrating neural crest cells during development.80,81
Therefore, FGF-3 and FGFR3 might participate in wound healing and cell migration. Their distribution in corneal epithelium may imply that a concomitant decrease of FGF-3 and FGFR3 in DR corneas may contribute to impaired corneal cell motility and delayed wound healing.
The laminin α4 chain is identified here for the first time in cornea. In corneal epithelial BM, it is probably part of laminin-8 (α4β1γ1).52
Laminin-8 is expressed in endothelial BM but is also found in skin epithelial BM.82
This laminin isoform is increased in human glioblastomas, participates in tumor invasion, and mediates cell migration.52,83,84
Therefore, it may mediate cell migration and corneal wound healing; its disruption in DR corneas () may thus impair these processes. Given that there was no reduction in mRNA for the laminin α4 chain by QPCR (not shown), one may suggest that its decrease in DR corneas was caused by degradation by proteinases, such as MMP-1022,23
or cathepsin F.
In summary, the analysis of gene microarrays validated by QPCR and immunostaining of tissue sections allowed us to identify distinct abnormalities in the expression of several proteinases and growth factors in DR corneas compared with normal ones. Induction of a more normal phenotype in organ-cultured DR corneas by cathepsin F inhibition and of a more diabetic phenotype in normal corneas by MMP-10 treatment were also demonstrated, suggesting functional significance of elevated proteolysis in diabetic corneal alterations. It would be important to show that changes in specific growth factors (see also Refs. 26-28
) would influence proteinase expression and to identify an exact role of select proteinases in epithelial BM degradation and impaired epithelial wound healing in DR corneas. Most of the growth factor, laminin, and proteinase changes described here primarily concern corneal epithelium, would lead to alterations of cell migration and wound healing, and may thus be directly related to clinically observed diabetic corneal abnormalities. The presented evidence may help elucidate the roles of distinct components in diabetic corneal alterations using antibodies, inhibitors, and purified proteins. It can also pave the way for an efficient future gene therapy85
for corneal diabetes. Select genes (MMP-10
, cathepsin F
) may be silenced using siRNA or antisense technology, whereas others (c-met
) may be upregulated using virally mediated gene transfer. In this respect, the corneal organ culture system would provide an ideal testing ground for these emerging therapies.