Abstract
Purpose
This study was designed to investigate the association between fibulin-1(FBLN1) and thyroid-associated ophthalmopathy (TAO).
Method
The plasma FBLN1 levels were measured in 80 participants, including 30 active TAO patients, 25 inactive TAO patients, and 25 Graves disease (GD) patients without TAO using enzyme-linked immunosorbent assay (ELISA).
Results
TAO patients had significantly higher TRAb level than GD patients (p < 0.05). The active TAO patients consumed more tobacco and had higher CAS than inactive TAO patients (all p < 0.05). No significant differences were found in age, sex, and the level of FT3, FT4, and TSH between TAO and GD patients, and between the active and inactive TAO patients (all p > 0.05).
The plasma FBLN1 level in TAO patients was higher than that in GD patients, and that in active patients was higher than that in inactive patients (all p < 0.05). Furthermore, the plasma FBLN1 level showed strong association with clinical activity score (CAS) of TAO (r = 0.67, p < 0.01). By receiver operator characteristic (ROC) curve analysis, FBLN1 demonstrated good efficiency for predicting disease activity at the cut-off value > 625.33 pg/ml with a sensitivity of 93.3% and a specificity of 88.0% (AUC:0.92, p < 0.01).
Conclusion
The plasma FBLN1 levels correlated with TAO activity and a value >625.33 pg/ml was associated with active disease. Our results suggest that the plasma FBLN1 level could be a novel biomarker for predicting disease activity of TAO.
Subject terms: Thyroid diseases, Predictive markers, Eye diseases
Introduction
Thyroid‐associated ophthalmopathy (TAO) is an orbital autoimmune inflammatory disease that occurs in 25% to 50% of Graves disease (GD) patients [1, 2]. Clinical manifestation includes oedema and erythema of the conjunctival and periorbital tissues, retracting eyelids, proptosis, and double vision [3, 4]. Severe TAO patients have a risk of blindness and disability, which occurs in 3–5% of all cases and is so-called sight-threatening TAO [5, 6].
The basic pathophysiological process of TAO includes the infiltration of immune cells in orbital tissues, the proliferation and differentiation of orbital fibroblasts(OFs), and the accumulation of extracellular matrix(ECM) such as glycosaminoglycans (GAG) and hyaluronan [7, 8]. Currently, OFs are thought to be the primary effector cells in TAO, which not only produce lots of ECM resulting in increased volume of orbital contents, but also produce a large amount of proinflammatory cytokines to mediate the recruitment of lymphocytes into orbital tissues [9].
Fibulin-1 (FBLN1) is a glycoprotein secreted mainly by fibroblasts and expressed in the orbital extracellular matrix (ECM) and blood [10, 11]. FBLN1 belongs to the eight-member fibulin protein family and contains tandem repeats of epidermal growth factor-like domains and a C-terminal fibulin-type module [12]. It is involved in many cellular functions, such as adhesion, migration, and differentiation by providing an intramolecular bridge that stabilise the organisation of supramolecular ECM structures [13]. However, the role of FBLN1 in TAO has not been reported.
In one of our proteomics analysis of blood (Supplementary Data), 151 proteins were identified as being deferentially expressed between TAO patients and GD patients. Among them, FBLN1 was higher in TAO patients than GD patients. While, the actual relationship between FBLN1 and TAO remains unclear. Therefore, to validate the proteomic results and clarify the actual association between FBLN1 and TAO, we measured the plasma level of FBLN1 in TAO and GD patients and analysed its association with the activity of TAO.
Methods
Patients
Between Jan 2019 and May 2020, TAO patients who visited the First Affiliated Hospital of Chongqing Medical University were enroled in our study. The inclusion criteria were TAO patients between 18 and 70 years old. Exclusion criteria were as follows: (1) previous treatment with orbital radiotherapy, decompression surgery, glucocorticoid therapy or other immunosuppressive therapy within three months; (2) abnormal T3 and T4 level; (3) incomplete data on ophthalmic assessments and/or important laboratory measurements; 4 co-morbidities including diabetes mellitus, SLE or other autoimmune diseases. Finally, 55 TAO patients, including 30 active and 25 inactive patients, were enroled. 25 age and sex matched GD patients without TAO were recruited (GD patients group).
The study was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Chongqing University and conformed to the Declaration of Helsinki. Written informed consent was obtained from all patients.
The diagnosis and assessment of GD and TAO
The diagnosis of GD was based on the WHO criteria [14]. The diagnosis and assessment of TAO followed the consensus statement of the European Group on Graves Orbitopathy criteria [15]. The activity of TAO was evaluated by the CAS score, which includes seven reference items: chemosis, redness of conjunctiva and eyelid, swelling of the eyelid, swelling of the caruncle, spontaneous ache behind the eyeball, ache on attempted upgaze. One symptom added one point, and the CAS score ≥3 was classified as an active stage.
Laboratory tests
FT3, FT4, and TSH were determined with electro-chemiluminescence method (Unicel DxI 800 Immunoassay System, Beckman Coulter, USA, normal range:FT3 2.01–4.82 pg/ml; FT4 0.59–1.25 ng/dl;TSH 0.560–5.91 μIU/ml). TRAb was examined by chemiluminescence immunoassay (Cobas601, Roche, Germany, normal range: 0.8–1.8 IU/L). The plasma FBLN1 level was measured by FBLN1 ELISA Kit (OKEH00229, the Aviva System Biology, USA, sensitivity: 41 pg/ml).
Statistical analysis
Statistical analysis was performed using the SPSS software package (v. 23.0, IBM, Armonk, NY, USA). Continuous variables with a normal distribution were presented as means ± SDs. Continuous data were compared by Independent-sample t test or Mann–Whitney test, whereas categorical variables were compared with the chi‐square test or Fisher exact test as appropriate. Spearman’s rank correlation test was used to evaluate the correlations between FBLN1 and CAS. Receiver-operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic efficiency of the identified variables for prediction. A P value <0.05 was considered to be significant.
Results
Clinical and biochemical characteristics of TAO patients and GD patients
As shown in Table 1, TAO patients had significantly higher TRAb levels than GD patients (p < 0.05). The active TAO patients consumed more tobacco and had higher CAS than the inactive TAO patients (all p < 0.05). No significant differences were found in age, sex, and the level of FT3, FT4, and TSH between TAO and GD patients, also between the active and inactive patients (all p > 0.05).
Table 1.
Basic characteristics of the TAO patients and GD patients.
| Variable | TAO | GD (n = 25) |
P value | ||
|---|---|---|---|---|---|
| Active TAO (n = 30) |
Inactive TAO (n = 25) |
TAO vs. GD | Active vs. Inactive | ||
| Age, y | 49.53 ± 1.87 | 45.48 ± 3.04 | 45.24 ± 3.16 | 0.49* | 0.35* |
| Female gender, n (%) | 19 (63.33) | 15 (60.00) | 15 (60.00) | 0.62# | 0.80# |
| Smoke, n (%) | 9 (30.00) | 1 (4.00) | 1 (4.00) | 0.18# | 0.03# |
| FT3 | 4.11 ± 0.65 | 3.95 ± 0.21 | 3.35 ± 0.67 | 0.24※ | 0.08※ |
| FT4 | 1.16 ± 0.20 | 1.14 ± 0.11 | 0.84 ± 0.08 | 0.59※ | 0.22※ |
| TSH | 2.75 ± 1.15 | 2.36 ± 1.16 | 2.14 ± 0.46 | 0.32※ | 0.29※ |
| TRAb | 13.56 ± 4.05 | 13.77 ± 2.33 | 3.67 ± 1.64 | 0.01※ | 0.65※ |
| CAS | 3.93 ± 0.19 | 1.44 ± 0.10 | NA | NA | <0.01※ |
GD Grave’s disease patients without ophthalmopathy, FT3 Free triiodothyronine, FT4 Free thyroxine, TSH Thyroid stimulating hormone, TRAb Thyrotrophin receptor antibody, NA no available.
*P compared by Independent-sample t test; ※P compared by Mann–Whitney test; #P compared by chi‐square test or Fisher exact test. Significant at p < 0.05.
The plasma Fibulin-1 level in TAO patients and GD patients
As shown in Table 2, the plasma FBLN1 level was significantly higher in TAO patients than GD patients, and higher in the active TAO patients than inactive TAO patients (all p < 0.05).
Table 2.
FBLN1 level in the TAO patients and GD patients.
| Variable | TAO | GD (n = 25) |
P value | ||
|---|---|---|---|---|---|
| Active TAO (n = 30) |
Inactive TAO (n = 25) |
TAO vs. GD | Active vs. Inactive | ||
| FBLN1 level, pg/ml | 1885.42 ± 154.82 | 346.37 ± 112.60 | 129.70 ± 11.25 | <0.01 | <0.01 |
GD Grave’s disease patients without ophthalmopathy, FBLN1 Fibulin-1, P compared by Mann–Whitney test. Significant at p < 0.05.
The plasma FBLN1 level and the CAS of TAO
The association between FBLN1 and CAS was analysed in Table 3. The plasma FBLN1 level was positively correlated with CAS (r = 0.67, p < 0.01).
Table 3.
The relationship between FBLN1 and CAS scores.
| CAS score | |
|---|---|
| Correlation coefficient | 0.67 |
| P value | <0.01 |
P compared by Spearman correlation analysis. Significant at p < 0.05.
Receiver-operating characteristic curve analysis
To define the optimal cut-off value for the active stage, we then performed receiver operator characteristic (ROC) curve analysis. As shown in Table 4, FBLN1 could discriminate the active TAO from inactive TAO at a cut-off value of 625.33 pg/ml with a sensitivity of 93.3% and a specificity of 88.0% (AUC:0.92, p < 0.01).
Table 4.
ROC curve analysis of active TAO.
| Variable | AUC | P value | Sensitivity | Specificity |
|---|---|---|---|---|
| FBLN1 = 625.53 pg/ml | 0.92 | <0.01 | 0.93 | 0.88 |
P calculated by Receiver-operating characteristic (ROC) analysis. Significant at p < 0.05
Discussion
FBLN1 is an ECM protein that plays an essential role in the maintenance of tissue structure. The previous studies on FBLN1 mainly focus on tumour and tissue development [13]. To date, it is not clear about the association between FBLN1 and TAO. Our study demonstrated that the plasma FBLN1 level correlated positively with the CAS in TAO, which suggests that the plasma FBLN1 level could be a novel biomarker for predicting disease activity of TAO. To our best knowledge, it is the first study to show the association between FBLN1 and TAO.
Firstly, our study showed a higher plasma FBLN1 level in TAO patients than GD patients, which may originate from activated orbital fibroblasts [16, 17]. Of course, further studies are needed to confirm this hypothesis.
Next, the plasma FBLN1 level in TAO patients was analysed. Our results demonstrated that the plasma FBLN1 level correlated positively with the CAS of TAO. Previous studies have shown that FBLN1 levels were closely correlated with tissue remodelling and fibrosis through activation of the TGF-b signalling pathway [17, 18], processes seen in inactive rather than active TAO. It is possible that fibroblasts response is variable in different diseases. In TAO, OFs are activated by TRAb and inflammatory cell, then produce FBLN1 and numerous cytokines [19],which induce further inflammatory cell infiltration. The degree of inflammation appears to correlate with OF activation and FBLN1 levels. The study in hepatocellular carcinoma also showed that up-regulated FBLN1 expression was associated with increased immune cell infiltration [20]. In some inflammatory diseases, such as asthma, serum and airway FBLN1 levels were increased [18, 21]. However, it is not clear whether the relationship between FBLN1 and inflammation is direct or indirect, and further cytological studies are needed. This study showed that a FBLN1 level of >625.33 pg/ml was diagnostic for active TAO with a sensitivity of 93.3% and specificity of 88%.
There are some study limitations. Although CAS is a commonly used instrument to measure TAO activity, it is subjective and imprecise. Further studies comparing FBLN1 level with multiple measures of disease activity may help validate our results. In addition, the normal function of FBLN1 and its role in TAO remains unknown.
Conclusions
The plasma FBLN1 levels correlated with TAO activity and a value > 625.33 pg/ml was associated with active disease. Our results suggest that the plasma FBLN1 level could be a novel biomarker for predicting disease activity of TAO.
Summary
What was known before
Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune inflammatory disease; while the role of FBLN1 in TAO has not been reported.
What this study adds
In the present study we found that the plasma FBLN1 level was significantly higher in TAO patients than in GD patients, especially in active TAO patients. The present study suggests that FBLN1 may be involved in the pathogenesis of TAO and can serve as a novel biomarker for the disease activity.
Supplementary information
Heatmap of the log2 Fold Change (logFC) of differential expression proteins in the plasma of TAO and GD group, through Tandem mass tag proteomics(TMT).
Author contributions
JL and CL contributed to the conception of the study. HH, XJ, and ZF performed the experiment. HH and JL performed the data analyses and wrote the paper. LL and XZ contributed to the diagnosis and assessment of TAO.
Funding
This study is supported by Key projects of Chongqing Natural Science Foundation (NO:cstc2020jcyj-zdxmX0016).
Data availability
The data that support the findings of this study are available from the corresponding author, upon reasonable request.
Competing interests
The authors declare no competing interests.
Ethics approval
The research was conducted ethically following the World Medical Association Declaration of Helsinki. The study was approved by the First Affiliated Hospital of Chongqing Medical University Ethical Committee.
Footnotes
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
The online version contains supplementary material available at 10.1038/s41433-022-02318-6.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Heatmap of the log2 Fold Change (logFC) of differential expression proteins in the plasma of TAO and GD group, through Tandem mass tag proteomics(TMT).
Data Availability Statement
The data that support the findings of this study are available from the corresponding author, upon reasonable request.