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Overexpression of Immediate Early Genes in Active Graves’ Ophthalmopathy

Department of Endocrinology (M.L., T.V., H.P., M.R., L.G., B.H.), and Departments of Ophthalmology (C.F., P.A.) and Plastic Surgery (M.A.), Malmö University Hospital, S-205 02 Malmö, Sweden

Address all correspondence and requests for reprints to: Mikael Lantz, M.D., Ph.D., Department of Endocrinology, Malmö University Hospital, S-205 02 Malmö, Sweden. E-mail: mikael.lantz{at}endo.mas.lu.se.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: In Graves’ ophthalmopathy a major problem is an increase in the intraorbital adipose tissue volume.

Objective: The aim of this work was to define mechanisms of orbital adipogenesis.

Design: This was an open-label prospective study.

Setting: The study was conducted at the Clinic of Endocrinology, University Hospital.

Participants: The study consisted of patients (n = 5) with severe ophthalmopathy with affection of the optic nerve and thyroid healthy controls (n = 5).

Interventions: We performed lateral decompression of orbital tissue in patients unresponsive to corticosteroids and restorative surgery of the upper eyelid in thyroid healthy controls.

Main Outcome Measure: We made large-scale measurements of gene expression, with microarray technique based on determination of fluorescence intensities in cases and controls.

Results: A marker of adipose tissue, stearoyl-coenzyme A desaturase, was overexpressed in ophthalmopathy, and selection criteria were set to favor identification of genes known to be expressed in normal adipogenesis. The immediate early gene, cysteine-rich, angiogenic inducer, 61 (CYR61), was overexpressed in addition to 15 other immediate early genes (IEGs), and the expression of selected IEGs was confirmed with RT-PCR: CYR61, cyclooxygenase-2, dual-specificity phosphatase 1, B cell translocation gene 2, and early growth response 1. CYR61-responsive genes, known to participate in inflammation, IL-1ß, matrix metalloproteinase-3, and vascular endothelial growth factor were also overexpressed. Patients showed greater expression of CYR61 in the active than the chronic phase of ophthalmopathy, indicating that CYR61 is a marker of disease activity. Cyclooxygenase-2, the target gene of IL-1ß, was also overexpressed, although all patients had been treated with corticosteroids.

Conclusion: Adipocyte-related IEGs are overexpressed in active ophthalmopathy, and CYR61 may have a role in both orbital inflammation and adipogenesis and serve as a marker of disease activity.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
GRAVES’ DISEASE IS an autoimmune disorder that causes overproduction of thyroid hormones with typical thyrotoxic symptoms. Whereas 10–25% of patients experience endocrine ophthalmopathy during some stage of the disease, a severe form of endocrine ophthalmopathy with optic neuropathy may occur in 5% of the patients with Graves’ disease (1, 2). The signs and symptoms of ophthalmopathy can be explained by an increase in the volume of the main orbital tissue components, i.e. the adipose and connective tissue as well as the eye muscles within the bony orbital walls resulting in increased intraorbital pressure. The pathogenesis of the disorder is poorly understood, but an autoimmune reaction has been assumed because the orbital tissue is usually infiltrated by immunocompetent T cells (3). Although the autoantigen is not known, the TSH receptor has been the prime suspect because it is expressed on both preadipocytes and adipocytes in affected orbital tissue as well as in other adipose/connective tissue depots (4, 5). The inflammation causes increased production and release of hygroscopic molecules, e.g. glycosaminoglycans, from orbital fibroblasts, which in part explains the orbital edema (6, 7). In parallel there is an increase in de novo adipogenesis or an enhanced accumulation of lipids in each adipocyte causing a rise in adipose tissue volume (8).

Adipocyte differentiation includes an initial proliferation phase during which growth arrested preadipocytes reenter the cell cycle and complete two rounds of cell division, a process known as mitotic clonal expansion (9). A terminal differentiation phase follows during which the specific genes that define the adipocyte phenotype are induced. During the initial proliferative phase of preadipocytes, immediate early genes (IEGs) are induced by adipogenic factors like growth factors, corticosteroids, and cAMP-increasing agents (10). IEGs function as triggers of the subsequent transcriptional cascade that leads to the adipocyte phenotype. In NIH-3T3 fibroblasts and 3T3-L1 preadipocytes, IEGs are induced during the initial proliferation phase in response to adipogenic factors (10, 11). Some of the IEGs studied in these in vitro models, e.g. cysteine-rich, angiogenic inducer, 61 (CYR61) and cyclooxygenase-2 (COX-2), have functions in both inflammation and adipogenesis.

Cyclooxygenases are required for conversion of arachidonic acid to prostaglandins, which mediate many effects of the inflammatory process (12). Of note, prostaglandin-J2 is the putative natural ligand of the nuclear receptor, peroxisome proliferator-activated receptor (PPAR), a key regulator of adipogenesis (13). Expression of PPAR is essential for adipogenesis to occur, making it an interesting pathogenic candidate in ophthalmopathy. PPAR expression was increased in biopsies from patients with endocrine ophthalmopathy, compared with healthy controls (8). Exacerbation of stable Graves’ ophthalmopathy during medication with the synthetic PPAR agonist pioglitazone for type 2 diabetes has been reported, and orbital fibroblasts from patients with ophthalmopathy subjected to rosiglitazone in vitro have also been shown to differentiate into mature adipocytes (14, 15, 16).

The precise sequence of events leading to endocrine ophthalmopathy is not known, nor is it established that the TSH receptor is the sole autoantigen. Neither is it known whether IEGs are overexpressed in orbital fibroblasts and preadipocytes in response to mitogens in patients with endocrine ophthalmopathy.

To shed light on these events, we studied gene expression in intraorbital tissue from patients with endocrine ophthalmopathy removed during decompressive surgery and compared it with expression levels in intraorbital tissues taken from controls during eye lid restorative surgery.

We provide evidence that overexpression of IEGs in orbital tissues is a characteristic feature of endocrine ophthalmopathy, regardless of treatment with corticosteroids.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Collection of patient samples and isolation of RNA

All tissue samples in this study were collected after informed consent with the approval of the Ethical Review Board of Lund University, Malmö/Lund, Sweden. Intraorbital adipose/connective tissue was collected from five patients undergoing lateral orbital decompression due to severe Graves’ ophthalmopathy affecting the optic nerve. All patients were in an active phase of their ophthalmopathy and had received corticosteroids for long periods of time before and including the day of surgery. For clinical characteristics, see Table 1. Control intraorbital tissue was obtained from five patients without thyroid disease (negative for TSH receptor and thyroid peroxidase antibodies and normal TSH) undergoing bilateral restorative surgery of the upper eyelids during which intraorbital tissue was obtained after cleavage of septum orbitale. This tissue is part of the same adipose/connective tissue as the retrobulbar adipose/connective tissue and was also collected from five Graves’ patients undergoing restorative surgery of the upper eyelid in the chronic phase of their ophthalmopathy. These patients were operated 31–34 months after the diagnosis of thyrotoxicosis and 31–47 months after the diagnosis of ophthalmopathy. The sc and visceral adipose/connective tissue from five obese individuals was also collected as a control for the study of stearoyl-CoA desaturase (SCD). To minimize the degradation of RNA, adipose/connective tissue was treated with RNA later (Ambion, Austin, TX) overnight before it was stored frozen at –80 C. RNA was extracted with RNeasy minikit (QIAGEN, Stockholm, Sweden), according to the manufacturer’s instructions. The concentration and purity was determined spectrophotometrically by the use of the ratio A260/A280 and found to be close to 1.5 when diluted in diethyl pyrocarbonate water. If water without diethyl pyrocarbonate was used, the ratio was close to 1.9. The integrity of RNA was determined by agarose gel electrophoresis.

Gene expression analysis was performed according to the Affymetrix expression analysis technical manual by the SWEGENE Microarray Resource Centre. The facility is situated at Department of Immunotechnology, Wallenberg Laboratory (Lund, Sweden) and is focused on commercially available high-density microarray analysis on a routine basis. Total RNA (1 µg) from five individuals with active endocrine opthalmopathy and five individual controls was concentrated by precipitation in ethanol to a final concentration of 1 µg/µl before reverse transcription to cDNA. The obtained cDNA was used as a template to generate biotinylated cRNA. After fragmentation the biotinylated cRNA was hybridized to HG-U133A gene chips, and one chip was used for analysis of each pool. Fluorescence intensities were measured with a gene array scanner and scaling was conducted as previously described in detail (17). The image and average difference expression and present/absent calls were analyzed with Affymetrix microarray suite user’s guide software version 5.0 (Affymetrix, Santa Clara, CA). The quality of the microarrays were evaluated with percent present-call (>10%) and glyceraldehyde-3-phosphate dehydrogenase 3'/glyceraldehyde-3-phosphate dehydrogenase 5' expression ratio less than 3. Average fluorescence intensity difference of greater than 100 accounts for reproducible and measurable gene expression levels (18, 19). Hence, we filtered out probe sets for which the expression level in either case or control was less than 100, and 6770 probe sets passed these filtering criteria. Gene expression changes between two arrays were calculated by comparing each probe pair on the case array with the corresponding probe pair on the control array.

Real-time RT-PCR

Total RNA, 0.3 µg, was reverse transcribed in a 20-µl reaction using Superscript II RT (Life Technologies, Gaithersburg, MD) and random hexamer primers (Life Technologies) according to the manufacturer’s protocol. After addition of random hexamer primers and deoxynucleotide triphosphate-mix, the PCR tubes were incubated at 65 C for 5 min and placed on ice for 1 min. Thereafter 5x first-strand buffer, 40 U RNase inhibitor, 0.1 M dithiothreitol, and 200 U Superscript II RT were added followed by incubation on a thermal cycler at 25 C for 10 min, 42 C for 50 min, and 70 C for 15 min.

The sequences for human CYR61, B cell translocation gene 2 (BTG2), dual-specificity phosphatase 1 (DUSP1), COX-2, and SCD were found using the National Center for Biotechnology Information sequence viewer, and specific primers were designed using the computer program Primer Express (Applied Biosystems, Foster City, CA). One of the primers or the probe was designed as intron-exon spanning to minimize the risk of genomic amplification. The probes were double labeled with 5'-reporter dye 6-carboxyl fluorescein and a 3'-quencher dye 6-carboxyl-tetramethyl-rhodamine.

Specific primers and probes (MWG-Biotech AG, Edsberg, Germany) used were as follows: CYR61, forward primer, 5'-CAGCTCCACCGCTCTGAAG-3', reverse primer, 5'-GGAAACTTTCCCCGTTTTGG-3', probe, 5'-CAGAGCTCAGTCAGAGGGCAGACCC-3'; BTG2, forward primer, 5'-GAGCGAGCAGAGGCTTAAGG-3', reverse primer, 5'-CTTGTGGTTGATGCGAATGC-3', probe, 5-'CGCTCCAGGAGGCACTCACAGAGC-3'; DUSP1, forward primer, 5'-AAAGGAGGATACGAAGCGTTTTC-3', reverse primer, 5'-CGCTGTCAGGGACGCTAGTAC-3', probe, 5'-CTGTGCAGCAAACAGTCGACCCCC-3'; COX-2, forward primer, 5'-GCTCAAACATGATGTTTGCATTC-3', reverse primer, 5'-GCTGGCCCTCGCTTATGA-3', probe, 5'-TGCCCAGCACTTCACGCATCAGTT-3'; and SCD, forward primer, 5'-GGGTGAGGGCTTCCACAACTA-3', reverse primer, 5'-CGGCCATGCAATCAATGAA-3', probe, 5'-CCTATGACTACTCTGCCAGTGAGTACCGCTG-3'.

For early growth response 1 (EGR1), vascular endothelial growth factor (VEGF), matrix metalloproteinase 3 (MMP3), IL-1ß, and cyclophillin A, 20x Assays-on-Demand gene expression assay mix (Applied Biosystems) was used.

PCRs were performed in 384-well optical plates in the ABI PRISM 7900HT sequence detection system (Applied Biosystems). The reactions contained cDNA equivalent to 30 ng in a reaction volume of 10 µl using the TaqMan Universal 2x PCR master mix (Applied Biosystems).

For CYR61, BTG2, DUSP-1, COX-2, and SCD, a final concentration of 900 nM of the forward and reverse primers and 50–200 nM of the probe were used. For EGR1, VEGF, MMP3, IL-1ß, and cyclophillin A, 10x Assays-on-Demand gene expression assay mix was used according to the manufacturer’s protocol.

The thermal cycling conditions were an initial 50 C for 2 min for optimal AmpErase UNG activity and 95 C for 10 min to activate the AmpliTaq Gold DNA polymerase, followed by 40 cycles of denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min.

All samples were run as duplicates with a nontemplate control in each batch. The housekeeping gene cyclophillin A was analyzed in parallel PCR with identical template concentrations in separate wells.

The standard curve method was used for quantification of gene expression. Values were normalized to cyclophillin A and expressed as the ratio of RNA (picograms) of the particular gene to RNA (picograms) of cyclophillin A. Results were confirmed in at least two independent experiments, and one representative experiment has been chosen for presentation.

Statistical analysis

The Mann-Whitney rank-sum test and one-way ANOVA was used to assess statistically significant differences between patient and control groups.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Gene expression in orbital tissue from patients with endocrine ophthalmopathy and controls

Gene expression in intraorbital tissue from five patients with severe endocrine ophthalmopathy and optic neuropathy (Table 1) was compared with gene expression in intraorbital tissue from five individuals without thyroid disease. Of the 22,000 probe sets on the Affymetrix chip, 6,770 exhibited an expression of 100 or more in either the patient or control group. Among these, 220 probe sets showed a relative expression ratio greater than 2 and 59 genes differed by a ratio greater than 4. Given the predominance of adipocytes and inflammatory cells in intraorbital tissue, we restricted our search to adipocyte or inflammatory-specific genes. Among the 59 probe sets with a difference in expression ratio greater than 4, the SCD was the only orbital adipocyte-specific gene. The expression of SCD was confirmed with real-time RT-PCR in all individuals (Fig. 1), and again orbital RNA from patients exhibited 2–3 times higher expression of SCD, compared with controls. Expression of SCD was also demonstrated in sc and visceral adipose tissue from individuals without thyroid disease (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Expression of the adipocyte-specific gene SCD in adipose tissue normalized to cyclophillin as measured by real-time RT-PCR. A, Endocrine ophthalmopathy orbital tissue and normal orbital tissue from five individual patients and five controls (left). Mean values for the case and control group are represented by horizontal lines (right); P < 0.05. B, The sc and visceral tissue from five obese individuals without thyroid disease (left). Mean values for the group of visceral and sc tissue are represented by horizontal lines.

We restricted our search for adipocyte-related genes to genes with a fluorescence intensity signal of 100 or more in both patients and controls on the microarray. By restricting our search to adipocyte-specific genes, many genes representing infiltrating inflammatory cells or contaminating glandular tissue are excluded because the biopsies of normal adipose/connective tissue lack or contain sparse numbers of these cells. Applying this criterion, we identified 107 probe sets with a relative expression ratio greater than 2 in patients, compared with controls. The greatest ratio was seen for the immediate early gene CYR61 closely followed by nine other IEGs (Table 2). The search for known adipocyte-related IEGs was then extended to the whole group of 6770 probe sets with expression of 100 or more in either the case or the control group on the microarray. The expression of 16 of 17 IEGs was increased in patients, compared with controls. In Table 3 the IEGs were divided into functional groups, and one gene from each subgroup was selected for replication by real time RT-PCR (except the metallothioneins). CYR61, COX-2, DUSP1, BTG2, and EGR1 showed a more than 4-fold increased expression in all individual cases, compared with controls (Fig. 2), which for all genes replicated the data from the microarray. Despite lengthy high-dose corticosteroid treatment, COX-2 was also overexpressed in patients when analyzed by both real-time RT-PCR and microarray. This was not true for COX-1, which in the microarray showed 50% lower expression in patients than in controls (data not shown). Also by real-time RT-PCR, the expression of COX-1 was suppressed in the patient group (Fig. 3).

View larger version (22K): [in this window] [in a new window]   FIG. 2. Expression of selected adipocyte-related immediate early genes in endocrine ophthalmopathy orbital and normal orbital tissue normalized to cyclophillin A as measured by real-time RT-PCR. A–E, Expressions of BTG2, COX-2, CYR61, DUSP1, and EGR1 in five individual patients and five controls previously analyzed on the oligonucleotide microarray chip. One control was replaced by another control not present on the chip due to lack of material. F–J, Mean values of the relative expressions of BTG2, COX-2, CYR61, DUSP1, and EGR1 for the group of patients and controls are represented by horizontal lines. The P value was less than 0.01 when ophthalmopathy orbital tissue was compared with normal orbital tissue for all analyzed genes.

View larger version (8K): [in this window] [in a new window]   FIG. 3. Expression of COX-1 in endocrine ophthalmopathy orbital and normal orbital tissue normalized to cyclophillin A as measured by real-time RT-PCR. A, Endocrine ophthalmopathy orbital tissue and normal orbital tissue from five individual patients and five controls previously analyzed on the oligonucleotide microarray chip. One patient and one control were replaced by samples not present on the chip due to lack of material. B, Mean values for the groups of patients and controls are represented by horizontal lines, and the P value was less than 0.05 when the patient and control groups were compared.

Inflammation and adipogenesis play important roles in the pathogenesis of endocrine ophthalmopathy. One key player involved in both processes is CYR61. We therefore specifically tried to identify CYR61-responsive genes on the microarray and found evidence for overexpression of IL-1ß, MMP3, and VEGF (data not shown). This was also confirmed by real-time RT-PCR; the expression of IL-1 and MMP3 was more than 5-fold increased and that of VEGF more than 2-fold in patients, compared with controls (Fig. 4). During the course of ophthalmopathy, inflammation and de novo adipogenesis gradually decrease. We were therefore interested to see whether the expression of CYR61 may serve as a marker for disease activity. To accomplish this, we studied expression of CYR61 in adipose/connective tissue from patients undergoing restorative eyelid surgery in the chronic phase of ophthalmopathy. In the intraorbital tissue, CYR61 expression was increased in the chronic phase of ophthalmopathy, compared with healthy individuals, but to a lesser degree, compared with patients with active ophthalmopathy (Fig. 5).

View larger version (20K): [in this window] [in a new window]   FIG. 4. Expression of CYR61-responsive genes in endocrine ophthalmopathy orbital and normal orbital tissue normalized to cyclophillin A as measured by real-time RT-PCR. A–C, Expressions of IL-1ß, MMP3, and VEGF in five individual patients and five controls previously analyzed on the oligonucleotide microarray chip. One patient and one control were replaced by another patient and control not present on the chip due to lack of material. D–F, Mean values for the groups of patients and controls are represented by horizontal lines and the P values are as follows: P < 0.01 for IL-1 and MMP3; P = 0.03 for VEGF when the patient and control groups were compared.

View larger version (9K): [in this window] [in a new window]   FIG. 5. Expression of CYR61 in patients with endocrine ophthalmopathy in acute phase and chronic phase and healthy controls normalized to cyclophillin A as measured by real-time RT-PCR. A, Active endocrine ophthalmopathy orbital tissue (active), chronic endocrine ophthalmopathy orbital tissue (chronic), and normal orbital tissue (control) in five separate individuals. B, Mean values for the groups of patients in active or chronic phase and controls are represented by horizontal lines, and P values between groups are as follows: *, P = not significant; **, P < 0.05; ***, P < 0.01.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

To preferentially select adipose tissue-related genes expressed in normal orbital tissue, the threshold for the fluorescence intensity signal of expression on the microarray was set to 100 in both the patient and control group. This limit has previously been used to identify genes with reproducible and measurable expression on the Affymetrix oligonucleotide microarray (19). Using this approach, a cluster of IEGs was noted to be markedly up-regulated in patients, compared with controls. This group of genes has earlier been shown to be expressed in differentiating preadipocytes and mitogen-stimulated NIH-3T3 fibroblasts (10, 11). In adipogenesis expression of IEGs initiates a 1- to 2-d-long mitotic clonal expansion phase of growth arrested preadipocytes. This is followed by a 3- to 4-d-growth arrest phase and a longer (4–10 d) terminal differentiation phase (9, 23).

But the key question remains. Do IEGs play a role in the pathogenesis of ophthalmopathy?

The identified IEGs can be divided into subgroups of adhesion factors; enzymes involved in prostaglandin synthesis; phosphatases inactivating MAPKs; transcription factors and their cofactors; and metallothioneins. Among these genes CYR61 and COX-2 function in both inflammation and adipogenesis and mediate intercellular effects. CYR61 mediates inflammatory signals by binding to integrins on monocytes, stimulates chemotaxis and adhesion of fibroblasts by binding to heparan sulfate, and promotes cell proliferation (24, 25). As an inducer of angiogenesis, CYR61 activates a complex genetic program of wound healing in skin fibroblasts by inducing expression of, for example, MMP3, VEGF, and IL-1ß, which have been demonstrated to be CYR61 responsive genes (26). IL-1ß has been shown to up-regulate COX-2 in orbital fibroblasts of patients with ophthalmopathy (27). In this study we could clearly demonstrate that selected CYR61-responsive genes, MMP3, VEGF, and IL-1ß were overexpressed in orbital tissue of patients with ophthalmopathy. Also, the target gene of IL-1ß COX-2 was up-regulated in patients, compared with controls. Cyclooxygenases seem to play a key role in adipocyte differentiation because one of their target genes, the prostaglandin-J2, has been suggested as a natural ligand for the key player in adipocyte differentiation, PPAR. Both COX-1 and -2 were expressed in orbital tissue of both patients and controls. Interestingly, COX-2 showed much higher expression in patients than controls despite the fact that they had been treated with corticosteroids known to decrease expression of COX-2. The expression of CYR61 and COX-2 in skin fibroblasts is decreased in response to corticosteroids (28).

The question thus arises whether patients with severe ophthalmopathy are resistant/insufficiently responsive to corticosteroids and whether COX inhibitors would provide an additional therapeutic alternative. In contrast to COX-2, corticosteroids are assumed not to affect the expression of COX-1. In this material the expression of COX-1 was, however, reduced in the patient group. The role of this observation is unclear, but in other tissues, like the inner medulla of the kidney, COX-1 mRNA is decreased in response to stimulation with lipopolysaccharide (29). Another stimulus for down-regulation of COX-1 mRNA in inner medulla collecting duct cells is lowered osmolality (30). Similar conditions can be found in Graves’ orbital tissue with abundance of inflammatory activators and/or changes in osmolality due to edema, which might explain the observed decrease in COX-1 mRNA.

Accumulation of preadipocytes and de novo adipogenesis or an enhanced accumulation of lipids in each adipocyte has been suggested in orbital tissue of Graves’ patients with ophthalmopathy (8, 31). In in vitro studies of orbital fibroblasts and/or preadipocytes, several factors including IGF-I can act as mitogens and stimulate proliferation and differentiation to mature adipocytes. Little, however, is known about orbital mitogens in vivo. In short-term studies using an inhibitor of IGF-I, octreotide, ophthalmopathy was partially improved, but long term data are lacking (32). In our study, IGF-I was expressed in orbital tissue, but we could not observe a difference between patients and controls. It is still possible that downstream targets of IGF-I could be involved in the process. The CYR61 gene product has several functionally specific domains. One of them binds IGF-I with low affinity while binding to heparin-like molecules (33). By binding IGF-I, CYR61 may function as a reservoir for IGF-I, which later could be released by binding to high-affinity IGF receptors located on fibroblasts and preadipocytes.

To complete differentiation of preadipocytes to adipocytes, it is necessary to not only induce proliferation but also cease proliferation in the mitotic clonal expansion phase. Antiproliferative factors have been studied in human sc preadipocytes, and one has been identified as a transcription factor, factor that binds to inducer of short transcripts-1, called FBI-1, which terminates proliferation in the mitotic clonal expansion phase (23). In our study a cofactor of transcription, BTG2, as well as a MAPK phosphatase with specificity for phosphothreonine and phosphotyrosine, DUSP1, were overexpressed in patients, compared with controls. Both these gene products are known to have antiproliferative effects in other cell systems, but future studies are needed to confirm such a role in orbital adipose tissue (34, 35).

Infiltration of orbital tissue with mononuclear leukocytes with subsequent release of cytokines has been considered to reflect disease activity. It has been shown that proinflammatory cytokines like IL-1ß, IL-6, and IL-8 predominate in the retrobulbar space in the active phase (20, 21). We investigated the possibility that IEGs might serve as markers of disease activity by comparing their expression between patients with active and chronic disease. In fact, expression of CYR61 in orbital tissue was lower in the chronic phase, compared with the active phase, but still increased when compared with normal orbital tissue.

Some caution is warranted in the interpretation of the data because the initial microarray analysis was based on only two pooled samples from patients and controls. However, the observed differences were large and confirmed by subsequent individual measurements using real-time RT-PCR.

In conclusion, adipocyte-related IEGs are overexpressed in active ophthalmopathy. CYR61 may have a role in both orbital inflammation and adipogenesis and thus serve as a marker of disease activity. The insufficient response of COX-2 to corticosteroid treatment may partially explain the failure of this treatment in endocrine ophthalmopathy. These findings should pave the way for further studies evaluating the potential role for, e.g. COX inhibitors, in the treatment of ophthalmopathy.

	Acknowledgments

 
The assistance by Gertrud Ahlqvist in handling tissue samples is gratefully acknowledged. We are also grateful to Joyce Carlson for valuable comments on the manuscript.

	Footnotes

 
This work was supported by grants from the Swedish Research Council, Research Funds at Malmö University Hospital, Anna Lisa and Sven-Eric Lundgren Foundation, and the Faculty of Medicine at Lund University.

First Published Online May 31, 2005

Abbreviations: BTG2, B cell translocation gene 2; COX-2, cyclooxygenase-2; CYR61, cysteine-rich, angiogenic inducer, 61; DUSP1, dual-specificity phosphatase 1; EGR1, early growth response 1; IEG, immediate early gene; MMP3, matrix metalloproteinase 3; PPAR, peroxisome proliferator-activated receptor; SCD, stearoyl-coenzyme A desaturase; VEGF, vascular endothelial growth factor.

Received November 19, 2004.

Accepted May 20, 2005.

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