SLE is a rheumatic immune disease with heterogeneous clinical symptoms that results from endothelial cell activation and immune disorders [25]. Endothelial cells can cause proliferative vasculopathy by regulating vascular tension, immune and coagulation systems, thereby contributing to autoimmune disease pathogenesis. When endothelial cells are activated under inflammatory stimulation, expression of surface adhesion molecules was increased, which promotes migration and accumulation of leukocytes to endothelial cells. Then, this induces vascular obstruction and tissue hypoxia, leading to apoptosis and tissue fibrosis [26]. Hence, measuring biomarkers related to endothelial cell activation is important for diagnosis of SLE. Endothelium-derived ET-1 plays a pathogenic role in connective tissue disease, pulmonary hypertension, and cancer by affecting angiogenesis, inflammation, and fibrosis [27]. In osteoarthritis, ET-1 induces an increase of IL-18 through the ET-1/ETAR axis and PI3K-dependent manner, promoting osteoblast proliferation and exacerbating the disease [28]. Previous study showed that compared with control group, increased ET-1 levels were observed in RA patients, particularly with kidney and cardiovascular system damage [29]. Moreover, ET-1 expression is elevated in other autoimmune diseases such as SSc, psoriasis and type 1 diabetes [30, 31]. According to our study, serum ET-1 levels were significantly higher in SLE patients in training cohort when compared with healthy controls, which was similar to previous findings [8, 32, 33]. Moreover, we found that serum ET-1 levels were associated with clinical symptoms of cylindruria, alopecia and laboratory indice (anti-Sm antibody). Similarly, Yoshio et al. proposed that serum ET-1 in SLE patients correlated with IgM antibody expression [33]. A study with small sample size suggested that serum ET-1 was higher in active SLE patients than in inactive SLE patients and controls, and ET-1 concentrations were higher in patients with visceral manifestation [32]. Consistently, we used a two-stage case-control study with a large sample size to confirm a positive correlation between serum ET-1 concentrations and SLEDAI score or disease activity, and that patients with active SLE had higher ET-1 levels. Urinary ET-1 may be a useful measurement of renal inflammatory activity and may serve as a marker of lupus nephritis disease activity [34]. In the present study, we also explored the potential of serum ET-1 as a biomarker for SLE. In training cohort, AUC of ET-1 was 0.912 (95% CI: 0.866–0.959), indicating that serum ET-1 could distinguish SLE patients from healthy subjects. In the validation cohort, ET-1 concentrations were higher in SLE patients than in other rheumatic diseases, including RA, OA, SS, AS, SSc. Compared with non-SLE diseases, the AUC of serum ET-1 in SLE was higher than or close to 0.900, and the AUC of SLE and OA patients was 0.979. Therefore, serum ET-1 has good discriminatory ability for SLE patients and is a promising marker for SLE disease.
CCN3, as a pro-angiogenic factor and fibrosis inhibitor, involves in numerous autoimmune diseases [14]. Serum CCN3 concentrations were elevated in RA patients and positively correlated with expression of IL-6 [35]. CCN3 expression was increased in SSc, multiple sclerosis patients [36, 37]. For osteoarthritis, CCN3 could inhibit PI3K/AKT/mTOR pathway by reducing HMGB1 levels and decrease extracellular matrix catabolism [38]. In our study, we found that serum CCN3 levels were significantly higher in SLE patients compared to normal subjects and were associated with thrombocytopenia. In addition, we observed a positive correlation between ET-1 levels and CCN3 levels in SLE patients. CCN3 inhibits expression of vascular adhesion molecules and reduces monocytes adhesion. CCN3 negatively regulates activation of NF-κB pathway, affecting endothelial cell inflammation and cardiovascular homeostasis [39]. Type III interferons (IFNs) and type I IFNs may promote THP-1 cell differentiation, which contributes to follicular B cell activation and participates in the pathogenesis of autoimmune diseases [40]. IL-28B, known as IFN-λ3, belongs to a subtype of type III IFNs. IL-28B regulates innate and adaptive immune responses. SSc patients with pulmonary fibrosis have higher IL-28B serum levels and IL-28B gene polymorphism (rs12979860) is associated with risk of pulmonary fibrosis in a Caucasian population with SSc [41]. With respect to SLE, IL-28B SNPs (rs8099917, rs12979860) are risk factors for lupus nephritis in Taiwanese. Moreover, serum concentrations of IL-28B were elevated in SLE patients compared to healthy controls, which were related to complement expression and SLE disease activity [42]. IL-28B expression is associated with lupus disease activity, such as skin involvement [18]. According to our study, IL-28B levels in SLE patients were positively associated with disease activity and ESR levels. Lupus patients with hematuria, proteinuria, cylindruria showed higher levels of IL-28B, which are typical clinical manifestations of lupus. We analyzed correlation between ET-1 and IL-28B levels and observed that expression of ET-1 was positively correlated with IL-28B expression. The above data suggest that ET-1 is associated with both CCN3 and IL-28B. High expression of ET-1 may regulate the expression of IL-28B, CCN3 and then contribute to the pathogenesis of lupus. However, more functional studies are needed to reveal the mechanism of ET-1 regulation of CCN3 and IL-28B.
It is now accepted that SNP as a new genetic marker can be used for discovery of high-risk patients. ET-1 gene polymorphisms are widely discussed in vascular-related diseases and cancer, such as hypertension, coronary atherosclerosis, and papillary thyroid cancer [43, 44]. However, ET-1 gene polymorphisms have been less studied in autoimmune diseases. In this study, we explored the relationship between ET-1 gene polymorphisms and SLE risk through a case-control study in a Chinese Han population. We found that genotypes of rs5370 (TG,TT + TG), rs1476046 (GG), rs2070699 (TT,TG), rs2071942 (GA), rs2071943 (GG), rs3087459 (CA + AA), rs6458155 (TC) and allele of rs2070699 (T) were associated with SLE susceptibility. For rs5370, TG, TT + TG genotype frequencies were higher in SLE patients, suggesting that rs5370 polymorphism may increase the risk of SLE in Chinese Han population. The GG genotype frequency of rs2071942 was increased in SLE patients compared to healthy subjects and was positively associated with SLE risk. Mantaka et al. investigated the association of ET-1 rs2071942 and rs5370 polymorphisms with primary biliary cirrhosis (PBC). The genotypes and alleles distribution of both loci were not significantly different from controls and PBC patients, but rs2071942 allele A and rs5370 allele T were associated with stage of disease progression [45]. This inconsistency may be due to differences in sample size, ethnicity, disease type, and duration. Similarly, in RA patients, frequencies of the genotypes and alleles of rs5370 and rs18000541 were not significantly different from those and healthy controls, but TT genotype of rs5370, rs18000541 was related to RA patients complicated with hypertension, indicating that rs5370 and rs18000541 loci were associated with cardiovascular risk in RA [46]. In our findings, rs5370, rs1476046, rs2070699, rs2071942, rs2071943 rs5369, rs3087459, and rs9369217 polymorphisms correlated with some clinical features and laboratory manifestations in SLE patients. Patients carrying rs2070699 T allele and TT genotype were more likely to develop symptoms of alopecia. Regarding rs2071942 and rs2071943, allele G was associated with pericarditis and pyuria symptoms, and genotype GA was related to fever. Moreover, significant correlation between rs5370 T allele, rs1476046 G allele and pericarditis, pyuria in SLE cases was observed. Rs3087459 (CC) and rs9369217 (TC) were associated with anti-SSB laboratory indicator. Rs5369 AA genotype correlated with IgG and CRP levels, suggesting that mutations at the rs5369 locus may affect the expression of these disease markers in SLE patients. Indeed, CRP is an acute phase protein produced by hepatocytes in response to inflammation and has important pathogenic significance in active lupus nephritis. IgG immune complexes are deposited in the spleen, causing damage to the immune barrier of the spleen, and then a large number of antibodies are produced [47]. Notably, one study proposed that in graves’ disease (GD), ET-1 gene polymorphisms (rs5370 and rs1800541) were not associated with disease susceptibility, but were associated with autoantibody production in GD patients [48]. To the best of our knowledge, this study is the first to discuss the relationship between ET-1 gene polymorphisms and lupus, which may offer new insights and basis for further discussion of ET-1 genetic mutation and SLE in the future.
Nevertheless, our study has several limitations. First, we measured cross-sectional serum ET-1 levels in a relatively limited number of samples. Therefore, a large number of multicenter clinical samples and longitudinal data are warranted to confirm serum ET-1 as a disease marker of SLE. Second, the related mechanism of ET-1 regulating CCN3 and IL-28B and then affecting the pathogenesis of SLE remains to be discussed.
In conclusion, high expression of ET-1 is associated with pathogenesis of SLE and may be a potential disease biomarker. ET-1 gene polymorphisms were related to SLE susceptibility in Chinese Han population.