Galectin-3: A Potential Biomarker of Rheumatic Diseases

Background: The purpose of our study was to assess the serum galectin-3 level and its potential association with disease activity and severity indexes in patients with rheumatic diseases. Methods: Eighty-two patients with rheumatoid arthritis (RA), 49 patients with systemic sclerosis (SSc), and 18 patients with systemic lupus erythematosus (SLE) were enrolled in this study. The control group comprised 30 healthy controls. Galectin-3 concentration in serum was measured using immunochemical method. Results: The galectin-3 concentration were signi�cantly elevated in the RA, SSc and SLE in comparison to the control group (P=0.000, P=0.000, P<0.001; respectively). But there were no signi�cant differences in the serum galectin-3 levels between rheumatic diseases (H=0.395, P=0.821). In RA and SSc patients, galectin-3 positively correlated with erythrocyte sedimentation rate (R=0.332, P=0.004; R=0.384, P=0.009; respectively). The diagnostic accuracy (ACC) of galectin-3 was high in all rheumatic diseases (87.5% for RA, 83.1% for SSc, 81.1% for SLE). ROC analysis revealed that galectin-3 had an excellent diagnostic power in RA (AUC=0.911) and SSc (AUC=0.903) and very good for SLE (AUC=0.859). Conclusions: We concluded that serum galectin-3 due to the high diagnostic power can be a very good laboratory marker in RA and SSc patients and a useful tool in the diagnosis of SLE.


Introduction
Rheumatoid arthritis (RA), systemic sclerosis (SSc) and systemic lupus erythematosus (SLE) are the most common rheumatic diseases worldwide.All of them are a chronic systemic autoimmune diseases characterized by in ammation and hyperplasia, but the pathogenesis of these diseases is still not fully understood [1][2][3].Previous studies have shown that rheumatic diseases are accompanied by a chronic in ammatory process, which is most often manifested by changes in the concentration of acute phase proteins [4].Consequently, an advanced in ammatory response promotes the development of intense brosis and accumulation of extracellular matrix molecules (e.g.chemokines, cytokines, growth factors, proteins) [5,6].
Galectin-3 is a member of the β-galactoside-binding lectins family [7][8][9].The galectin-3 molecule has a domain structure.There are two structural domains: the N-terminal domain containing the phosphorylation site and the C-terminal domain with the carbohydrate recognition domain -CRD.In human genome this lectin is coded by a single gene LGALS3 situated on chromosome 14.Galectin-3 is predominantly located in the cytoplasm, but it has also been detected in the nucleus of cell, what suggesting a multifunctionality of this protein.Consequently, galectin-3 is involved in many biological processes, such as cell and cell recognition, cell and extracellular matrix adhesion, cell growth and differentiation, cell cycle and signaling, apoptosis and angiogenesis.Besides, galectin-3 is widely expressed protein in human cells and tissues [8][9][10].Expression of galectin-3 has been shown in many types of cells (e.g.: neutrophils, monocytes, macrophages, dendritic cells, mast cells, osteoclasts, broblasts, cancer cells), and what is most importantly -in all types of immune cells.The presence of galectin-3 has been demonstrated also in the tissues of the lungs, spleen, stomach, and also in the heart, kidneys, pancreas and liver.Thus, a signi cant increase of galectin-3 level in the blood serum is observed in many pathological processes which take place in various tissues.Previous studies have already shown that galectin-3 plays an important role in the development of liver or pulmonary brosis [9,11,12].
Taking into account the presence of galectin-3 in immune cells, and the role of immune system in rheumatic diseases the purpose of our study was to assess the serum galectin-3 concentration and its potential association with disease activity and severity indexes in patients with selected rheumatic diseases, including rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus.

Subjects
The study group consisted of 149 patients with rheumatic diseases (124 females and 25 males), aged 19-85 years (median age: 51 years) admitted to the Department of Rheumatology and Internal Diseases, Medical University of Bialystok.The patients were divided into subgroups according to the diagnosis of rheumatic diseases: rheumatoid arthritis (RA) − 82 patients (69 females and 13 males), systemic sclerosis (SSc) − 49 patients (39 females and 10 males), and systemic lupus erythematosus (SLE) − 18 patients (16 females and 2 males).The diagnosis of RA was con rmed according to the ACR 2010 classi cation criteria [13].In these criteria, the diagnosis of RA is based on the presence of synovitis in at least 1 joint, and achievement of a total score of ≥ 6 from the individual scores in 4 domains.RA activity was evaluated by disease activity score (DAS28) calculated by a following complex formula which included the number of tender (t28) and swollen (s28) joints, erythrocyte sedimentation rate (ESR) and visual analog scale (VAS): DAS28 = 0.56 x sqrt(t28) + 0.28 x sqrt(s28) + 0.7 x ln(ESR) + 0.014 x VAS DAS28 in RA patients ranges from 2.32 to 8.0 (median: 6.0).The recognition of systemic sclerosis was made on the ACR/EULAR 2013 classi cation criteria [14].According to them, patients with a total score of ≥ 9 are classi ed as having de nite SSc.In turn, the diagnosis of systemic lupus erythematosus was based on the SLICC 2012 classi cation criteria [15].These require ful llment of at least 4 of the criteria, including at least one clinical and one immunologic criterion.To eliminate of an effect of cardiomyopathy on the level of galectin-3, all patients had to carry out the heart ultrasonography.
The control group consisted of 30 healthy subjects recruited from hospital workers (19 females and 11 males) aged 21-54 years (median age: 25 years).Informed consent was obtained from all individual participants (healthy and sick) included in the study.This study was in accordance with Helsinki declaration and was approved by the Bioethical Committee working at the Medical University of Bialystok (Approval No. R-I-002/416/2018).

Blood sampling
Blood samples from patients with rheumatic diseases and healthy subjects were collected by peripheral vein puncture.The sera were separated by centrifugation at 1500 x g for 10 min at room temperature and stored at -86 °C until assayed.Besides serum, a part of each blood samples was collected into tubes containing liquid sodium citrate for determination of ESR and EDTA-2 for hematological analysis.
Another biochemical assay such as CRP was measured by the immunoturbidimetric method on the Architect ci 8200 analyzer.ESR was assayed by Westergren method on the Sediplus S 2000 (Sarstedt, Germany).Hemoglobin level and PLT were determined by using routine methods on the Sysmex XS-800i analyzer (Sysmex Corporation, Singapore).

Statistical analysis
Statistical analysis was performed using Statistica 13.1 PL (StatSoft, Poland).The results were given as medians and ranges.The differences between study and control groups were evaluated by Mann-Whitney U test.To test the hypothesis about the differences between rheumatic diseases, ANOVA rank Kruskal-Wallis test was done.The correlation between variables was assessed by Spearman's rank correlation coe cient.The P values less than 0.05 were considered as signi cant.To calculate the diagnostic accuracy of galectin-3 in rheumatic diseases the area under the receiver operating characteristic curve (AUROC) was calculated.Diagnostic sensitivity, speci city, accuracy, positive (PPV) and negative predictive values (NPV) were counted using a cut-off point suggested by the ROC analysis.

Results
The demographic and laboratory data of all study groups (patients with rheumatic diseases and healthy subjects) are summarized in the Table 1.The median of serum galectin-3 concentration was signi cantly elevated in RA (18.75 ng/mL), SSc (19.4 ng/mL), and SLE (19.2 ng/mL) in comparison to the healthy subjects (9.45 ng/mL) (Z = 5.710, P = 0.000; Z = 5.323, P = 0.000; Z = 3.596, P < 0.001; respectively).The median levels of ESR, CRP, and PLT in RA, SSc, and SLE were also signi cantly increased compared to the control group (with exception of PLT level in SLE), whereas the median of HGB was decreased in comparison to the controls (Table 1).

Table 1
The demographic and laboratory data of rheumatic patients and control group.Data are median and ranges.The differences between tested groups and controls were estimated by Mann-Whitney U test.The differences between rheumatic subgroups estimated by ANOVA rank Kruskal-Wallis test.Signi cant differences at P < 0.05.* -when comparing tested groups and controls; # -when comparing RA and SSc.
The diagnostic power of galectin-3 in rheumatic diseases (RA, SSc, and SLE) is presented in Table 2 and Fig. 1.The results of this study showed that galectin-3 has a high diagnostic sensitivity in the detection of RA and SSc.In SLE it was low, but is accompanied by 100% speci city.The diagnostic accuracy (ACC) of galectin-3 was high and similar in all rheumatic diseases (over 80%).The positive predictive value (PPV) of was highest in SLE, while the negative predictive value (NPV) of galectin-3 was lowest in RA.ROC analysis revealed that galectin-3 had an excellent diagnostic power in RA and SSc and very good diagnostic power for SLE, because the area under curves (AUCs) were as follows in decreasing order: for RA − 0.911, for SSc − 0.903, and for SLE − 0.859 (Fig. 1).

Discussion
Throughout the past decade, galectin-3 has attracted the attention of researchers due to its regulatory role in immune response, in ammation and brosis [9,16].Thus, several studies already showed that galectin-3 plays an important role in the development of different pathological conditions.Some studies proved that increased galectin-3 concentration is associated with high heart failure risk [17].There are evidences that galectin-3 concentration increases signi cantly, both in chronic and acute heart failure, according to the progression of disease [18,19].Also in pulmonary and liver brosis serum concentration of galectin-3 was found to be elevated [9,11,12,20].Accordingly, mentioned studies proposed that determination of serum galectin-3 concentration could be a useful marker of active brosis in the course of these diseases.Similar studies suggested association of high serum galectin-3 levels with in ammatory disorders and autoimmune diseases, including rheumatic diseases [8,16].The diagnosis of rheumatic conditions, especially in early stages of the disease is still very di cult.Therefore, in recent years, interest in nding useful circulating biomarkers for early diagnosis and which may re ect the progression of rheumatic diseases, does not diminish.
Due to these facts, we try to assess the serum galectin-3 levels, its diagnostic values and potential association with progression of disease in the most common rheumatic diseases.[22,23].Lee et al. showed a higher serum galectin-3 concentration in patients with Behcet's disease than the healthy ones, and that the active BD patients had higher galectin-3 levels than these with the inactive BD.While, the levels of galectin-3 binding protein were not different between BD patients and controls, but it was higher in active BD than in non-active BD [24].The mechanism by which galectin-3 is released into the extracellular space is still not fully understood.There are ndings which suggested that galectin-3 molecule on its own has the capacity to traverse the lipid bilayer [25].Against, galectin-3 has a direct effect on immune system and in ammatory responses by modulating cell adhesion of various type of immune cell.The most probably mechanism is that upon tissue injury, galectin-3 which is normally stored in the cytoplasm, is actively secreted to the blood and other biological uids by activated and damaged cells.Therefore, in ammation and dysfunction of immune response may increase serum galectin-3 concentration in patients with different rheumatic diseases.There is well known that galectin-3 plays an important role in the development of in ammation by interacting with various cytokines and chemokines.Moreover, galectin-3 has been suggested to play a key role in inducing brosis in different tissues [9,11,12].The effects of galectin-3 clearly depend on cellular or tissue localization.
In the most of diseases mentioned above the concentrations of galectin-3 were signi cantly associated with the C-reactive protein (CRP) and disease activity scores.It's suggested that determination of serum galectin-3 may be utilized marker for the diseases prognosis.Although the increased level of galectin-3 is not speci c for example in RA patients, as showed Ohshima et al. [6].Also in our study we did not reported signi cantly correlation between galectin-3 concentration and CRP values, and also there were no correlation with DAS28.On the other hand, we observed positively correlation of galectin-3 with ESR in RA and SSc patients.Moreover, we denoted positive correlation with age of RA patients.
While increased serum levels of galectin-3 have been previously reported in patients with rheumatic diseases, this is probably the rst study investigating the diagnostic values of serum galectin-3 in the diagnosis of selected rheumatic diseases.Our study showed that galectin-3 has the high diagnostic sensitivity (92.1% and 95.6%; respectively), PPV (92.1% and 82.7%; respectively) and diagnostic accuracy (87.5% and 83.1%; respectively) in rheumatoid arthritis and systemic sclerosis patients.For the SLE patients the sensitivity was lower than in RA and SSc.Besides, SLE patients has 100% speci city and PPV values.It is a consequence of the lack of false positive results.Moreover, we showed that galectin-3 had a very high diagnostic power (area under the ROC curve) for all tested rheumatic diseases, making galectin-3 a good diagnostic marker.Diagnostic power was excellent for RA and SSc patients (ACC over 0.9) and very good for SLE patients (ACC over 0.8).

Figures
Figure 1 ROC curves for serum galectin-3 in rheumatic diseases.

Table 2
The diagnostic power of galectin-3 in rheumatic diseases.
RA -rheumatoid arthritis, SSc -systemic sclerosis, SLE -systemic lupus erythematosus, ACCdiagnostic accuracy, PPV -positive predictive value, NPV -negative predictive value, AUC -area under ROC curve, SE -standard error [21][22][23][24]hese study has shown increased serum concentration of galectin-3 in all studied rheumatic diseases: rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus in comparison to the healthy subjects.The results of our study were comparable with those reported in literature.Previous studies documented the changed levels of galectin-3 in patients with RA, juvenile idiopathic arthritis (JIA), SSc, ankylosing spondylitis (AS) or Behcet's diseases (BD) [6,[21][22][23][24].For example,Ohshima etal.has demonstrated increased levels of galectin-3 both in RA synovial uid and serum comparison to patients with osteoarthritis and healthy subjects [6].Similar results obtained Ezzat et al. in JIA patients [21].Also Koca et al. reported higher serum galectin-3 in systemic sclerosis and Cao et al. in patients with ankylosing spondylitis