September 2021
Volume 62, Issue 12
Open Access
Biochemistry and Molecular Biology  |   September 2021
The Potential Role of Osteopontin in the Pathogenesis of Graves’ Ophthalmopathy
Author Affiliations & Notes
  • Heng Lou
    Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China
  • Lian-Qun Wu
    Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China
  • Hao Wang
    Department of Ophthalmology, Shanghai Changzheng Hospital, Shanghai, China
  • Rui-Li Wei
    Department of Ophthalmology, Shanghai Changzheng Hospital, Shanghai, China
  • Jin-Wei Cheng
    Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China
    Department of Ophthalmology, Shanghai Changzheng Hospital, Shanghai, China
  • Correspondence: Jin-Wei Cheng, Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China; jinwei_cheng@yeah.net
Investigative Ophthalmology & Visual Science September 2021, Vol.62, 18. doi:https://doi.org/10.1167/iovs.62.12.18
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      Heng Lou, Lian-Qun Wu, Hao Wang, Rui-Li Wei, Jin-Wei Cheng; The Potential Role of Osteopontin in the Pathogenesis of Graves’ Ophthalmopathy. Invest. Ophthalmol. Vis. Sci. 2021;62(12):18. doi: https://doi.org/10.1167/iovs.62.12.18.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The aim of this study is to evaluate the expression of osteopontin (OPN) and its relationship with relative cytokines in patients with Graves’ ophthalmopathy (GO), and to observe the effect of OPN on orbital fibroblasts (OFs) proliferation, migration, and the expression of relative cytokines, as well as the signaling pathways involved in its effect.

Methods: The orbital adipose connective tissue was obtained from 24 patients with GO (12 cases of active GO, and 12 cases of inactive GO) and 12 healthy controls. OFs were isolated from orbital tissues obtained from patients with active GO who were undergoing orbital decompression surgery. Quantitative PCR and Western blot were performed to detect RNA and protein expression. The proliferation and cell migration rates of OFs were measured by methylthiazol tetrazolium (MTT) and the cell scratch test. Signaling pathway inhibitors, such as OPN monoclonal antibody 1A12, ERK1/2 inhibitor PD98059, and PI3K inhibitor LY294002, were applied to determine the involved pathways.

Results: The mRNA and protein levels of OPN were increased in orbital adipose connective tissue from patients with active GO than those from patients with inactive GO (2.83-fold increase, P < 0.001; 1.91-fold increase, P < 0.05). The OPN mRNA level was positively correlated with CD40 ligand (CD40L) and hyaluronan synthases 2 (HAS2) mRNA in patients with GO. OPN promoted proliferation and migration rate of OFs and induced vascular endothelial growth factor (VEGF) and collagen I mRNA expression, and the effects were inhibited by 1A12 or LY294002.

Conclusions: OPN in orbital adipose connective tissues were significantly increase in active GO, and there were significant correlations of OPN with CD40L and HAS2 mRNA levels in patients with GO. OPN promoted proliferation and migration of OFs and induced VEGF and collagen I mRNA expression in OFs through PI3K/Akt signaling pathway. This suggested a role for OPN in the pathogenesis of GO through the activation of OFs.

Graves’ ophthalmopathy (GO) is an autoimmune inflammatory disorder whose pathogenesis is very complicated and needs to be further explored. The natural clinical process follows a biphasic course with an active phase followed by a stable period. The orbital inflammatory response was intense in the active period, leading to collagen and glycosaminoglycan accumulation. Then, the orbital tissue expansion and extraocular muscle thickening as well as fibrosis were the results.1 Current evidence suggests that orbital fibroblasts (OFs) play a vital role in orbital inflammation and tissue remodeling in GO. Enhanced proliferative activity, generation of inflammation mediators and excessive extracellular matrix (ECM), and differentiation into fat cells and myofibroblasts occur in OFs once activated by direct interaction with immune cells or by soluble cytokines.2,3 Non-sulfated glycosaminoglycans (especially hyaluronic acid) and collagen content are increased in GO orbital tissue leading to tissue expansion and fibrosis. Hyaluronan synthases 2 (HAS2) is the main regulator of the hyaluronic acid synthesis in OFs from patients with GO.4,5 The main objective of current clinical management is to reduce inflammation response in order to promote the transition from active to stable phase. 
As a multifunctional cytokine, osteopontin (OPN) plays a part in normal physiology and participates in pathological process. It is expressed in fibroblasts,6 osteoblasts, and T cells,7 and has been proved to be a proinflammatory cytokine,8 which is closely related with tissue fibrosis.9 OPN also plays a pathogenetic role in many autoimmune diseases10,11 (i.e. Grave's disease12 and multiple sclerosis [MS]).13,14 In the in vitro experiment, it has shown that OPN activated hepatic stellate cells and initiated the increase of collagen I leading to liver fibrosis, via activation of the PI3K/pAkt/NF-κB signaling pathway.15 Recent studies had found that the level of serum OPN in patients with GD was significantly increased, as well as the OPN receptors (i.e. αν, β1, β3, and CD44), and many proinflammatory factors, such as CCL2, CCL3, CCL20, IL-1β, and IL-8.12,16 
CD40/CD40 ligand (CD40L, also known as CD154, can act as a soluble cytokine) represents as an important activation pathway initially implicated in T cell/B cell interactions.17 Recently, it was reported that CD40 was overexpressed in OFs from patients with GO, and the CD40/CD40L co-stimulating pathway induced the activation of OFs.18 The expression of plasma CD40L was increased in patients with GD and positively correlated with plasma OPN protein.19 
An independently developed monoclonal antibody against OPN is 1A12 (patent application No. 200710039873.3). In the in vitro experiments, it was confirmed that 1A12 blocked the OPN-induced high expression of vascular endothelial growth factor (VEGF).20 Animal experiments had also shown that 1A12 effectively inhibited the formation of new blood vessels and the growth and metastasis of tumors.20,21 Therefore, 1A12 can alter OPN function and potentially has therapeutic value. 
We hypothesize that OPN also plays a role in the pathogenesis of GO through the activation of OFs. This study aims to detect OPN in orbital adipose tissue in GO and to correlate OPN with GO disease activity, and to determine the effect of OPN on orbital fibroblasts through in vitro study. 
Materials and Methods
Materials
The reagents used were as follows: OPN, OPN monoclonal antibody 1A12 (Institute of Oncology, Second Military Medical University, China); anti-vimentin, anti-CD45, anti-cytokeratin, and anti-factor VIII antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA); ERK1/2 inhibitor PD980592, and PI3K inhibitor LY294002 (Sigma-Aldrich, St. Louis, MO, USA). 
Subjects
Orbital adipose connective tissue explants were collected from 36 participants, which were recruited consecutively from the Department of Ophthalmology, Shanghai Changzheng Hospital, after informed consent was approved by the institutional review board. 
Twenty-four patients were diagnosed with GO, based on Bartley diagnosis criteria.22 Twelve of them were diagnosed with active GO who accepted the emergency orbital decompression to save the sight of the patients due to dysthyroid optic neuropathy or exposure keratitis. The rest of them were patients with inactive GO. The activity of GO was evaluated by the clinical activity score (CAS), with active GO being defined as CAS≥3/7 and inactive GO defined as CAS≤2/7.23 Twelve individuals undergoing plastic surgery were included as normal controls. The clinical characteristics of these patients are shown in Table 1. Three different OF culture strains were obtained from three patients diagnosed with active GO (see Table 1, No. 1–3). 
Table 1.
 
Clinical Characteristic of the Patients
Table 1.
 
Clinical Characteristic of the Patients
The following conditions were excluded: patients with other systemic autoimmune or inflammatory diseases and patients with diabetes mellitus. 
All 12 patients with active GO were initially diagnosed and treated for hyperthyroidism, 6 of them were women, median age 49 years (range = 32–64 years). Five of the patients were current smokers, one quit smoking 12 months ago, the other 6 had never smoked. Time from onset of Graves’ disease to decompression surgery ranged from 10 to 72 months. Three patients had received radioiodine treatment, one had received thyroid surgery 6 months ago. Nine patients were taking antithyroid drugs at the time of the surgery, and the other patients stopped taking antithyroid drugs 8 to 36 months ago. One of the 12 patients had never received glucocorticoids, the remaining 11 patients had been treated with glucocorticoids in the past but had discontinued use several months before the surgery. Two patients had received orbital radiation 2 months before decompression surgery. 
All 12 patients with inactive GO were initially diagnosed and treated for hyperthyroidism, 7 of them were women, median age 43 years (range = 20–60 years). One of the patients quit smoking 24 months ago, and the rest of them had never smoked. Time from onset of Graves’ disease to decompression surgery ranged from 12 to 132 months. One patient had received radioiodine treatment. All of them were taking antithyroid drugs at the time of the surgery. Three of them had been treated with glucocorticoids in the past but had discontinued use 6 to 12 months before the surgery, the other 9 patients had never received glucocorticoids. One patient had received orbital radiation 36 months before decompression surgery. 
All the subjects included were euthyroid and had normal TSH levels within 6 months at the time of orbital fat harvesting. There was no significant difference in gender ratio or age between the groups. These activities were undertaken after informed consent was obtained from the participants, and the research protocol adhered to the tenets of the Declaration of Helsinki. 
RNA Isolation and Quantitative RT-PCR
Orbital adipose connective tissue explants were washed by normal saline immediately upon harvesting, and then frozen in liquid nitrogen within 5 minutes until use. Total RNA was extracted from orbital specimen and OFs using TRIpure Reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) and was solubilized by RNase free water. RNA was converted into first-strand cDNA with the reverse transcription kit (Takara, Shiga, Japan). Real time (RT)-PCR was performed using 2 × Real Time PCR Mix (Probe; Aidlab Biotechnologies Co., Ltd.) on an Applied Biosystems 7500HT Real-Time PCR system according to the manufacturer's protocol. PCR array data were calculated by the ΔΔCt method. The endogenous reference gene GAPDH was used as the negative control for normalization treatment, and there was no difference in GAPDH gene expression levels among the three groups. 
All PCR primers and probes (TaqMan Gene Expression Assay) for OPN, CD40L, CD44, NF-κB, CCL20, collagen I, HAS2, and VEGF were designed by Sangon Biotech (Sangon, Shanghai, China; Table 2). 
Table 2.
 
All Primers and Probes Sequences
Table 2.
 
All Primers and Probes Sequences
Western Blot Analysis of OPN Protein
Orbital samples were washed in pre-cold saline and the vascular tissue was cut off as much as possible, then solubilized by RIPA Lysis Buffer (Aidlab Biotechnologies Co., Ltd.) and every 10 mL was supplemented with a protease inhibitor, then protein concentrations in the extracts were measured with BCA Protein Assay Kit (Aidlab Biotechnologies Co Ltd.). Equal volumes of extracts were separated by SDS-PAGE, and then transferred onto the polyvinylidene fluoride (PVDF) membranes. The membranes were sealed using 5% non-fat milk solution for 2 hours at 24°C. The blots were then incubated with primary antibodies for 24 hours at 4°C and then incubated with secondary antibodies for 2 hours at 4°C. Scans of the full films used to generate Western blot data were obtained by Amersham Imager 600 (GE Healthcare Bio-Sciences AB, Sweden). The protein expression levels were quantified by measuring band intensities using Image J software (National Institutes of Health [NIH], Bethesda, MD, USA). The values were normalized to β-actin. 
Cell Culture
Surgical tissue explants were transported to the laboratory cell room for cell culture as soon as possible (within 6 hours) in DMEM (Hyclone Co., Logan UT, USA) under low temperature. Primary culture cells were adhered and proliferated in 10% FBS (Hyclone Co., USA). The cells need to be identified by immunostaining and flow cytometry with positive staining of vimentin and collagen, as well as negative staining of CD45, cytokeratin, or factor VIII tested by anti-vimentin, anti-CD45, anti-cytokeratin, and anti-factor VIII antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Cells then passaged by trypxin (Sigma Co., USA) and stable passages (passage 4 to 7) were used for following experiments. 
The following cell experiments were divided into five groups, the control group (normal medium), OPN treatment group (5 µM), OPN + LY294002 (5 µM + 20 µM) treatment group, OPN + PD980592 (5 µM + 20 µM) treatment group, and OPN + 1A12 treatment group (5 µM + 20 µg/mL). 
Cell Proliferation
Approximately, 1000 cells per well were seeded in 96-well plates in media containing 10% FBS and incubated for 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours. On the indicated days, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5- dip-henytetrazoliumromide (MTT) reagent (Sangon, China) was added to each well, and cells were incubated for 4 hours at 37°C. Supernatants were then removed and 100 µL of DMSO (Sangon, China) was added per well to dissolve formazan crystals. Optical densities (ODs) were measured at 490 nm using an automatic microplate reader (Gene Co., Ltd., Hong Kong). The OD is proportional to the number of OFs. Relative cell proliferation rates were calculated by the following formula: cell proliferation rate (%) = OD day N / OD day 0 × 100%. 
Cell Scratching
Cultures of OFs cells were seeded at a density of 1 × 106 cells/well in 12-well plates and incubated until confluence. Thereafter, the incubated cell cultures were scratched with a p200 pipette tip in the middle of the wells and the suspension cells were washed off with PBS. The scratch area was analyzed by microscope and images were captured at times 0 hours, 12 hours, and 24 hours. The scratch areas were quantified using Image J software. 
Statistical Analysis
SPSS 22.0 was used to analyze the experimental data, Shapiro-Wilk Test was used to determine whether the data follow normal distribution. The Levene test was used to analyze the homogeneity of variance. Kruskal-Wallis H with the Bonferroni's post hoc test was used to analyze data with non-normal distribution or nonheterogeneity variance from three groups. Multiple logistic regression analysis was used to test whether smoking has a significant effect on OPN, CD40L, collagen I, and HAS2 mRNA expression. Spearman correlation analysis was used in the study of correlations among OPN mRNA and the other cytokines. One-way ANOVA followed by Tukey's post hoc test was used to compare multiple groups with normal distribution and heterogeneity variance. 
Results
The mRNA Levels of OPN in Orbital Adipose Connective Tissue
There was a statistical difference among the three groups (H = 20.02, P < 0.001). The mRNA level of OPN in orbital adipose connective tissues from patients with active GO was significantly higher than those from patients with inactive GO (2.83-fold increase, P < 0.001, n = 12:12), as well as from healthy subjects (1.94-fold increase, P < 0.05, n = 12:12). There was no significant difference of the mRNA levels of OPN in orbital adipose connective tissues between from patients with inactive GO and from healthy subjects (0.69-fold increase, P > 0.05, n = 12:12; Fig. 1). 
Figure 1.
 
The relative expression (fold change) of OPN RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, ***P < 0.001: statistically significant difference compared with the active GO group.
Figure 1.
 
The relative expression (fold change) of OPN RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, ***P < 0.001: statistically significant difference compared with the active GO group.
The Protein Levels of OPN in Orbital Adipose Connective Tissue
The previous experiment demonstrated the increased mRNA level of OPN in orbital tissue from patients with active GO when compared with those from patients with inactive GO and healthy controls. In the next step, we tested the protein level of OPN in orbital tissue by Western blot (Fig. 2). 
Figure 2.
 
Western blot (A) and densitometric analysis (B) (fold change) of the expression of OPN protein in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The Western blot assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05: statistically significant difference compared with the patients with active GO group.
Figure 2.
 
Western blot (A) and densitometric analysis (B) (fold change) of the expression of OPN protein in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The Western blot assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05: statistically significant difference compared with the patients with active GO group.
There was a statistical difference of OPN protein levels among the three groups (H = 9.641, P < 0.01). OPN protein expression in orbital adipose connective tissue from patients with active GO was significantly higher than those from patients with inactive GO (1.91-fold increase, P < 0.05, n = 12:12). 
There was no significant difference of OPN protein expression in orbital adipose connective tissue from patients with active GO (1.25-fold increase, P > 0.05, n = 12:12) and patients with inactive GO (0.65-fold increase, P > 0.05, n = 12:12), when compared with those from healthy subjects. 
The mRNA Levels of Inflammatory Cytokines and Correlation with OPN Level
Then, the mRNA levels of several inflammatory cytokines, such as CD40L, collagen I, HAS2, NF-κB, CCL20, and CD44 were detected, and the correlation coefficients between logarithmically transformed OPN levels and the inflammatory cytokines of GO were calculated. There were statistical differences among the three groups of CD40L, collagen I, and CD44 mRNA levels (H = 20.02, P < 0.001; H = 12.907, P < 0.01; H = 20.122, P < 0.001; Fig. 3). 
Figure 3.
 
The relative expression (fold change) of CD40L, collagen I, HAS2, NF-κB, CCL20, and CD44 RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, **P <0.01, ***P < 0.001: statistically significant difference compared with the patients with active GO group. #P < 0.05, ##P <0.001: statistically significant difference compared with the control group.
Figure 3.
 
The relative expression (fold change) of CD40L, collagen I, HAS2, NF-κB, CCL20, and CD44 RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, **P <0.01, ***P < 0.001: statistically significant difference compared with the patients with active GO group. #P < 0.05, ##P <0.001: statistically significant difference compared with the control group.
The mRNA levels of CD40L and collagen I in orbital tissue from patients with active GO were significantly higher than those from patients with inactive GO (3.14-fold increase, P < 0.01, n = 12:12; 2.75-fold increase, P < 0.01, n = 12:12) and healthy subjects (5.03-fold increase, P < 0.01, n = 12:12; 1.87-fold increase, P < 0.05, n = 12:12). There was no significant difference of the mRNA levels of CD40L and collagen I between patients with inactive GO and healthy subjects (1.60-fold increase, P > 0.05, n = 12:12; 0.68-fold increase, P > 0.05, n = 12:12). 
The mRNA levels of CD44 in orbital adipose connective tissues from healthy subjects were higher than those from patients with active (1.51-fold increase, P < 0.05, n = 12:12) and inactive (2.48-fold increase, P < 0.001, n = 12:12) GO, and there was no significant difference between patients with active GO and patients with inactive GO (P > 0.05). 
There was no significant difference of the mRNA level of HAS2, NF-κB, and CCL20 in orbital adipose connective tissues among the three groups (P > 0.05). 
The Spearman correlation analysis revealed that the expression of OPN mRNA positively correlated with CD40L (r = 0.80, P < 0.001), collagen I (r = 0.61, P < 0.01), and HAS2 mRNA (r = 0.73, P < 0.001) in orbital adipose connective tissue from patients with active and inactive GO (n = 24; Fig. 4). 
Figure 4.
 
Spearman correlation analysis of the mRNA levels between OPN and CD40L (r = 0.80, P < 0.001) (A), OPN and collagen I (r = 0.61, P < 0.01) (B), OPN and HAS2 mRNA (r = 0.73, P < 0.001) (C), in orbital adipose connective tissue from all patients with active and inactive GO (n = 24).
Figure 4.
 
Spearman correlation analysis of the mRNA levels between OPN and CD40L (r = 0.80, P < 0.001) (A), OPN and collagen I (r = 0.61, P < 0.01) (B), OPN and HAS2 mRNA (r = 0.73, P < 0.001) (C), in orbital adipose connective tissue from all patients with active and inactive GO (n = 24).
The Relationship Among Smoking Status, TRAB Levels CAS Score, and Cytokine Levels
Smokers have a greater chance of developing GO and more severe forms of the ophthalmopathy. Six out of the 12 patients with active GO were current or former smokers, and 1 out of the 12 patients with inactive GO smoked before (see Table 1). Multiple logistic regression analysis showed that smoking had no significant effect on OPN (β = -0.205, 95% confidence interval [CI] = -1.159 to 0.369, P =0.293), CD40L (β = -0.102, 95% CI = -1.407 to 0.774, P =0.551), collagen I (β = 0.325, 95% CI = -0.470 to 2.282, P =0.184), and HAS2 (β = -0.287, 95% CI = -2.076 to 0.639, P =0.281) mRNA expression levels in patients with GO. 
There was no significant difference of the TSHR antibody (TRAB) levels between patients with active and inactive GO (P > 0.05; see Table 1). Because all patients with inactive GO had a CAS score of 0, the Spearman correlation analysis was only performed on patients with active GO. There was no linear correlation of TRAB levels, and CAS score with OPN (r = −0.064, P = 0.768; r = 0.165, P = 0.608) and other inflammatory cytokines mRNA (P > 0.05). 
OPN Promotes Proliferation Rate of OFs
The results showed enhanced OFs proliferation rate starting from day 1 in the OPN group, and a dramatic reduction of OFs proliferation with the addition of LY294002 and 1A12 to the level of the control group (P < 0.001). The OFs proliferation rate of the OPN + PD980592 treatment group was higher than that of the other three groups (P < 0.001), except of the OPN treatment group. There was no significant difference among the OPN + 1A12, OPN + LY294002 treatment group, and the control group (P > 0.05, Fig. 5). 
Figure 5.
 
OFs proliferation rate curve of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time of day 0, day 1, day 2, day 3, and day 4. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.001: statistically significant difference compared with the other four groups. #P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 5.
 
OFs proliferation rate curve of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time of day 0, day 1, day 2, day 3, and day 4. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.001: statistically significant difference compared with the other four groups. #P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
OPN Promotes Migration Rate of OFs
At 0 hours, 12 hours, and 24 hours after the scratch, cell migration photographs and relative migration distance in the low-power field were shown in Figure 6-1 and Figure 6-2, respectively. At the 12th and 24th hours, the OFs migration distance of OPN and the OPN + PD980592 treatment group was significantly higher than that of the other three groups (P < 0.001), whereas the difference between OPN + 1A12, OPN + LY294002, and the control group was not statistically significant (P > 0.05). 
Figure 6-1.
 
Representative images of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours.
Figure 6-1.
 
Representative images of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours.
Figure 6-2.
 
Percentage of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ###P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 6-2.
 
Percentage of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ###P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
OPN Induces VEGF and Collagen I mRNA Expression of OFs
After 48 hours of the culture, the VEGF mRNA expression in the control group was onefold. When OPN was introduced to the cell culture, VEGF mRNA expression increased by 2.25-fold (P < 0.001), the increase of VEGF mRNA was completely eliminated by the addition of an PI3K inhibitor (LY294002) and monoclonal antibody toward OPN (1A12), but not by the addition of the ERK1/2 inhibitor (PD980592; Fig. 7). 
Figure 7.
 
The relative expression (fold change) of VEGF mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ##P < 0.01: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 7.
 
The relative expression (fold change) of VEGF mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ##P < 0.01: statistically significant difference compared with the other three groups except the OPN treatment group.
After 48 hours of the culture, the collagen I mRNA expression in the control group was onefold, the expression was significantly increased when OPN was introduced to cell culture (2.66-fold increase, P < 0.001). The effect was completely eliminated by the addition of a PI3K inhibitor (LY294002) and monoclonal antibody toward OPN (1A12), but not by the addition of the ERK1/2 inhibitor (PD980592; Fig. 8). 
Figure 8.
 
The relative expression (fold change) of collagen I mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. #P < 0.05: statistically significant difference compared with the control and OPN + LY294002 treatment groups.
Figure 8.
 
The relative expression (fold change) of collagen I mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. #P < 0.05: statistically significant difference compared with the control and OPN + LY294002 treatment groups.
Discussion
As an autoimmune inflammatory disorder, GO has a complicated management and its immunopathogenesis is poorly understood. Currently, the main treatments for active GO have been systemic corticosteroids and orbital radiation to control disease progression. However, due to lack of specificity and limitation of side effects, it is easy to relapse. Therefore, further studies on the pathogenesis of GO are needed to find newly effective treatments. 
In early active GO, T cells infiltrate into the orbital tissue and activate OFs via the secretion of inflammatory mediators. T-helper 1 (Th1)-lymphocytes dominated in the active phase of GO, whereas in the stage of tissue remodeling and fibrosis at the later stage of disease, T-helper 2 (Th2)-lymphocytes and associated cytokines gradually became predominant.2427 OPN was reported to be a proinflammatory cytokine, which promoted Th1 response and inhibited Th2 response.8,28,29 The binding of OPN and CD44 mediated chemotaxis and adhesion of fibroblasts, T cells, and bone marrow cells.3032 Serum OPN protein levels was elevated among patients with Grave's disease and correlated with fT3 and fT4 strongly.16 
The proliferation of OFs, production of extracellular matrix, and differentiation into adipocytes and myofibroblasts are important factors for the expansion and fibrosis of the orbital tissues in GO.3335 CD40-CD40L binding is involved in physical interactions between OFs and T-lymphocytes in GO. In studies performed by our research group, it was found that intercellular adhesion molecular-1 (ICAM-1), vascular cell adhesion molecular-1 (VCAM-1), and E-selectin expression in cultured OFs from patients with GO were enhanced by CD40L stimulation.36,37 
In the present study, we detected the overexpression of OPN, CD40L, and collagen I mRNA as well as OPN protein in orbital tissues from patients with active GO, and the levels of OPN mRNA from patients with GO have significant correlations with CD40L and collagen I mRNA. The gene expression of OPN was positively correlated with the expression of HAS2. However, there appeared to be no significant difference of HAS2 mRNA expression between active and inactive GO. As the main regulator of the hyaluronic acid synthesis in OFs from patients with GO,38,39 HAS2 may be expressed in both active and inactive GO. Otherwise, the limitations of the present study, such as small sample size, might be a potential reason for the negative result. 
We also did not find any significant difference of CD44 and NF-κB mRNA expression between patients with active and inactive GO. Previous study had shown that OPN upregulated collagen I via ανβ3 engagement in liver fibrosis.15 OPN increased the expression of CCL20 in CD4+T cells from patients with GD via the β3 integrin receptor.40 Therefore, the binding receptor of OPN, which exerted its function in OFs from patients with GO need to be further explored. In patients with GD, the expression of cytokine genes downstream of NF-κB was elevated in peripheral blood mononuclear cells (PBMCs), and OPN could activate NF-κB signaling in vitro.12 Previous experiments had shown that cytokines played pathophysiological roles through NF-κB signaling in vitro.4143 However, in this present study, we found that the levels of NF-κB mRNA did not increase in orbital adipose connective tissues from patients with GO. We hypothesized that OPN might not play its effect through the NF-κB pathway in orbital adipose connective tissues of active GO, and an in vitro study confirmed that OPN activated OFs through PI3K/Akt signaling pathway. 
VEGF, as a potent stimulator of glycosaminoglycan accumulation and edema formation,45 has been reported to be increased in serum and orbital tissues among patients with active GO, which could reflect the degree of ocular inflammatory activity.46,47 In vitro experiments had confirmed that OPN enhanced the expression of collagen I in hepatic stellate cells to expand liver fibrosis and promoted the expression of VEGF and induce angiogenesis through the PI3K/Akt signaling pathway.15,48 In the present study, we found that OPN activated OFs and induced VEGF and collagen I mRNA expression in OFs through the PI3K/Akt signaling pathway. 
Smoking, as a major risk factor for GO, is related to the occurrence and severity of GO. However, our study was too small to show an effect of smoking, and there was not a positive association of smoking status or cigarette pack years with OPN and other inflammatory cytokines’ mRNA expression levels. 
TRAB plays a unique role in the development of autoimmune hyper- and hypothyroidism.44 In this study, we found that there was no significant difference of the TARB levels between patients with active and inactive GO, and there was no linear correlation between TRAB levels and OPN and other inflammatory cytokines mRNA. Larger sample size experiments are needed to verify the results. 
There were various data in this study that supported OPN's role in the pathogenesis of GO, perhaps not the correlation between OPN mRNA/protein level and CAS, which might also be because of the small sample size. 
There are several limitations to the current study that merit consideration. First of all, the sample size of each group was small. Second, the OFs were only obtained from three patients with active GO. Further studies with larger sample size may be needed in the future. 
Conclusion
Our study has shown that the mRNA and protein levels of OPN in orbital adipose connective tissues, as well as CD40L and collagen I mRNA, increased significantly among patients with active GO. Furthermore, there were significant linear correlations of OPN with CD40L, collagen I, and HAS2 mRNA levels in patients with GO. Additionally, we also found that OPN promoted proliferation and migration of OFs and induced VEGF and collagen I mRNA expression in OFs through the PI3K/Akt signaling pathway. Overall, these findings indicated that OPN might play an important role in the pathogenesis of GO, especially in the active period through the activation of OFs, to promote orbital inflammation and the remodeling and fibrosis in orbital tissue, through the PI3K/Akt signaling pathway. 
Acknowledgments
Supported by the National Natural Science Foundation of China (81170874). 
Disclosure: H. Lou, None; L.-Q. Wu, None; H. Wang, None; R.-L. Wei, None; J.-W. Cheng, None 
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Figure 1.
 
The relative expression (fold change) of OPN RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, ***P < 0.001: statistically significant difference compared with the active GO group.
Figure 1.
 
The relative expression (fold change) of OPN RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, ***P < 0.001: statistically significant difference compared with the active GO group.
Figure 2.
 
Western blot (A) and densitometric analysis (B) (fold change) of the expression of OPN protein in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The Western blot assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05: statistically significant difference compared with the patients with active GO group.
Figure 2.
 
Western blot (A) and densitometric analysis (B) (fold change) of the expression of OPN protein in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The Western blot assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05: statistically significant difference compared with the patients with active GO group.
Figure 3.
 
The relative expression (fold change) of CD40L, collagen I, HAS2, NF-κB, CCL20, and CD44 RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, **P <0.01, ***P < 0.001: statistically significant difference compared with the patients with active GO group. #P < 0.05, ##P <0.001: statistically significant difference compared with the control group.
Figure 3.
 
The relative expression (fold change) of CD40L, collagen I, HAS2, NF-κB, CCL20, and CD44 RNA in orbital adipose connective tissue from patients with active GO, patients with inactive GO and healthy subjects. The RT-PCR assay was performed three times for each sample. All the results were shown as mean ± SD. Statistical analysis has been performed by Kruskal-Wallis H with the Bonferroni's post hoc test (n = 12:12:12). *P < 0.05, **P <0.01, ***P < 0.001: statistically significant difference compared with the patients with active GO group. #P < 0.05, ##P <0.001: statistically significant difference compared with the control group.
Figure 4.
 
Spearman correlation analysis of the mRNA levels between OPN and CD40L (r = 0.80, P < 0.001) (A), OPN and collagen I (r = 0.61, P < 0.01) (B), OPN and HAS2 mRNA (r = 0.73, P < 0.001) (C), in orbital adipose connective tissue from all patients with active and inactive GO (n = 24).
Figure 4.
 
Spearman correlation analysis of the mRNA levels between OPN and CD40L (r = 0.80, P < 0.001) (A), OPN and collagen I (r = 0.61, P < 0.01) (B), OPN and HAS2 mRNA (r = 0.73, P < 0.001) (C), in orbital adipose connective tissue from all patients with active and inactive GO (n = 24).
Figure 5.
 
OFs proliferation rate curve of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time of day 0, day 1, day 2, day 3, and day 4. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.001: statistically significant difference compared with the other four groups. #P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 5.
 
OFs proliferation rate curve of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time of day 0, day 1, day 2, day 3, and day 4. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. *P < 0.001: statistically significant difference compared with the other four groups. #P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 6-1.
 
Representative images of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours.
Figure 6-1.
 
Representative images of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours.
Figure 6-2.
 
Percentage of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ###P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 6-2.
 
Percentage of scratch areas of OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures at time 0 hours, 12 hours, and 24 hours. All the results were shown as mean ± SD (n = 3), which were three separate experiments performed in triplicate. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ###P < 0.001: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 7.
 
The relative expression (fold change) of VEGF mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ##P < 0.01: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 7.
 
The relative expression (fold change) of VEGF mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. ##P < 0.01: statistically significant difference compared with the other three groups except the OPN treatment group.
Figure 8.
 
The relative expression (fold change) of collagen I mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. #P < 0.05: statistically significant difference compared with the control and OPN + LY294002 treatment groups.
Figure 8.
 
The relative expression (fold change) of collagen I mRNA in OFs of control, OPN, OPN + LY294002, OPN + PD980592, and OPN + 1A12 cultures after 48 hours. The cells were harvested after stimulation was completed, and RT-PCR analysis was performed with the total mRNA extracted from the OFs. Values are presented as the mean ± SD of three separate experiments. Statistical analysis has been performed by 1-way ANOVA followed by Tukey's post hoc test. ***P < 0.001: statistically significant difference compared with the other three groups except the OPN + PD980592 treatment group. #P < 0.05: statistically significant difference compared with the control and OPN + LY294002 treatment groups.
Table 1.
 
Clinical Characteristic of the Patients
Table 1.
 
Clinical Characteristic of the Patients
Table 2.
 
All Primers and Probes Sequences
Table 2.
 
All Primers and Probes Sequences
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