Proteomics of Orbital Tissue in Thyroid-Associated Orbitopathy.

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Proteomics of Orbital Tissue in Thyroid-Associated Orbitopathy N. Matheis, M. Lantz, F. H. Grus, K. A. Ponto, D. Wolters, H. Brorson, T. Planck, B. Shahida, S. Pitz, N. Pfeiffer, and G. J. Kahaly Molecular Thyroid Research Laboratory (N.M., G.J.K.), Department of Medicine I, Experimental Ophthalmology (N.M., F.H.G., D.W.), and Department of Ophthalmology (F.H.G., K.A.P., S.P., N.P.), Johannes Gutenberg University Medical Center (J.G.U.), Mainz 55101, Germany; Departments of Endocrinology (M.L., T.P., B.S.) and Plastic Surgery (H.B.), Lund University, 221 00 Lund, Sweden; and Skåne University Hospital, 214 28 Malmö, Sweden

Context: A potentially altered protein expression profile in orbital tissue from patients with thyroid-associated orbitopathy (TAO) is suspected. Objective: To detect for the first time changes in proteomic patterns of orbital connective tissue in TAO and compare these with control tissue using mass spectrometry. Design: Proteomics cross-sectional, comparative study. Setting: Two academic endocrine institutions. Samples: A total of 64 orbital and peripheral adipose tissue samples were collected from 39 patients with TAO and 25 control subjects. Methods: Samples were analyzed and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. Main Outcome Measures: Mean intensity values of all identified peptides per protein. Results: Thirty-one proteins were identified, of which 16 differentiated between controls and patients with TAO. Different protein patterns between orbital and peripheral adipose tissue were observed. Compared to controls, 10 proteins were markedly up-regulated (ⱖ2-fold) in the orbital tissue of untreated patients: beta IV spectrin (6.2-fold), GTP binding G protein 2 (5.6-fold), POTE ankyrin domain family member F (5.4-fold), xylulokinase (4.1-fold), kinesin family member 1A and lipocalin 1 (both 3.6-fold), semicarbazide-sensitive metalloproteinase amine oxidase 3 and polymerase I transcript release factor (both 3.4-fold), cell-cycle protein elongin A binding protein 1 (3.3-fold), annexin A2 and cavin (both 3-fold), protein pointing to cell proliferation histone H4 (2.8-fold), and ADAM metallopeptidase with thrombospondin type 1 motif 14 (2.7-fold). The highest protein up-regulations were noted in the orbital tissue of medically untreated patients. Steroid therapy markedly reduced up-regulation of these proteins, foremost in nonsmokers. Conclusions: Proteins involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling were up-regulated in orbital tissue of untreated patients with TAO. Steroids decreased the expression of these proteins, whereas smoking attenuated such effect. (J Clin Endocrinol Metab 100: E1523–E1530, 2015)

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in USA Copyright © 2015 by the Endocrine Society Received July 24, 2015. Accepted October 6, 2015. First Published Online October 9, 2015

doi: 10.1210/jc.2015-2976

Abbreviations: ANXA2, annexin A2; AOC3, semicarbazide-sensitive amine oxidase 3; ATS14, ADAM metallopeptidase with thrombospondin type 1 motif 14; CAV1, caveolin-1; GTPB2, GTP binding protein 2; H4, histone H4; KIF1A, kinesin family member 1A; LCN1, lipocalin 1; MALDI, matrix-assisted laser desorption/ionization; MYH2/3/6, myosin heavy chain 2/3/6; POTEE/F, POTE ankyrin domain family, member E/F; PTRF, polymerase I and transcript release factor or cavin 1; REXO1, elongin A binding protein 1; SPTN4, beta IV spectrin; TAO, thyroid-associated orbitopathy; TOF, time-of-flight; XYLB, xylulokinase homolog.

J Clin Endocrinol Metab, December 2015, 100(12):E1523–E1530

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Proteomics in Orbitopathy

hyroid-associated orbitopathy (TAO) is an inflammatory disorder of the orbit and is the most frequent extrathyroidal manifestation in patients with autoimmune thyroid disease (1). The orbital tissue is infiltrated by activated mononuclear cells releasing proinflammatory cytokines that stimulate target orbital fibroblasts to secrete hydrophilic mucopolysaccharides leading to edema, swelling of retrobulbar tissue, and proptosis (1, 2). Recent gene expression studies have shown that markers of adipogenesis (ie, the stearoyl-coenzyme A desaturase) and inflammation (cyclooxygenase-2) in orbital tissue are related to disease activity in TAO (3). Furthermore, immediate early genes, ie, the cysteine-rich, angiogenic inducer 61 (CYR61), are overexpressed in orbital tissue of patients with active TAO (4), and this is induced by smoking (5). Also, both smoking and gene polymorphisms of CYR61 rs 12756618 interact to increase the risk of TAO (6). Proteomic analysis and mass spectrometry are ideally suitable for mass screening of peptides in body fluids and various tissues (7–9). However, very scarce data pertaining to proteomics in patients with autoimmune thyroid diseases are available. In a pilot project, we detected a set of protein biomarkers using the surface-enhanced laser desorption/ionization time-of-flight (TOF) mass spectrometry technology (10). Using these proteins, peptides were identified in the tear fluid of TAO patients with a specificity of 99% (11). Proteins were further characterized with matrix-assisted laser desorption/ionization (MALDI) TOF mass spectrometry (11). A subsequent proteomics and microarray study revealed a significantly different protein panel in TAO vs dry eye syndrome and/or controls (12). Now, in the present multicenter study, we aimed to look for dysregulated proteins in the orbital connective tissue of patients with TAO and compare the protein profile to orbital and peripheral adipose tissue of subjects devoid of autoimmune and thyroid disorders. We looked for the impact of specific steroid treatment and smoking on the orbital protein pattern.

T

Subjects and Methods Subjects and tissue samples A total of 64 tissue samples, 39 orbital tissue samples of patients with TAO collected during orbital decompression surgery (six nonsmoker patients without previous steroid treatment, 16 previously steroid-treated nonsmokers, and 17 steroid-treated smokers), 12 orbital samples collected during blepharoplasty or translid fat resection from nonsmoker subjects devoid of autoimmune and thyroid disorders, and 13 peripheral adipose tissue samples obtained from the healthy arm of patients with lymphedema (seven smokers and six nonsmokers) were included. Thirteen orbital TAO samples and the peripheral tissue samples were collected in Malmö, Sweden. All patients and con-


trols gave their written informed consent. The study protocol was approved by the Ethics Committee of the state of Rhineland Palatinate, Germany, in accordance with the ethical standards in the Declaration of Helsinki. All patients underwent complete endocrine and ophthalmic investigation. Diagnosis and definition of clinical activity and severity of TAO were based on the criteria recommended by the Consensus Statement of the European Group on Graves’ Orbitopathy, EUGOGO (13).

Tissue collection and lysis Biopsies of adipose tissue samples from patients and controls were performed exclusively during routine surgeries, independently of the current study. No additional tissue was removed during surgery. Samples were immediately frozen in liquid nitrogen (Mainz) or treated with the substance RNAlater (Ambion) overnight (Malmö) before being frozen and stored until use. To extract the proteins, samples were thawed on ice and dissociated with the Proteo Extract Dissociation Buffer Kit (Calbiochem). The manufacturer’s protocol was modified as follows: tissue ⬍500 mg was dissociated with only 1.5 mL tissue dissociation buffer, no cell strainer was used, and centrifugation was performed at 7000 rpm for 30 minutes. Lysis of samples was performed using 200 ␮L PBS containing 0.1% SDS and a pestle for Eppendorf tubes. Samples were vortexed, treated with ultrasound, and then subjected to a freeze-thaw cycle with liquid nitrogen. Afterward, the samples were centrifuged at 14 000 rpm for 30 minutes. This was conducted two times for each sample, and the supernatant was unified. Subsequently, samples were precipitated with 100% ice-cold acetone. Samples of each study collective were pooled (using the same amount of protein of each sample). Per pool group, 50 ␮g of protein was applied on an SDS gel.

MALDI mass spectrometry and data acquisition Samples were separated on a one-dimensional SDS-PAGE. Per lane and pool group, 50 ␮g were applied on the gel. An overnight in gel digestion and an elution of the peptides followed. Samples were evaporated and adjusted to pH ⱕ 4 with 0.5% trifluoroacetic acid for fractionation with zip tips (Zip Tip Pipette Tips; Merck Millipore) according to the manufacturer’s protocol. Samples where eluted in five steps with 15, 25, 35, 45, and 50% acetonitrile solution and then directly spotted to a steel target. The sample was co-crystallized with an energy-absorbing matrix (cinnamic acid). Data acquisition was accomplished using MALDI TOF/TOF mass spectrometer (Ultraflex II TOF/ TOF; Bruker Daltonics) with a nitrogen laser. After acquiring the digest spectra with 100 laser shots averaged from five sample positions in the linear mode, peptides below m/z 4000 Da with good peak intensity were selected for fragmentation analysis using a reflector mode. Peptide fragmentation was performed using collision-induced dissociation, and 50 laser shots from five sample positions were summed up for each parent ion. All spectra were externally calibrated by using the peptide calibration standard (Angiotensin II 1047, 19 Angiotensin I 1297.49, Substance P 1348.64, Bombesin 1620.86, ACTH clip 1–17 2093.08, ACTH clip 18 –39 2465.19, Somatostatin 28 3147.47; Bruker Daltonics). Data processing of raw spectra, peak detection, and protein identification was performed using Bruker software (Flex analysis 2.4 and BioTools 3.1) and MASCOT. The MALDI spectra obtained were used for database searches with MASCOT using SwissProt release 11_1 (Swiss Institute of Bioinformatics)


doi: 10.1210/jc.2015-2976


database. MASCOT compares the peptide and lift spectra against peptide patterns in the database and searches for homologies. BioTools Software, which is linked with the MASCOT server, was used. If the data of the spectra were matching the data in the database, probability if this was a contingency was determined. Protein queries were run under Mudpit Scoring conditions with a significance threshold of P ⬍ .05 for protein identification.

Data analysis With the help of the P2M software (Proteomics Pipeline Mainz; Experimental Ophthalmology), intensities of identified proteins were normalized and clustered. Corresponding cluster lists containing the normalized peak intensity values and the identifications for each sample were exported to Statistica (version 6.2; StatSoft). Peptide intensity values for each protein were averaged, and tables with the mean intensities were constructed. Differences of mean intensities ⱖ2-fold or ⱕ0.5-fold were considered as significantly up- or down-regulated, respectively.

Results Demographic, clinical, and serological data The demographic, clinical, and serological data of the 39 patients with TAO and the 25 control subjects are summarized in Table 1. All baseline serum TSH levels of Table 1.

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controls were within the normal range. Nonsmoker patients who did not require immunosuppressive treatment before sample collection showed a more prevalent mild clinical TAO phenotype vs smokers. In this group, two of six patients had a euthyroid TAO and an autoimmune thyroiditis. Eight, 15, and 11 patients with TAO and Graves’ hyperthyroidism have been treated with a 12-month course of antithyroid drugs, radioactive iodine, or thyroidectomy, respectively. In patients with clinically active and severe TAO, glucocorticoids were administered iv according to a published treatment protocol (14 –16) 5 months (median; range, 1–22 mo) before decompression surgery. Proteome analysis Patients’ orbital tissue vs control tissue Mean intensity values (and SD) of all measured peptides per protein and the calculated protein ratios between the various patient and control groups are presented in Tables 2 and 3, respectively. Thirty-one proteins were identified, of which 16 differentiated between patients with TAO and controls. Ten proteins are up-regulated in patients’ orbital adipose tissue vs control orbital adipose tissue: polymerase I and transcript release factor (PTRF or caveolin-1, 3.1-fold), beta IV spectrin

Demographic Data

No. of patients Age, median (range), y Females Nonsmoker Duration of TAO, median (range), mo Active TAO Clinical activity score of TAO, median (range, 0 –7 points) Severity classes Mild Moderate-to-severe Sight-threatening NOSPECS-score of TAO, median (range, 0 –18 points) Duration of autoimmune thyroid disease, median (range), mo Thyroid involvement Graves’ disease Hashimoto’s thyroiditis Euthyroid TAO Thyroid treatment Antithyroid drugs Radioactive iodine Thyroidectomy Thyroid function at orbital decompression surgery Euthyroid Hyperthyroid TSH receptor antibody positive

Untreated Nonsmokers

Steroid-Treated Nonsmokers

Steroid-Treated Smokers

Control Orbital Tissue

Control Peripheral Tissue

6 30 (20 –73) 5 (83.3) 6 (100) 47.5 (10 –162) 0 1 (0 –2)

16 55.1 (20 –76) 11 (68.8) 16 (100) 22.5 (8 – 646) 11 (68) 3.5 (0 –5)

17 48.5 (27–71) 10 (58.8) 0 (0) 28 (6 –131) 13 (76.5) 4 (1–5)

12 67.2 (48 – 85) 8 (66.7) 12 (100)

13 61 (16 –78) 12 (92.3) 6 (46.2)

2 (33.3) 4 (66.7) 0 5.2 (2.5–7)

2 (12.5) 13 (81.3) 1 (6.3) 6 (4 –10.5)

2 (11.8) 15 (88.2) 0 (0) 6 (2–10.5)

90 (10 –332)

86 (10 – 646)

90 (10 –332)

4 (66) 1 (16.7) 1 (16.7)

16 (100) 0 0

17 (100) 0 0

0 1 (16.7) 2 (16.7)

2 (12.5) 10 (62.5) 3 (18.8)

6 (35.3) 4 (23.5) 6 (35.5)

6 (100) 0 5 (83.3)

14 (87.5) 2 (12.5) 14 (87.5)

15 (88.2) 2 (11.8) 14 (82.4)

Demographic, clinical, and serological data of the 39 patients with TAO and the 25 euthyroid control subjects. Data are expressed as number (percentage) unless stated otherwise.


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Table 2.



Mean Intensity Peptide Values

Patient Orbital Adipose Tissue Untreated Nonsmokers

SPTN4 XYLB AOC3 PTRF REXO1 ANXA2 CAV1 KIF1A GTPB2 H4 ATS14 POTEE MYH6 MYH3 POTEF MYH2 LCN1

Control Tissue

Steroid-Treated Nonsmokers

Steroid-Treated Smokers

Orbital Adipose Tissue

Peripheral Adipose Tissue, Smokers

Peripheral Adipose Tissue, Nonsmokers

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

18 312.70 9599.00 5939.43 6289.80 9194.50 8109.48 9186.00 7683.30 13 848.29 7723.92 5240.50 4443.50 3419.00 3448.25 7371.00 1880.45 2961.50

14 423.07 5394.98 5161.36 2770.53 6347.05 6512.78

6492.55 4134.63 3595.50 6401.80 5917.75 3281.56 2693.00 2715.70 15 900.29 4619.42 1955.88 2211.19 5354.50 5750.88 3438.50 2641.64 1973.25

2167.21 825.72 921.06 4600.15 3369.01 91.36 1105.92 1450.98 18 557.92 683.77 131.70 57.54 874.69 4151.95 4862.77 219.72 2189.56

6300.30 4170.13 4257.39 4945.20 8399.83 4289.68 4076.00 4025.20 8005.21 5490.25 4891.75 3795.44 3964.00 3247.88 5303.50 4023.05 4564.75

4725.17 2920.17 3246.88 4288.74 8213.05 2537.10 1538.66 1526.36 1748.88 4446.41 3967.93 2289.52 1083.29 1753.09 362.75 2489.47 2813.93

2908.95 2287.88 1719.61 1833.70 2812.58 2616.52 2982.00 2550.85 4800.29 2711.33 1901.38 2000.56 1898.50 2012.50 6551.50 1984.27 3221.50

113.07 194.28 108.14 2.69 160.63 229.89 902.27 387.85 614.58 361.80 95.28 363.19 197.28 160.16 2339.82 21.73 750.95

2877.30 28 138.50 6488.93 9625.00 26 849.67 4304.88 4695.00 1130.10 2106.14 19 523.67 4470.75 1621.75 3714.00 9121.25 1114.00 1487.45 770.00

1604.11 25 479.12 5869.02 3755.09 31 574.58 2912.26

1334.35 34 138.63 3405.76 3661.38 45 149.88 3459.34

1348.41 1027.16 25 289.09 2833.80 1271.62

3109.60 37 726.25 4007.21 10 777.40 39 105.67 4842.80 7291.00 1129.10 1948.71 25 901.17 2761.00 1575.13

5271.69

8936.00

5242.75

1566.95 130.11

682.18 1061.00

888.65 452.55

4942.38 8619.58 5170.54 3850.98 2672.91 0.00 1209.61 521.75 409.41

1707.69 1151.34 34 631.17 2016.05 977.66

Data are expressed as mean intensity values, MALDI measurements ⫾ 1 SD, of all identified peptides per protein and patient/control pooled groups.

(SPTN4, 3-fold), elongin A binding protein 1 (REXO1, 2.9fold), the metalloproteinase semicarbazide-sensitive amine oxidase 3 (AOC3, 2.6-fold), myosin heavy chain 6 (MYH6, 2.4fold). Furthermore, GTPB2, a G protein; XYLB, a xylulokinase homolog; and myosin heavy chain 3 (MYH3) were all 2.3-fold up-regulated. The protein pointing to cell proliferation histone H4 (H4) and the ADAM metallopeptidase with thrombospondin type 1 motif 14 (ATS14 or ADAMTS14) were 2.13- and 2.07-fold up-regulated, respectively (Figure 1A). The deregulated proteins are involved in adipogenesis, lipid metabolism, Table 3.

Protein Ratios

Patient Orbital Adipose Tissue vs Control Orbital Adipose Tissue

SPTN4 XYLB AOC3 PTRF REXO1 ANXA2 CAV1 KIF1A GTPB2 H4 ATS14 POTEE MYH6 MYH3 POTEF MYH2 LCN1

andinflammatoryresponse,determinedwiththepathwayanalysis software Ingenuity 2012 (Ingenuity Systems Inc). Comparing patients’ orbital tissue vs peripheral subcutaneous fat, the following proteins were up-regulated: GTPB2 (5.6-fold), POTE ankyrin domain family member F (POTEF; 5.4-fold), kinesin family member 1A (KIF1A; 3.6-fold), lipocalin 1 (LCN1; 3.6-fold), MYH2 (3.5-fold), SPTN4 (2.9fold), and MYH6 (2.5-fold), whereas the proteins XYLB (ratio, 0.15), REXO (0.24), and H4 (0.25) were down-regulated (Figure 1B).

Patient Orbital Adipose Tissue vs Control Peripheral Adipose Tissue

Untreated Nonsmoker

SteroidTreated Nonsmoker

SteroidTreated Smoker

Untreated TAO Nonsmoker vs Control Smoker

Untreated TAO Nonsmoker vs Control Nonsmoker

Steroid-Treated TAO Nonsmoker vs Control Smoker

Steroid-Treated TAO Nonsmoker vs Control Nonsmoker

6.295 4.196 3.454 3.430 3.269 3.099 3.080 3.012 2.885 2.849 2.756 2.221 1.801 1.713 1.125 0.948 0.919

2.232 1.807 2.091 3.491 2.104 1.254 0.903 1.065 3.312 1.704 1.029 1.105 2.820 2.858 0.525 1.331 0.613

2.166 1.823 2.476 2.697 2.987 1.639 1.367 1.578 1.668 2.025 2.573 1.897 2.088 1.614 0.810 2.027 1.417

6.365 0.341 0.915 0.653 0.342 1.884 1.957 6.799 6.575 0.396 1.172 2.740 0.921 0.378 6.617 1.264 3.846

5.889 0.254 1.482 0.584 0.235 1.675 1.260 6.805 7.106 0.298 1.898 2.821

2.190 0.148 0.656 0.514 0.313 0.996 0.868 3.562 3.801 0.281 1.094 2.340 1.067 0.356 4.761 2.705 5.928

2.088 0.110 0.897 0.594 0.151 0.678 0.369 2.405 8.159 0.178 0.708 1.404

0.386 2.757 2.791

0.644 3.872 1.860

Steroid-Treated TAO Smoker vs Control Smoker

Steroid-Treated TAO Smoker vs Control Nonsmoker

2.256 0.147 0.554 0.665 0.220 0.762 0.574 2.403 7.549 0.237 0.437 1.363 1.442 0.630 3.087 1.776 2.563

2.088 0.110 0.897 0.594 0.151 0.678 0.369 2.405 8.159 0.178 0.708 1.404 0.644 3.872 1.860

Protein ratios between the various patient and control groups are shown. Ratios, either higher than 2 or lower than 0.5, were regarded as significantly up- or down-regulated. Missing protein ratios were due to the absence of measured proteins in the corresponding sample. Significant up or downregulations are in bold.


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There was no relationship between the thyroid metabolic status and the proteome expression profiles. When comparing orbital tissue of previously steroid-treated nonsmoker patients with control orbital tissue of nonsmokers, seven up-regulated proteins were noted, ie, PTRF (3.4-fold), GTPB2 (3.3-fold), SPTN4 (2.2-fold) and REXO1 (2.1-fold). When looking at orbital tissue of steroid-treated TAO smokers vs control orbital tissue eight up-regulated proteins were detected; ie, REXO (2.9-fold), PTRF (2.6-fold), ATS14 (2.5-fold), AOC3 (2.4-fold), SPTN4 (2.1-fold), and H4 (2-fold). Control orbital adipose tissue vs control peripheral adipose tissue When comparing control orbital vs control peripheral connective tissue, Figure 1. A, TAO patients’ orbital adipose tissue vs control orbital adipose tissue (mean intensity different protein patterns were obratios). Ratios, either higher than 2 or lower than 0.5, were regarded as significantly up- or down-regulated. B, TAO patients’ orbital adipose tissue vs control peripheral subcutaneous served. As shown in Figure 3, LCN1 adipose tissue (mean intensity ratios). Ratios, either higher than 2 or lower than 0.5, were (3.6-fold) and GTPB2 (2.3-fold) were regarded as significantly up- or down-regulated. up-regulated. However most proteins were down-regulated: XYLB (14-fold; The most marked protein up-regulations were noted in ratio, 0.07), REXO (11-fold; ratio, 0.09), PTRF (5.5-fold; the orbital tissue of TAO patients not previously treated ratio, 0.18), and AOC3 (3-fold; ratio, 0.35). with steroids vs control orbital tissue (Figure 2): SPTN4 (6.2-fold); XYLB (4.1-fold); AOC3 (3.4-fold); PTRF or caveolin-1 (3.4-fold); REXO1 (3.3-fold); the structural Discussion protein annexin A2 (ANXA2), cavin (CAV1), and KIF1A, all 3-fold; the G protein GTPB2 (2.9-fold); H4 (2.8-fold); For the first time, proteome analysis of the orbital conATS14 or ADAM14 (2.7-fold); and POTEE (2.2-fold). nective adipose tissue of patients with TAO was compared

Figure 2. Protein ratios in orbital tissue of patients with TAO. Black columns, untreated; striped columns, steroid-treated nonsmokers; gray columns, steroid-treated smokers. Ratios, either higher than 2 or lower than 0.5, were regarded as significantly up- or down-regulated.


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Figure 3. Control orbital adipose tissue vs control peripheral subcutaneous adipose tissue (mean intensity ratios). Ratios, either higher than 2 or lower than 0.5, were regarded as significantly up- or down-regulated.

to both control orbital adipose tissue and peripheral subcutaneous fat. The multicenter design of this comparative study allowed us to include a relatively large number of samples and to look for the impact of both immunosuppressive treatment and smoking on the regulation of the identified proteins. The deregulated proteins were peptides involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling. Steroid treatment decreased the expression of these proteins, whereas smoking attenuated such effect. The importance of testing control orbital tissue became apparent because different protein expressions, ie, tissue inflammation and remodeling, were registered in control orbital vs peripheral connective tissue. Very scarce proteomic data and only one similar study with as few as six tissue samples are available in the literature. In this single report (17), comparison of orbital fat proteins from TAO patients with controls showed significant differences in the proteome, and up-regulation of specific proteins in orbital tissue from TAO was associated with biochemical mechanisms or capacities against endoplasmic reticulum stress, mitochondria dysfunction, and cell proliferation as well as apoptosis in TAO orbital tissue. Also, a correlation between the decreased protein concentrations of proline-rich proteins and uridine diphosphate-glucose-dehydrogenase with smoking was shown. This is in line with findings that smoking is a risk factor for development of TAO and has an impact on orbital tissue composition (5). This is also in line with our previous studies showing a significant reduction of protective proteins in the tear fluid of patients with TAO (11, 12). The findings of overexpression of proteins that appear to be relevant to the observed TAO pathology are quite interesting and could suggest a molecular (protein) basis

for the pathogenesis of TAO (18). Because most tissues examined were collected from previously treated patients with a TAO of relatively long duration, an overexpression of proteins involved in lipid metabolism and tissue remodeling, ie, fibroblast and adipocyte proliferation, as well as orbital fibrosis is not surprising. However, inflammatory proteins also were up-regulated in untreated patients, and with this respect, we focused on the up-regulated metalloproteinase AOC3 (19 –21) which is involved in leukocyte tethering (22) and rolling during inflammatory processes (23–25). In addition to its inflammatory involvement, AOC3 contributes to glucose uptake and fat cell differentiation through insulin-mimicking effects in adipocytes (26 –28). Additional proteins involved in adipogenesis and pathophysiology of metabolic diseases (29) were the up-regulated G protein GTP binding protein 2 and xylulokinase, the final enzyme in the glucuronatexylulose pathway (30), which produces xylulose 5-phosphate, a key regulator of lipogenesis (31, 32). Also up-regulated were structural proteins involved in the remodeling of orbital tissue; beta IV spectrin, an actin/ plasma membrane crosslinking and molecular scaffold protein that links the plasma membrane to the actin cytoskeleton, is involved in the determination of cell shape, the arrangement of transmembrane proteins, and the organization of organelles. Also, annexin A2, a member of a calcium-dependent phospholipid-binding protein family that plays a role in the regulation of cellular growth, in signal transduction pathways, and in cell– cell adhesion via membrane/cytoskeletal organization, was up-regulated in the single published study dealing with orbital protein expression in TAO (17). Furthermore, the up-regulated protein ADAMTS14 is a disintegrin-like and metalloproteinase domain with thrombospondin type 1


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modules. This is in line with a recently published overexpression of another member of the ADAMTS family, ADAMTS 1 motif 1 (4). Expression of matrixins can be increased by inflammatory cytokines, growth factors, and cell– cell interaction (33). Finally, KIF1A, a member of the kinesin family, is an anterograde motor protein and carries membranous organelles along axonal microtubules in neurons (34 –36). Remodeling of the orbital tissue explains its up-regulation best. As in tear fluid of patients with TAO (11), proteins that are involved in inflammatory processes were up-regulated in the current study, ie, POTEE and POTEF both paralogs of POTEI, an isoform of POTE-2a-actin, which is elevated in Hela cells treated with proapoptotic factors, eg, the FAS ligand, antibodies to FAS ligand, and TRAIL. Also, overexpression of POTEI was demonstrated to induce apoptosis (29). Thus, up-regulation of POTEI paralogs in TAO may point to eventual apoptosis of infiltrative mononuclear cells during the course of the disease and/or apoptosis of target cells within the process of tissue remodeling in the orbit. Limitations of this study have to be acknowledged. Proteomic studies such as this cannot by themselves discern whether the differentially expressed proteins are indeed involved pathophysiologically in the generation of the morbid process or are simply epiphenomena. Furthermore, although samples were processed separately, they were pooled for the analysis. This may eventually impact the reproducibility of the findings. In conclusion, proteomics revealed up-regulated proteins of lipid metabolism, inflammation, and tissue remodeling in orbital adipose tissue of patients with TAO. In addition, smoking attenuated the effects of steroids.


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Acknowledgments 20.

Address all correspondence and requests for reprints to: Prof George J. Kahaly, Department of Medicine I, Johannes Gutenberg University Medical Center, Mainz 55101, Germany. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.

References 1. Bahn RS. Graves’ ophthalmopathy. N Engl J Med. 2010;362:726 – 738. 2. Orgiazzi J, Ludgate M. Pathogenesis. In: Wiersinga WM, Kahaly GJ, eds. Graves’ Orbitopathy: A Multidisciplinary Approach–Questions and Answers. Basel, Switzerland: Karger; 2010:40 –53. 3. Vondrichova T, de Capretz A, Parikh H, et al. COX-2 and SCD, markers of inflammation and adipogenesis, are related to disease activity in Graves’ ophthalmopathy. Thyroid. 2007;17:511–517. 4. Lantz M, Vondrichova T, Parikh H, et al. Overexpression of im-

21.

22.

23.

24.

25. 26.

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mediate early genes in active Graves’ ophthalmopathy. J Clin Endocrinol Metab. 2005;90:4784 – 4791. Planck T, Shahida B, Parikh H, et al. Smoking induces overexpression of immediate early genes in active Graves’ ophthalmopathy. Thyroid. 2014;24:1524 –1532. Planck T, Shahida B, Sjögren M, Groop L, Hallengren B, Lantz M. Association of BTG2, CYR61, ZFP36, and SCD gene polymorphisms with Graves’ disease and ophthalmopathy. Thyroid. 2014; 24:1156 –1161. Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis. 1998;19: 1853–1861. Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003;422:198 –207. Grus FH, Podust VN, Bruns K, et al. SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci. 2005;46:863– 876. Okrojek R, Grus FH, Matheis N, Kahaly GJ. Proteomics in autoimmune thyroid eye disease. Horm Metab Res. 2009;41:465– 470. Matheis N, Okrojek R, Grus FH, Kahaly GJ. Proteomics of tear fluid in thyroid-associated orbitopathy. Thyroid. 2012;22:1039 –1045. Matheis N, Grus FH, Breitenfeld M, et al. Proteomics differentiate between thyroid-associated orbitopathy and dry eye syndrome. Invest Ophthalmol Vis Sci. 2015;56:2649 –2656. Bartalena L, Baldeschi L, Dickinson A, et al. Consensus statement of the European Group on Graves’ orbitopathy (EUGOGO) on management of GO. Eur J Endocrinol. 2008;158:273–285. Zang S, Ponto KA, Kahaly GJ. Clinical review: Intravenous glucocorticoids for Graves’ orbitopathy: efficacy and morbidity. J Clin Endocrinol Metab. 2011;96:320 –332. Kahaly GJ. Management of moderately severe Graves’ orbitopathy. In: Wiersinga WM, Kahaly GJ, eds. Graves’ Orbitopathy: A Multidisciplinary Approach–Questions and Answers. Basel, Switzerland: Karger; 2010:120 –158. Kahaly GJ, Pitz S, Hommel G, Dittmar M. Randomized, single blind trial of intravenous versus oral steroid monotherapy in Graves’ orbitopathy. J Clin Endocrinol Metab. 2005;90:5234 –5240. Cheng KC, Huang HH, Hung CT, et al. Proteomic analysis of the differences in orbital protein expression in thyroid orbitopathy. Graefes Arch Clin Exp Ophthalmol. 2013;251:2777–2787. Iyer S, Bahn R. Immunopathogenesis of Graves’ ophthalmopathy: the role of the TSH receptor. Best Pract Res Clin Endocrinol Metab. 2012;26:281–289. Zorzano A, Abella A, Marti L, Carpéné C, Palacín M, Testar X. Semicarbazide-sensitive amine oxidase activity exerts insulin-like effects on glucose metabolism and insulin-signaling pathways in adipose cells. Biochim Biophys Acta. 2003;1647:3–9. Salmi M, Jalkanen S. Developmental regulation of the adhesive and enzymatic activity of vascular adhesion protein-1 (VAP-1) in humans. Blood. 2006;108:1555–1561. Salmi M, Jalkanen S. A 90-kilodalton endothelial cell molecule mediating lymphocyte binding in humans. Science. 1992;257:1407– 1409. Jaakkola K, Nikula T, Holopainen R, et al. In vivo detection of vascular adhesion protein-1 in experimental inflammation. Am J Pathol. 2000;157:463– 471. Jalkanen S, Karikoski M, Mercier N, et al. The oxidase activity of vascular adhesion protein-1 (VAP-1) induces endothelial E- and Pselectins and leukocyte binding. Blood. 2007;110:1864 –1870. Koskinen K, Vainio PJ, Smith DJ, et al. Granulocyte transmigration through the endothelium is regulated by the oxidase activity of vascular adhesion protein-1 (VAP-1). Blood. 2004;103:3388 –3395. Ley K, Deem TL. Oxidative modification of leukocyte adhesion. Immunity. 2005;22:5–7. Morin N, Lizcano JM, Fontana E, et al. Semicarbazide-sensitive amine oxidase substrates stimulate glucose transport and inhibit lipolysis in human adipocytes. J Pharmacol Exp Ther. 2001;297: 563–572.


E1530

Matheis et al


27. Bour S, Daviaud D, Gres S, et al. Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes. Biochimie. 2007;89:916 –925. 28. Fontana E, Boucher J, Marti L, et al. Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A cells. Biochem J. 2001;356:769 –777. 29. Blad CC, Tang C, Offermanns S. G protein-coupled receptors for energy metabolites as new therapeutic targets. Nat Rev Drug Discov. 2012;11:603– 619. 30. Touster O. Essential pentosuria and the glucuronate-xylulose pathway. Fed Proc. 1960;19:977–983. 31. Kabashima T, Kawaguchi T, Wadzinski BE, Uyeda K. Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver. Proc Natl Acad Sci USA. 2003;100:5107–5112. 32. Bunker RD, Bulloch EM, Dickson JM, Loomes KM, Baker EN.


33.

34. 35.

36.

Structure and function of human xylulokinase, an enzyme with important roles in carbohydrate metabolism. J Biol Chem. 2013;288: 1643–1652. Yoon JS, Chae MK, Jang SY, Lee SY, Lee EJ. Antifibrotic effects of quercetin in primary orbital fibroblasts and orbital fat tissue cultures of Graves’ orbitopathy. Invest Ophthalmol Vis Sci. 2012;53:5921– 5929. Hirokawa N, Takemura R. Kinesin superfamily proteins and their various functions and dynamics. Exp Cell Res. 2004;301:50 –59. Okada Y, Higuchi H, Hirokawa N. Processivity of the single-headed kinesin KIF1A through biased binding to tubulin. Nature. 2003; 424:574 –577. Ikegami K, Heier RL, Taruishi M, et al. Loss of ␣-tubulin polyglutamylation in ROSA22 mice is associated with abnormal targeting of KIF1A and modulated synaptic function. Proc Natl Acad Sci USA. 2007;104:3213–3218.


Effect of orbital decompression on diplopia in thyroid-related orbitopathy.

Piezosurgery for orbital decompression surgery in thyroid associated orbitopathy.

Surgical complications in orbital decompression for Graves' orbitopathy.

connective tissue of patients with Graves' orbitopathy: clinical implications.

Idiopathic sclerosing orbital inflammation and active Graves' orbitopathy.

Pitfalls associated with orbital decompression for thyroid-related orbitopathy.

Mesenchymal Stem Cell-Like Properties of Orbital Fibroblasts in Graves' Orbitopathy.

Surgical effects of various orbital decompression methods in thyroid-associated orbitopathy: computed tomography-based comparative analysis.

Graded decompression of orbital fat and wall in patients with Graves' orbitopathy.

Differential Adipose Tissue Proteomics.

Orbital Volumetry in Graves' Orbitopathy: Muscle and Fat Involvement in relation to Dysthyroid Optic Neuropathy.

Transcranial orbital decompression for progressive compressive optic neuropathy after 3-wall decompression in severe graves' orbitopathy.

Spontaneous medial orbital decompression associated with medial wall remodeling in Graves' orbitopathy.

Evaluation of the association between subconjunctival orbital fat prolapse and thyroid-associated orbitopathy.

Long-term outcome of Graves' orbitopathy following high-dose intravenous glucocorticoids and orbital radiotherapy.

Characterization and outcomes of repeat orbital decompression for thyroid-associated orbitopathy.

Fat-removal orbital decompression for thyroid associated orbitopathy: The right procedure for the right patient.

Orbital decompression for thyroid-associated orbitopathy in England: trends over time and geographical variation.

Torsional diplopia after transantral orbital decompression and extraocular muscle surgery associated with Graves' orbitopathy.

Thyrotropin receptors in adipose tissue, retro-orbital tissue and lymphocytes.

Subconjunctival orbital fat prolapse and thyroid associated orbitopathy: a clinical association.

Tissue proteomics of splenic marginal zone lymphoma.

Which factors are associated with quality of life in patients with Graves' orbitopathy presenting for orbital decompression surgery?

Evaluation of the Retro-Orbital Fatty Tissue Volume in Delayed Orbital Blow-Out Fractures.