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(Investigative Ophthalmology and Visual Science. 2002;43:2143-2151.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Microarray Analysis of Corneal Fibroblast Gene Expression after Interleukin-1 Treatment

Vinit B. Mahajan1, Cui Wei2 and Peter J. McDonnell, III2,3

1 From the Departments of Microbiology and Molecular Genetics and 3 Ophthalmology, University of California Irvine, Irvine, California; and the 2 Department of Ophthalmology, University of Southern California Keck School of Medicine, Los Angeles, California.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To identify changes in gene expression in human corneal fibroblasts after exposure to interleukin-1{alpha}.

METHODS. RNA was isolated from cultured human corneal fibroblasts after treatment with interleukin-1{alpha} and subjected to DNA microarray analysis. Changes in gene expression were determined by comparison with untreated cells in three independent experiments after a Bayesian statistical analysis of variance.

RESULTS. Changes in gene expression were reproducibly observed in 165 genes representing previously identified and novel chemokines, matrix molecules, membrane receptors, angiogenic mediators, and transcription factors that correlated with pathophysiological responses to inflammation. Dramatic increases in gene expression were observed with exodus-1 (CCL20), MMP-12, and RhoA.

CONCLUSIONS. DNA microarray analysis of the corneal fibroblast response to interleukin-1{alpha} provides important insight into modeling changes in gene expression and suggests novel therapeutic targets for the control of corneal inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cornea is a transparent, five-layered tissue that serves as a critical barrier to ocular infection and injury. The stromal layer constitutes 90% of the corneal thickness and is a frequent site of inflammation after infection, trauma, or surgery. Although the inflammatory response may initially help control an infection or seal a wound, all too often the process leads to edema, collagen disorganization, neovascularization, ulceration, and scarring. Current anti-inflammatory treatment is frequently inadequate to prevent destruction of tissue and loss of corneal transparency.1 2 In severe cases, the only option is corneal transplantation, which is itself subject to failure after excessive inflammation. Thus, uncontrolled inflammation within the cornea may significantly decrease visual acuity or end in blindness.3 Understanding the endogenous inflammatory response is critical for developing improved treatment options for the more than 40,000 patients annually who undergo corneal transplantation because of cornea-related blindness.4

Interleukin-(IL)-1{alpha} is an important mediator of early inflammation in the cornea.5 6 7 8 9 The cytokine IL-1{alpha} is produced early by epithelial cells and resident corneal fibroblasts and later by invading leukocytes.7 10 Expression of IL-1{alpha} is upregulated after corneal grafting,11 infection,12 epithelial trauma,6 and alkali burns.13 As one of the earliest cytokines released into the corneal stroma, IL-1{alpha} amplifies the inflammatory response through membrane receptors expressed by fibroblasts.7 14 During dynamic phenotypic changes, these cells alter their shape, become motile, elaborate additional inflammatory factors, and regulate stromal matrix components.8 15 16 This response is in part due to altered gene expression mediated by IL-1{alpha}, yet only a limited number of these genes are known.

With the advent of gene therapy and the increased number of small-molecule pharmaceuticals, it is increasingly important to discover which genes might serve as targets in controlling excessive inflammation. DNA microarrays offer a powerful method to identify potential targets by simultaneously screening expression changes in thousands of genes.17 In a culture model of corneal inflammation, we applied this technique to monitor early changes in gene expression in corneal fibroblasts exposed to IL-1{alpha}. Using novel statistical software, a model for genes involved in corneal inflammation was developed with the hope that this approach may eventually identify specific therapeutic avenues for the reduction of corneal opacification.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells Culture and RNA Isolation
Cell culture reagents were purchased form Gibco-BRL (Grand Island, NY) and IL-1{alpha} from Calbiochem (La Jolla, CA). A fresh human cornea was obtained from the Lions Doheney Eye Bank (Los Angeles, CA). A central section measuring 6 mm in diameter was excised, and epithelial and endothelial layers were scraped away. The remaining stromal layer was minced, and explants were cultured in Eagle’s minimum essential medium with 20% fetal bovine serum, 100 IU penicillin G, and 100 µg/mL streptomycin in 35-mm dishes (Corning, Inc., Corning, NY). After 14 days, corneal fibroblasts became confluent and were subcultured. Fifth-passage corneal fibroblasts were then grown on 100-mm dishes in DMEM supplemented with 10% fetal bovine serum until 80% to 90% confluent. Cells were then rinsed and cultured in cell culture medium (Opti-MEM; Gibco) for an additional 72 hours, as previously described.18 Corneal fibroblasts were treated for 24 hours with either vehicle (EtOH) or IL-1{alpha} (10 ng/mL) in three independent experiments. Afterward, cells were trypsinized, and approximately 20 µg total RNA was isolated with an RNA extraction system (RNeasy; Qiagen, Valencia, CA), according to the manufacturer’s protocol. The RNA concentration was determined by spectroscopy, and the quality was confirmed by gel electrophoresis.

cRNA Preparation and Array Hybridization
Total RNA was converted into double-stranded cDNA using a custom kit (SuperScript II Double-Stranded cDNA Synthesis Kit; Life Technologies, Gaithersburg, MD). After ethanol extraction, in vitro transcription reactions were performed (BioArray HighYield RNA Transcript Labeling Kit; Enzo Biochemicals, Inc., Farmingdale, NY) according to the manufacturer’s protocol. Purified, labeled cRNA was quantified by spectrophotometric analysis, qualitatively analyzed for size distribution by gel electrophoresis, and fragmented to 30 to 60 base fragments with Tris-acetate (pH 8.1; 40 mM), KOAc (100 mM), and MgOAc (30 mM) in a 20-µL volume heated for 35 minutes at 94°C. Protocols and instrumentation setups, including total RNA samples, hybridization to U95A human microarrys, washing, staining, and scanning were followed as recommended in the manufacturer’s technical manual (GeneChip Microarrays; Affymetrix, Santa Clara, CA).

Data Analysis
The resultant arrays were scanned twice in the system’s confocal scanner (HP GeneArray Scanner; Hewlett Packard, Palo Alto, CA). Data analysis was first performed with the software accompanying the microarrays (GeneChip Expression Analysis Software, ver. 3.3; Affymetrix) to obtain average difference intensities. The Cyber-T software package developed at the University of California, Irvine, California, (provided in the public domain by the National Center for Genome Resources, Santa Fe, NM, at http://genomics.biochem.uci.edu/genex/cybert/) was then used to determine average intensity values of genes across three independent experiments and to compare values for control- and IL-1{alpha}–treated cells. This list was further restricted based on whether genes were consistently present or consistently absent in three independent experiments. By using a Bayesian statistical analysis, significant changes were ranked by the assignment of probabilities.19 Genes displaying intensities lower than 30 were empirically considered absent. Multiples of changes in gene expression were calculated by comparing average intensities in control with IL-1{alpha} treatment. For genes that were entirely absent in either control or IL-1{alpha} treatment but present in the other condition, relative multiples of change were assigned by comparing intensities with a normalized value determined by the software (Affymetrix).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The U95A human gene microarray is composed of oligonucleotides representing approximately 12,000 full-length, partially sequenced, and expressed sequence tag genes. Of the sequences represented, approximately 7700 were detected in cRNA produced from cultured human corneal fibroblasts, and 3532 of these correlated with complete cDNAs. Pair-wise comparison of gene expression levels in three independent control experiments displayed an overall correlation coefficient of R2 = 0.95, suggesting excellent reproducibility (data not shown).

Corneal fibroblasts were treated with IL-1{alpha} for 24 hours, as in previous studies,18 and cRNA was generated for microarray analysis. A common approach to identifying significant changes in gene expression is based on selection by multiples of change. After IL-1{alpha} treatment, 1115 genes displayed a greater than threefold expression change (data not shown) on average. Yet, many of these were changes with high interexperimental variance and probably represented experimental artifacts. An alternative approach is based on applying the t-test within a Bayesian statistical framework and using probabilities rather than multiples of change to rank significant expression changes. This method was preferred, because it more conservatively identifies genes with expression changes and reduces false-positive findings in microarray studies, with few replicates.19 20

After statistical analysis, significant changes in messenger RNA levels were detected in only 14 genes with P < 0.01 (Table 1 , asterisks). Expanding the selection of genes to those with a P < 0.05 generated an additional 118 genes (Table 1) . Some of the genes were especially noteworthy for their inflammatory roles and are described in the results (Table 1) . In addition, 22 genes absent in untreated cells were detected after IL-1{alpha} treatment (Table 2) , and 25 genes became undetectable (Table 3) . Several unknown or incomplete sequences were also identified. This included 55 sequences with a P < 0.05: 6 detected only after IL-1{alpha} treatment and 16 that became undetectable after IL-1{alpha} treatment (data not shown). Altogether, these results suggest that the number of highly reproducible changes in gene expression were limited to approximately 2% of the sequences sampled. This is in spite of the extremely sensitive detection system and application of a highly potent cytokine.


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  		Table 1. Genes Expression Changes after IL-1{alpha} Treatment

 

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  		Table 2. Genes Expressed after IL-1{alpha} Treatment

 

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  		Table 3. Genes Undetectable after IL-1{alpha} Treatment

 
Identification of Control Genes
Studies demonstrate that gene detection by microarrays correlates with standard methods such as Northern analysis and RT-PCR.21 In untreated corneal fibroblasts, for example, the microarray experiment detected 521 corneal genes previously catalogued by various methods.22 At the same time, genes specifically not expressed by passaged corneal fibroblasts, such as the type 2 IL-1 receptor, collagenase, and stromelysin were appropriately absent.23 24 To further confirm whether previously described changes in gene expression in IL-1{alpha}–treated corneal fibroblasts were evident in these microarray studies, we examined genes for IL-1{alpha}, regulated on activation normal T-cell expressed and secreted (RANTES), and granulocyte-macrophage–colony-stimulating factor (GM-CSF)-1.18 25 These genes served as internal controls, and, consistent with previous studies, each of these genes was upregulated. In treated cells IL-1{alpha} gene expression increased sixfold, RANTES eightfold, and GM-CSF-1 sevenfold (Tables 1 2) . These findings lend strong support to the statistical methods used for gene selection and the validity of the microarray analysis within the biological context. Some IL-1{alpha}–responsive genes such as hepatocyte growth factor,26 monocyte chemotactic protein-1,18 and GRO-{alpha},27 were not represented on the microarray and could not be tested.

IL-1{alpha}–responsive genes have also been studied in cells of different tissue origin. The microarray analysis identified similar expression changes not previously described in the corneal fibroblasts. Some of these were the modulation of interferon-ß2, endothelin-1, platelet-derived growth factor (PDGF)-{alpha} receptor, thrombospondin, manganese superoxide dismutase, Gro-ß, Gro-{gamma}, plasminogen activator-inhibitor (PAI) 2, c-myc, and IL-6.10 No expression changes were observed in inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-1, metalloproteinase (MMP)-1, IL-8, or keratinocyte growth factor. This may reflect either a tissue-specific expression or response, or in the case corneal fibroblast genes, differences in the experimental conditions.24 28

Secreted Inflammatory Genes
As one of the earliest expressed cytokines, IL-1{alpha} propagates the expression of additional cytokines that amplify the inflammatory process. Genes for both IL-1{alpha} and IL-1ß were upregulated (Table 1 2) . It is important to note that the increased IL-1ß RNA detected may reflect increased stabilization of its mRNA rather than increased transcription and that some IL-1–responsive genes, such as keratinocyte growth factor, may depend specifically on the IL-1ß isoform.26 29 Interferon-ß2a, which is identical with IL-6, was upregulated and may have a protective role during corneal infection.30 Interferon-{alpha}, which displays antiviral, antiparasitic, and antiproliferative activities,31 was downregulated. This could create a permissive environment for fibroblast mitogenesis and viral infection.

Chemokines are cytokines that recruit leukocytes to sites of inflammation.32 In response to IL-1{alpha}, for example, corneal fibroblasts upregulate and secrete MCP-1 (CCL2), RANTES (CCL5), and GRO{alpha} (CXCL1).6 18 The microarray experiments identified two other growth-regulated oncogenes (GRO) genes: GRO-ß (XXCL2) and GRO-{gamma} (CXCL3). The chemokine exodus-1/MIP-3{alpha}/LARC (CCL20),33 34 displayed a dramatic 29-fold increase in expression. Monocyte chemotactic protein-2 (CCL8) and ENA-78 (CXCL5) were upregulated as well.35 Together, these chemokines bind to various G-protein–coupled chemokine receptors expressed by neutrophils, eosinophils, monocytes, and T cells and could attract a full range of leukocytes from the limbal region into the corneal stroma.36

Cellular Inflammation Genes
IL-1{alpha} regulation of genes expressed at the membrane fundamentally alters the fibroblast responsiveness to extracellular inflammatory factors. Membrane receptors for IL-7 and IL-15 were upregulated. Expression of gp130, a membrane receptor/signal transducer for IL-6,37 38 increased fivefold, and together with an increase in IL-6, suggests the coordinated expression of a signaling pathway. Receptor responses to the cytokine glia-derived neurotrophic factor (GDNF) are modified by RETL-2, which increased threefold.39 In contrast, the cytokine receptor for IL-11 was downregulated, suggesting a possible decreased responsiveness to the antiapoptotic signal generated by IL-11.40 In addition, the decreased level of major histocompatibility complex (MHC) class II HLA-DR ß-1 suggests that antigen presentation mediated by the fibroblasts may be diminished.41

Modulation of gene transcription is an essential feature of cytokine signaling, and expression changes in several transcriptional factors were observed (Tables 1 2 3) . Of particular note was the increased expression of signal transducer and activator of transcription (STAT)-4 and R-{kappa}B. STAT proteins have dual functions: first binding to cytokine receptors and then migrating into the nucleus and directly activating transcription. STAT4 is essential for IL-12 signals that lead to production of interferon-{gamma}.42 43 The recently identified R-{kappa}B is a homologue of nuclear factor (NF)-{kappa}B, the prototypical inflammatory transcription factor. Although R-{kappa}B is known to regulate interleukin-2 receptor {alpha}-chain gene expression,44 its conceivably broad transcriptional and inflammatory effects remain to be explored. In contrast to transcription factors, an alternative mechanism for gene regulation is the degradation of newly formed mRNA. The observed increase in the nuclear protein HEM45 may have this effect, as suggested by its putative role in degrading viral mRNA.45

An important mechanism of IL-1{alpha}–mediated inflammation is the activation of eicosanoid signaling by modulating arachidonic acid metabolism.10 No effects were observed on phospholipase A2 or COX-2 expression. In contrast to the cyclooxygenase pathway, the lipoxygenase pathway appeared to be activated early with the observed increase in leukotriene C4 synthase.46 Among other effects, its secreted product leukotriene-C4 (LTC4) increases microvascular permeability.47

Corneal Transparency and Extracellular Matrix Genes
One of the remarkable features of the cornea is its essential transparency to light—a transparency that is diminished after injury. This transparency depends on the extracellular collagen composition and organization, and corneal fibroblasts are a primary source of extracellular matrix proteins in the stroma. TGFß, for example, regulates collagen synthesis and deposition and is an immunosuppressant in the anterior chamber.48 49 Activation of TGFß is dependent on thrombospondin-1, which increased fourfold after IL-1{alpha} treatment. This may affect both the matrix structure and regulation of inflammation.50 An observed decrease in the TGFß type III receptor suggests IL-1{alpha} may modulate specific TGF signaling pathways.51 52 bone morphogenic protein (BMP)-7, which regulates specific collagen deposition, displayed a decreased expression.53 In addition to these signaling molecules, IL-1{alpha} induced a threefold increase in {alpha}-1 type VII collagen (COL7A1), which has a specific role in anchoring the basal cells of the corneal epithelium to the stroma.54 Also observed was a threefold decrease in {alpha}-5 collagen type IV (COL4A5), which helps form corneal basement membranes.55

Collagen matrix organization is regulated by accessory proteins. Proteoglycans maintain corneal transparency by regulating collagen fibril spacing and corneal hydration. A sevenfold increase in chondroitin 6-sulfotransferase–like protein was detected. Expression of this enzyme may be critical for maturation of the keratan sulfate proteoglycans, which are the major proteoglycans in the cornea.56 57 At the same time, a fivefold decrease in the chondroitin sulfate proteoglycan versican was also detected. Other accessory proteins identified were matrilin-3, which forms collagen-dependent and -independent fibrils,58 and thrombospondin-1.59

Secreted proteases are known to play a major role in remodeling the stromal extracellular matrix during corneal wound healing.60 61 62 In response to IL-1{alpha}, fibroblasts displayed a 10-fold increase in human metalloelastase (HME/MMP-12), which was originally identified in macrophages.63 MMP-12 has multiple extracellular matrix substrates and disrupts basement membranes.64 65 No expression changes in membrane type matrix metalloproteinase-1, -2, or -4 were detected. Although MMP-1 gene regulation was not detected under these experimental conditions, the observed increase in manganese superoxide dismutase, and leukotriene C4 synthase may eventually signal an increase in MMP-1.66 67 Secreted proteases are regulated in turn by a various inhibitors. RECK is a membrane protein that inhibits MMP-9 activity.68 Decreased RECK suggests a concomitant increase in MMP-9 proteolytic activity, which is associated with corneal tissue destruction.61 62 In contrast, IL-1{alpha} induced the increased expression of two protease inhibitors: squamous cell carcinoma antigen (SCCA) 2 (leupin) and PAI-2, which may help control the degradation of the stromal matrix.69 70 There were no observed expression changes in tissue inhibitor of metalloproteinase-2, -3, or -4.

The avascular and relatively acellular quality of the stroma is essential to maintaining corneal transparency. Stromal neovascularization during inflammation and subsequent wound healing interferes with corneal light transmission, and IL-1 is implicated in this process.71 72 Several angiogenic factors were identified after IL-1{alpha} treatment including endothelin (EDN)-1,73 74 heparin-binding epidermal growth factor (HB-EGF),75 GM-CSF-1,76 and connective tissue growth factor (CTGF).77 At the same time, changes in the potent angiogenic factor VEGF, which is not detected in stromal fibroblasts, was appropriately absent.78 79 Similar to stromal neovascularization, proliferation of stromal fibroblasts interferes with corneal clarity. Some of the angiogenic factors, such as HB-EGF and CTGF, are also fibroblast mitogens.80 81 82 Enhanced expression of the PDGF receptor may prime fibroblasts for PDGF mitogenic effects.83 Increased expression of the follistatin-related protein (FLRG), a BMP-2 inhibitor, may protect specifically against BMP-2’s proliferative effects.84 85

Cell Cytoskeletal and Motility Genes
Corneal fibroblasts normally lie flat between collagen lamella and extend filopodia to adjacent fibroblasts. After injury, however, filopodial extensions retract, actin networks form, and the cells become motile.15 16 IL-1{alpha} is known to have a chemorepulsive effect on corneal fibroblasts in vitro and at the site of IL-1{alpha} injection in vivo.83 86 The microarray identified several genes that may contribute to these processes.

One of the most dramatic changes was the 46-fold increase in RhoA gene expression. The small guanosine triphosphatase (GTPase) RhoA is a signaling molecule that provides a link between extracellular signals and dynamic changes in the actin cytoskeleton.87 It is associated with membrane retraction by the formation of actin stress fibers and focal adhesions and was recently identified in IL-1 receptor signal transduction.88 Several intracellular proteins that directly regulate the cytoskeleton were recognized. These included actin-binding proteins, such as the LIM and the LIM domain binding protein (LDB)-1; the short form of ß-II spectrin, {alpha}-catenin-like protein; and T-platstin.89 Expression changes in microtubule-related proteins included alternatively spliced microtubule-associated protein (MAP)2 and the tubulin-binding protein RP-1.90

The transformation of quiescent keratocytes to mobile fibroblasts involves modulation of cellular interactions with extracellular matrix. Enzymes that degrade the matrix as previously described lead to loss of cell anchorage, whereas other matrix proteins, such as thrombospondin and PDGF signals, may promote motility.83 91 BMP-2 and -4 are also chemoattractive signals, but no significant changes in gene expression were observed other than the increased BMP-2 inhibitor FLRG. Another component is the differential expression of cell adhesion molecules. A threefold decrease in HNK-1 sulfotransferase, which alters the cell adhesion activity of HNK-1, was detected along with a decreased expression of protocadherin 68. In contrast, increased expression of Nr-CAM/hBRAVO and syndecan-1 was observed.92 93 Finally, transmembrane integrin proteins connect matrix molecules such as collagen and laminin to intracellular actin and transmit cell motility signals.94 95 After IL-1{alpha} treatment, integrin-{alpha} expression increased, whereas integrin ß-5 and laminin {alpha}-4 decreased. Together, these changes in gene expression may influence fibroblast cytoskeletal reorganization and motility during IL-1{alpha} mediated inflammation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The pathophysiological mechanisms of corneal inflammation and how best to treat patients are poorly understood. Activation of the endogenous inflammatory response by IL-1 has both beneficial and detrimental effects.5 After stromal injury, some degree of inflammation is essential for wound healing and control of infection. At the same time, however, inhibition of IL-1 may be favorable under certain conditions.96 97 We hypothesized that identifying IL-1–responsive genes may help develop models for more specific therapeutic interventions. Studies with cultured cells are a well-established method for the initial identification of cytokine-responsive genes. Which corneal fibroblast phenotype represents the best therapeutic target remains to be determined,8 98 and although the complex leukocyte effects are not represented, cytokine studies have nevertheless provided important insights.6 The application of DNA microarray technologies, we expected, would identify additional cytokine responsive genes and help develop models for further investigation.17 99

A model for the response to IL-1{alpha} in corneal fibroblasts is presented in Figure 1 . Genes thought to contribute to inflammation, maintenance of corneal transparency, and fibroblast motility and cytoskeletal rearrangement are emphasized and organized by their predicted protein localization. Such a model represents an exciting starting point for further investigation, yet it is critical to be aware of the limitations imposed by the model and inherent in microarray studies. The observed gene expression may reflect either increased transcription or increased mRNA stability, and whether the mRNA is translated into protein is not certain. Distinguishing tissue culture artifact from genuine expression changes requires in vivo studies. For example, Il-1{alpha}–responsive genes may be regulated by serum starvation or by an intrinsic IL-1{alpha} autocrine loop activated after trypsin-passage.28 The expense associated with commercial DNA microarray analysis precludes the comprehensive study of time and dose responses, but the generation of custom gene chips containing only genes in the model will now allow such studies to be conducted at far less cost. Appropriate statistical tools have been lacking to interpret the seemingly thousands of changes in gene expression revealed by microarrays in the context of low replication, errors from multiple measurements, and determining significance by multiples of change. These pitfalls were avoided in this study with the use of the Cyber-T statistical software. Finally, it is important to recall that DNA microarray results do not reveal other important mechanisms that regulate proteins directly rather than through gene expression.



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  		Figure 1. Model for IL-1{alpha}–mediated inflammatory response by corneal fibroblasts. IL-1{alpha} responsive genes with putative activities during inflammation, maintenance of corneal transparency, and modulation of the cytoskeleton and cellular motility are presented. Genes are arranged according to their predicted protein localization in various cellular compartments. Genes with increased expression (bold type) and decreased expression (normal type).

 
These caveats notwithstanding, DNA microanalysis was an effective and efficient screening method. Several previously known IL-1–responsive genes were identified, supporting the statistical and experimental methods. Some novel genes representing chemokines, chemotactic factors, cell adhesion molecules, interleukin receptors, matrix metalloproteinase, protease inhibitors, proteoglycans, angiogenic factors, cytoskeletal regulators, and transcription factors were identified. Each of these molecules represent potential targets, but what genes warrant further investigation? Regulation of MMPs is promising,60 and this study correctly identified MMP-9 as an important candidate. Inhibition of MMP-9 has already shown some success as an adjunctive therapy in clinical studies,100 and results suggest that targeting MMP-12, identified in this study, may also be important. Even with multiple redundant chemokine expression, targeting single chemokines or their receptors can dramatically reduce inflammation.36 We identified chemokine targets not previously described in the cornea. Perhaps more important are the G-protein receptors (CXCR2 and CCR1, -2, -3, -5, and -6) implicated by this array of chemokines. It is interesting to speculate that therapies may be narrowed toward receptors expressed during leukocyte reactions specific to allergic, bacterial, or viral inflammation. Regulation of RhoA may control fibroblast motility and eventually wound contraction and corneal scarring, and specific inhibitors are available to test such a hypothesis. Finally, corneal gene therapy makes possible the targeting of specific transcription factors, such as STAT4 and R-{kappa}B, which have conceivably broad roles in inflammation.101 102 Taken together, the observations based on this DNA microarray experiment provide fertile ground for developing and testing novel hypotheses to reduce cornea-related blindness.


   Acknowledgements
 
The authors thank G. Wesley Hatfield and She-pin Hung for assistance with the statistical software and J. Denis Heck and Kim Nguyen for assistance with the microarrays.


   Footnotes
 
Supported in part by a grant from the Dumont Foundation, Los Angeles, California (PJM).

Submitted for publication August 22, 2001; revised February 14, 2002; accepted March 1, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Peter J. McDonnell III, 118 Med Surg I, Department of Ophthalmology, University of California Irvine, Irvine, CA 92697; pjmcdonn{at}uci.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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