|Molecular Vision 2004;
Received 1 September 2004 | Accepted 1 November 2004 | Published 8 November 2004
Gene expression profile in corneal neovascularization identified by immunology related macroarray
Tomohiko Usui, Satoru
Yamagami, Seiichi Yokoo, Tatsuya Mimura, Kyoko Ono, Shiro Amano
Department of Ophthalmology, Faculty of Medicine, University of Tokyo, Tokyo, Japan
Correspondence to: Tomohiko Usui, M.D, Department of Ophthalmology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113-8655; Phone: 81-3-3815-5411; FAX: 81-3-3817-0798; email: firstname.lastname@example.org
Purpose: To identify differentially expressed genes in corneal neovascularization using cDNA macroarray.
Methods: Mechanical denudation of corneal and limbal epithelium was performed to induce corneal vascularization in mice. Corneas were harvested 4 days after operation. Total RNA was isolated from both normal and vascularized corneas and used for the synthesis of cDNA probes. 32P labeled exponential cDNA probes were hybridized to mouse cDNA immunology arrays. To validate the gene expression patterns revealed by the cDNA expression array analysis, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and statistical analysis were performed to compare the normal and vascularized corneal samples.
Results: Of 545 immunology related genes on the arrays, 6 genes were upregulated and 1 gene was downregulated in the vascularized corneas compared with the normal corneas. Semi-quantitative RT-PCR was performed on the 6 genes selected in the arrays study, and showed that calreticulin (calregulin), apolipoprotein E, HSP84 (HSP90β), and pleiotrophin were upregulated while interferon regulatory factor-1 was downregulated in the vascularized corneas compared with the normal corneas. These genes possess a number of biological functions including molecular chaperon, growth factor, and transcriptional factors.
Conclusions: The differentially expressed genes newly identified in the context of corneal neovascularization represent novel candidate factors for further functional studies of the mechanisms of corneal neovascularization. Our data may provide new insight into the biological process of inflammation induced corneal neovascularization.
The cornea is normally avascular, but under certain conditions capillaries invade from the limbal plexus. Therefore, corneal neovascularization (NV) represents a central feature in the pathogenesis of many blinding corneal disorders and major sight-threatening conditions.
Since corneal NV is usually associated with inflammatory disorders, various inflammatory cells are recruited in vascularized corneas. These leukocytes play an essential role in stimulating corneal vascular growth [1-3]. The recruited inflammatory cells have the capability to influence each phase of the angiogenic process, such as alterations of the local extracellular matrix, induction of endothelial cells to migrate or proliferate, and even inhibition of vascular growth with formation of differentiated capillaries . The recruitment of leukocytes to inflammatory sites is mediated by growth factors and chemokines. Genetic ablation of CCR2 (the principle receptor of monocyte chemoattractant proteins [MCPs]), or CCR5 (the receptor of macrophage inflammatory proteins [MIPs]), inhibited corneal NV, suggesting that these inflammatory mediators are essential components in corneal NV [5,6]. Yoshida et al.  showed that MCP-1 and the proinflammatory cytokine IL-1β is involved in inflammatory corneal NV. Although vascular endothelial growth factor (VEGF) is well known as a potent endothelial growth factor, VEGF itself is also a chemotactic factor for monocyte cell lineage and stimulated the inflammatory responses in corneal NV [8,9]. These emerging data suggest that multiple inflammatory mediators are involved in the angiogenic process, and that mechanistic link between angiogenesis and inflammation is important for the understanding of corneal NV.
Gene array technology allows the screening of simultaneous gene expression and large scale comparison of many genes by a single hybridization. In the present study, an immunology related cDNA array was used to investigate differential gene expression in a clinically relevant ocular model of corneal injury by studying the neovascular response of animals. We identified novel genes that were differentially regulated during vascular development in the inflammed cornea.
Model of corneal neovascularization
All animal experiments were approved by the animal care committee of the University of Tokyo Hospital and conformed to the Association for Research in Vision and Ophthalmology guidelines for animal use. Male C57 BL/6 mice (weighing 20-25 g; Saitama Jikken Doubutsu, Saitama, Japan) were used in the experiments. All procedures were performed with the animals under general anesthesia by xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (35 mg/kg). The animals were allowed free access to food and water. A 12 h day-night cycle was maintained.
After general anesthesia, topical proparacaine (Santen, Osaka, Japan) and 2 μl of 0.15 N NaOH were applied to the corneas of each mouse. The corneal and limbal epithelium was totally removed using a Tooke corneal knife (Katena Products, Denvelle, NJ) in a rotary motion parallel to the limbus as described previously [5,6]. Erythromycin ophthalmic ointment was instilled immediately following epithelial denudation. The corneas were harvested 4 days after injury for gene expression analysis.
Gene expression analysis with cDNA arrays
Gene expression profile of mouse corneal neovascularization was analyzed by macroarray, as described previously . Briefly, a mouse cDNA expression array (Toyobo, Osaka, Japan), in which 545 known immunology related genes are represented, was used in this experiment. Corneas were collected from normal and injured vascularized mice (8 corneas per sample). Total corneal RNA from each mouse group was isolated using an extraction reagent (Isogen; Nippon Gene, Toyama, Japan) according to the manufacture's protocol. RNA was treated with DNase to remove genomic DNA. After DNase treatment, 32P labeled cDNA probe was synthesized from total RNA. Incorporation of the label was assessed by scintillation counting, and the cDNA probes from both groups were adjusted to the same radioactive concentration. Each probe was hybridized in a hybridization solution (Express Hyb; Clontech, Palo Alto, CA) with the mouse gene immunology array membranes overnight at 68 °C with continuous agitation. The array membranes were washed with sodium citrate containing sodium dodecylsulfate. The array membranes were exposed to a phosphorescence imager (Eastman Kodak, Rochester, NY). Phosphorescent imaging was analyzed by comparison with each membrane using another phosphorescence imager (Storm; Amersham Bioscience, Sunnyvale, CA). The data obtained from the phosphorescence imaging analysis system reflects the accumulated x-ray energy and provides accurate quantification in comparison with densitometric analysis of spot size. After nomalization to the levels of all housekeeping genes included on the membrane, the average intensity differences were calculated.
The results of gene profiles obtained from macroarray were confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) as described elsewhere . Total RNA was isolated from normal and injured corneas as mentinoned above, and cDNA was produced using reverse transcriptase (SuperScript II; Invitrogen, San Diego, CA). The PCR reaction mixtures comprised 1% cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 20 μM oligonucleotides, and 2.5 U Taq polymerase (Ampli-Taq Gold; Perkin Elmer, Wellesley, MA). The PCR conditions were as follows; 30 s at 94 °C, 30 s at 60 °C, and 45 s at 72 °C, with an initial 9 min denaturation step. The oligonucleotide primers, selected to discriminate between cDNA and genomic DNA by using primers specific for different exons are summarized in Table 1. PCR products were separated in a 1% agarose gel. Band densities were normalized to those for glyceraldehydes-3-phosphate dehydrogenase (G3PDH). Within the linear range of amplification, four sets of PCR products were prepared under appropriate cycling conditions, and the band densities were compared between samples from normal and vascularized corneas. The Mann-Whitney U test was used to compare the band densities on RT-PCR.
To identify the genes that are expressed in the process of corneal neovascularization, we compared phosphorescent images of the arrays hybridized with 32P labeled cDNA probes generated from RNA preparations of normal and vascularized corneas. The array membrane that we used in this study contained 11 housekeeping gene controls and two negative controls. All the housekeeping genes were detected on the macroarray. No signals were observed for the negative control spots, indicating that the hybridization was highly specific. Intensity differences greater than 3 arbitary units between normal and vascularized corneas were accepted as significant and selected for further study. Among the 545 immune related genes tested, 6 genes were upregulated and 1 gene was downregulated in the vascularized corneas compared with normal cornea (Table 2). In the vascularized corneas, the expression of calreticulin (calregulin), HMGI(Y), apolipoprotein E (apo E), HSP84 (HSP90β), pleiotrophin, and IGFBP-5 were upregulated. In contrast, interferon regulatory factor-1 (IRF-1) was decreased in vascularized corneas compared with normal corneas.
To confirm the results of gene array analysis, semi quantitative RT-PCR was performed. There were no significant differences in G3PDH between the two groups. After linear amplification of PCR products, the gene expression levels of all 7 genes were compared (Figure 1). The mRNA levels of calreticulin, apo E, HSP84, and pleiotrophin were significantly higher in the vascularized corneas than those in the normal corneas. IRF-1 mRNA levels in the vascularized corneas were significantly lower than those in the normal corneas. These findings were consistent with the results of macroarray. However, there was no significantly differences in the mRNA levels of IGFBP-5 and HMGI(Y) between the vascularized and normal corneas (data not shown). Similar results were obtained with total RNA derived from another set of animal groups for semi-quantitative RT-PCR.
In the current study, we investigated the gene expression profile in the process of corneal NV by means of cDNA immunology macroarray. Several unexpected genes were upregulated or downregulated. Although these molecules have been defined as important factors associated with tumorgenesis and/or inflammatory responses, they have not been investigated in the context of corneal NV and thus represent novel candidate factors involved in corneal NV.
Corneal NV is mediated by inflammatory cytokines and growth factors such as MCP-1, MIPs, IL-1β, and VEGF listed on the immunology related arrays [1,5-7]. Unexpectedly, the expression levels of those genes were not predominant in our experiment. This may be due to the time point difference because most of inflammatory cytokines and chemokines are usually upregulated in early phase of corneal NV. For example, the expression of VEGF, which is thought to be essential for corneal NV, at the mRNA level is stimulated only in the early phase . MCP-1 and IL-1β are high 12 h to 2 days after injury although model of corneal NV is different from this study . In our study the corneas were harvested 4 days after injury. In that phase, vessels have been already grown into the cornea. Therefore, we may have detected more specific molecules involved in vessel growth.
Calreticulin belongs to the family of heat shock proteins . This protein has been shown to associate with peptides transported into endoplastic reticulum. It binds antigen processing molecules and MHC class I-β2 microgloblin molecules to aid antigen presentation . The peptide-bound calreticulin purified from tumor extracts has been shown to elicit an anti-tumor effect specific to the source of the tumor . Further, calreticulin and its protein fragment, called vasostatin, have been reported to be endothelial cell inhibitors of tumor growth [13,14]. Anti-cancer cancer vaccines employing calreticulin also successfully generate anti-tumor effects through enhanced anti-tumor immune responses and anti-angiogenic effects . Therefore, the calreticulin in vascularized cornea may be upregulated to protect and maintain corneal transparency in corneal neovasculoarization through its anti-angiogenic effect.
HSP84 (HSP90β) is also responsible for chaperoning proteins involved in cell signaling, proliferation, and survival . Although the functions of HSP90β have not been fully elucidated, HSP90β is involved in oncogenic and pro-angiogenic activities . HSP90β binds many client proteins such as hypoxic inducible factor 1 alpha, Akt kinase, MEK, and nitric oxidase synthase . Brouet et al.  reported that HSP90β plays a key role in the proangiogenic action and this molecule promotes neovascularization in pathological conditions. Geldanamycin, a potent inhibitor of HSP90β, is effective to induce tumor regression . We expect that agents that inhibit HSP90β might be useful for inhibiting corneal NV.
Pleiotrophin (PTN, also termed HB-GAM) is an 18 kDa heparin binding cytokine that signals diverse functions including differentiation of neural cells, neurite outgrowth, oncogenic activity, and angiogenesis . PTN is normally expressed during embryogenesis but is expressed at very low levels or not at all in healthy adult tissues . However, the expression of PTN is stimulated in human tumors [20-25], brain ischemia , and rheumatoid arthritis [27,28]. The PTN gene is also upregulated in newly formed blood vessels and in surrounding activated macrophages and mesenchymal cells in a brain ischemic angiogenesis model . This is the first report of PTN gene expression in corneal NV. PTN might be the major pro-angiogenic growth factor in corneal NV, and is a potential therapeutic target for the treatment of corneal NV.
Apolipoprotein E (ApoE), a 34 kDa glycoprotein that coats VLDLs, IDLs, and HDLs, plays a central role in serum cholesterol homeostasis through its ability to bind cholesterol and other lipids and to mediate their transport into cells [29,30]. ApoE also influences angiogenesis and lines of evidence have indicated that mice lacking apoE have impaired angiogenesis [31-33]. Impaired angiogenesis in ApoE-deficient mice was dependent on a reduced capacity to upregulate VEGF in response to stimuli [32,33]. Therefore, ApoE appears to have important effects on angiogenesis. Although the functions of apoE on corneal angiogenesis have not been elucidated, it is important to investigate the functional significance of apoE in corneal NV.
IRF-1 is the only molecule that is decreased in vascularized corneas compared with normal corneas in this study. This molecule is identified as a regulatory transcriptional factor of IFN-gamma genes, resulting in negative regulation of cell proliferation and growth . Recently, Liu et al.  showed that IRF-1 activated tryptophanyl-tRNA synthethase (TrpRS) promotor activity. TrpRS links protein synthesis to signal transduction pathways in angiogenesis, and the fragment of TrpRS containes anti-angiogenic (angiostatic) activity . We speculate that high expression of IRF-1 in normal cornea may be interpreted as one of mechanism to keep the cornea transparent through activation of a fragment of TrpRS.
It is interesting that corneal inflammatory angiogenesis shares the molecules involved in tumor angiogenesis and oncogenesis, such as PTN and HSP90β. The responses of neoplastic disorders are associated with a defective capacity to mount inflammatory reactions at sites other than the tumor itself . This systemic tumor related effect has been attributed to many factors including production of chemokines and cytokines with anti-inflammatory activities, continuous leukocyte recruitment in the tumor microenviroment, and defective functions of leukocyte exposed to tumor products . Furthermore, even ischemic pathological angiogenesis is involved in inflammatory processes . Therefore, investigation and understanding of inflammatory angiogenesis might provide certain therapeutic approachs to corneal NV, and also contribute to elucidating tumor and pathological ischemic angiogenesis.
In summary, our data showed that gene expressions of calreticulin, HSP84 (HSP90β), pleiotrophin, and apoE were significantly upregulated while IRF-1 was significantly downregulated in vascularized corneas compared with normal corneas. These molecules are candidate genes closely associated with corneal NV. Our findings provide a novel insight into the complex gene expression in the whole genome, concerning the underlying mechanism of corneal NV. Functional analysis is required to determine the definitive roles of these genes in corneal NV.
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