Abstract
Objective
Previous studies by our research group demonstrated that interstitial lung disease (ILD) associated with idiopathic inflammatory myopathy (IIM) correlates with endothelial cell injury and neutrophil extracellular trap (NETs) formation. This study aimed to investigate the clinical relevance of serum endothelin‐1 (ET‐1) and NETs markers, such as cell‐free DNA (cfDNA), in IIM‐associated ILD (IIM‐ILD).
Methods
Peripheral blood samples were collected from 60 patients with IIM (44 with dermatomyositis and 16 with polymyositis) and 20 healthy controls. Among the patients with IIM, 33 were diagnosed with ILD. Serum ET‐1 and cfDNA levels were measured, and differences in ET‐1 levels across groups, correlations between ET‐1 and cfDNA, and diagnostic performance via logistic regression models and receiver operating characteristic (ROC) curves were analyzed.
Results
Serum ET‐1 levels were significantly elevated in patients with IIM compared to healthy controls (10.89 ± 3.49 pg/mL vs 2.09 ± 0.56 pg/mL, P < 0.0001). The subgroup with IIM‐ILD exhibited a 36.3% increase in ET‐1 levels compared to the subgroup with IIM without ILD (12.17 ± 3.67 pg/mL vs 9.34 ± 2.53 pg/mL, P = 0.0012), with a positive correlation between ET‐1 and cfDNA (r = 0.374, P = 0.003). Multivariate analysis identified ET‐1 as an independent risk factor for ILD (odds ratio 1.442, 95% confidence interval 1.082–1.923). A combined model incorporating ET‐1, albumin–globulin ratio, lactate dehydrogenase, and complement C4 achieved an area under the ROC curve of 0.888 (sensitivity 78.8% and specificity 88.9%).
Conclusion
Serum ET‐1 levels were elevated in patients with IIM, particularly in those with ILD, suggesting its potential role in the pathogenesis and progression of IIM‐ILD.
INTRODUCTION
Idiopathic inflammatory myopathy (IIM) is a group of autoimmune diseases characterized primarily by inflammatory cell infiltration in skeletal muscles and necrosis of muscle fibers. Subtypes of IIM include dermatomyositis (DM), polymyositis (PM), and others. 1 , 2 Epidemiologic studies have shown that approximately 40% to 70% of patients with IIM develop comorbid interstitial lung disease (ILD), termed IIM‐associated ILD (IIM‐ILD). Characterized by the inflammation of the alveolar walls and progressive pulmonary fibrosis, IIM‐ILD has emerged as the leading cause of respiratory failure and death in affected individuals. 3 , 4 Currently, there is a lack of specific biomarkers in clinical practice, leading to difficulties in early diagnosis. Moreover, existing treatments exhibit limited efficacy in reversing pulmonary fibrosis. Therefore, investigating the pathogenesis of IIM‐ILD and identifying serum biomarkers hold significant clinical value.
SIGNIFICANCE & INNOVATION.
Serum endothelin‐1 (ET‐1) levels are significantly higher in patients with idiopathic inflammatory myopathy (IIM), particularly those with interstitial lung disease (ILD), suggesting its potential role in disease pathogenesis and progression.
A positive correlation between serum ET‐1 and cell‐free DNA levels highlights a synergistic mechanism linking neutrophil extracellular trap–mediated endothelial injury to fibrotic processes in IIM‐associated ILD (IIM‐ILD).
A combined biomarker panel (ET‐1, albumin‐to‐globulin ratio, lactate dehydrogenase, and complement C4) demonstrates superior diagnostic accuracy for IIM‐ILD (area under the receiver operating characteristic curve 0.888), outperforming individual parameters.
Multivariate analysis identifies serum ET‐1 as an independent risk factor for ILD in patients with IIM, emphasizing its clinical utility for early detection and risk stratification.
Endothelin‐1 (ET‐1), a potent vasoactive peptide composed of 21 amino acids, is widely expressed in endothelial cells, epithelial cells, and immune cells. It exerts strong vasoconstrictive effects and is critically involved in pathophysiologic processes such as inflammatory responses, cellular proliferation, and tissue fibrosis. 5 In addition to regulating vascular tone, ET‐1 activates ETA or ETB receptors to promote fibroblast proliferation, enhances the activity of the transforming growth factor β (TGFβ) signaling pathway, and mediates epithelial–mesenchymal transition (EMT), thereby playing a pivotal role in the progression of tissue fibrosis. 6 Recent studies have revealed aberrant expression of ET‐1 in multiple autoimmune diseases. 7 , 8 These findings suggest its potential diagnostic value in inflammatory diseases; however, the role of ET‐1 in IIM‐ILD remains incompletely elucidated.
Previous studies by our research group 9 , 10 , 11 , 12 , 13 have demonstrated that neutrophil extracellular traps (NETs) contribute to the pathogenesis of IIM‐ILD by modulating fibroblast phenotypic transformation, inducing endothelial cell pyroptosis, releasing substantial cytokines, and activating the inflammasome. As a major source of cell‐free DNA (cfDNA), NETs not only promote collagen deposition via the TLR9–miR‐7–Smad2 pathway 9 but also induce alveolar EMT by activating the cGAS–STING signaling axis. 12 Notably, TLR9 can promote ET‐1 synthesis via mediating endothelial injury. 14 , 15 However, the interplay between cfDNA and ET‐1 in IIM‐ILD, as well as their clinical implications, remains to be further elucidated. This study aimed to investigate the correlation between serum ET‐1 and cfDNA levels and clinical characteristics in patients with IIM. By comparing the expression differences of ET‐1 and cfDNA between patients with IIM‐ILD and IIM without ILD (IIM‐non‐ILD), and integrating logistic regression and receiver operating characteristic (ROC) curve analyses, we seek to evaluate the predictive value of ET‐1 for IIM‐ILD.
MATERIALS AND METHODS
Study participants
A total of 60 patients with IIM, including 44 with DM and 16 with PM, were randomly enrolled from the department of rheumatology, Lanzhou University Second Hospital between January 2022 and November 2024. These patients were further divided into two subgroups: IIM‐ILD (n = 33) and IIM‐non‐ILD (n = 27). The diagnosis of IIM was based on the 2017 EULAR/American College of Rheumatology classification criteria for adult and juvenile IIM. 16 ILD was defined according to the 2013 American Thoracic Society/European Respiratory Society updated classification criteria for interstitial pneumonia. 17 Computed tomography (CT) scores for patients with IIM‐ILD were evaluated using the Ichikado scoring system. 18 Additionally, 20 healthy individuals undergoing routine physical examinations during the same period were selected as controls. This study was approved by the ethics committee of Lanzhou University Second Hospital (approval 2020A‐017), and written informed consent was obtained from all participants.
Measurement methods
Fasting venous blood samples (5 mL) were collected from all participants in the morning. After standing for 30 minutes, the samples were centrifuged at 3,000 revolutions per minute for 10 minutes to separate serum, which was then stored at −80°C until analysis. Serum ET‐1 levels were measured using a commercially available enzyme‐linked immunosorbent assay kit (Shanghai Jianglai Biology Co, Ltd., catalog JL19687‐96T) following the manufacturer's instructions. Serum cfDNA levels were quantified using the Quant‐iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific Inc., catalog P11496) according to the manufacturer's protocol.
Clinical data collection
Demographic information (age, sex, and disease duration), laboratory parameters (erythrocyte sedimentation rate, immunoglobulins, complement levels, neutrophil count, blood glucose, etc), and radiologic findings (eg, high‐resolution CT of the chest) were collected from hospital records.
Statistical analysis
Data were analyzed using IBM SPSS Statistics 30.0 and GraphPad Prism 10.1.2 software. Continuous variables are expressed as mean ± SD. Intergroup comparisons were performed using independent Student's t‐test. Pearson and Spearman correlation analyses were applied to assess associations between variables. Linear regression and binary logistic regression models were employed to identify factors influencing ET‐1 levels and ILD occurrence in patients with IIM. Two‐tailed P <0.05 was considered statistically significant. Data supporting the findings of this study are available from the corresponding author upon reasonable request.
RESULTS
Clinical characteristics of the study cohort
Among the 60 patients with IIM enrolled, 44 were diagnosed with DM and 16 with PM. A total of 33 patients with IIM were complicated with ILD, whereas 27 had no ILD. The IIM group comprised 18 male and 42 female participants, with comparable baseline characteristics across subgroups (Table 1).
Table 1.
Clinical characteristics of the study cohort, including patients with IIM, DM, and PM*
| Characteristic | IIM (n = 60), mean ± SD | DM (n = 44), mean ± SD | PM (n = 16), mean ± SD |
|---|---|---|---|
| Male/female | 18/42 | 13/31 | 5/11 |
| Age, y | 44.97 ± 14.06 | 45.59 ± 14.8 | 43.25 ± 12.07 |
| Infection‐related indicators | |||
| PCT, ng/mL | 0.09 ± 0.15 | 0.07 ± 0.07 | 0.14 ± 0.29 |
| IL‐6, pg/mL | 18.76 ± 48.13 | 20.44 ± 54.16 | 14.14 ± 26.51 |
| Autoantibody spectrum | |||
| ANA(+/−) | 43/17 | 30/14 | 13/3 |
| nPNP/Sm(++/+/±/−) | 2/3/0/55 | 2/2/0/40 | 0/1/0/15 |
| Sm(++/+/±/−) | 1/0/2/57 | 1/0/1/42 | 0/0/1/15 |
| SSA(++/+/±/−) | 3/3/1/53 | 2/1/1/40 | 1/2/0/13 |
| Ro‐52(++/+/±/−) | 12/6/4/38 | 9/3/3/29 | 3/3/1/9 |
| SSB(++/+/±/−) | 0/1/0/59 | 0/1/0/43 | 0/0/0/16 |
| Scl‐70(++/+/±/−) | 1/0/0/59 | 1/0/0/43 | 0/0/0/16 |
| PM‐scl(++/+/±/−) | 0/1/0/59 | 0/1/0/43 | 0/0/0/16 |
| Jo‐1(++/+/±/−) | 4/0/0/56 | 1/0/0/43 | 3/0/0/13 |
| CENP B(++/+/±/−) | 1/0/0/59 | 1/0/0/43 | 0/0/0/16 |
| PCNA(++/+/±/−) | 0/0/1/59 | 0/0/1/43 | 0/0/0/16 |
| AMA‐M2(++/+/±/−) | 3/2/1/54 | 1/1/0/42 | 2/1/1/12 |
| Rib.P(++/+/±/−) | 0/0/2/58 | 0/0/1/43 | 0/0/1/15 |
| Ig and complement‐related indicators | |||
| IgA, g/L | 2.55 ± 1.2 | 2.51 ± 1.15 | 2.65 ± 1.38 |
| IgG, g/L | 12.5 ± 3.96 | 12.45 ± 3.77 | 12.66 ± 4.57 |
| IgM, g/L | 1.34 ± 0.76 | 1.38 ± 0.82 | 1.23 ± 0.55 |
| Complement C3, g/L | 1.21 ± 0.25 | 1.19 ± 0.23 | 1.26 ± 0.29 |
| Complement C4, g/L | 0.33 ± 0.1 | 0.33 ± 0.1 | 0.32 ± 0.11 |
| Blood cell‐related indicators | |||
| WBC count, 1 × 109/L | 7.98 ± 3.31 | 7.93 ± 3.44 | 8.11 ± 3.01 |
| NE count, 1 × 109/L | 5.71 ± 2.83 | 5.75 ± 3.05 | 5.59 ± 2.18 |
| LY count, 1 × 109/L | 1.55 ± 0.86 | 1.44 ± 0.73 | 1.86 ± 1.12 |
| MO count, 1 × 109/L | 0.59 ± 0.27 | 0.61 ± 0.26 | 0.52 ± 0.28 |
| RBC count, 1 × 1012/L | 4.56 ± 0.59 | 4.5 ± 0.6 | 4.72 ± 0.53 |
| HGB, g/L | 134.57 ± 21.15 | 132.59 ± 21.26 | 140 ± 20.51 |
| PLT, 1 × 109/L | 258.6 ± 89.78 | 255.14 ± 96.98 | 268.13 ± 67.94 |
| Inflammatory and acute‐phase response indicators | |||
| ESR, mm/hr | 19.8 ± 18 | 21.52 ± 19.68 | 15.06 ± 11.44 |
| CRP, mg/L | 7.61 ± 11.8 | 7.07 ± 10.05 | 9.03 ± 15.8 |
| SF, ng/mL | 958.37 ± 2,024.18 | 1,217.26 ± 2,308.45 | 218.7 ± 135.45 |
| Indicators related to myocardial and muscle enzymes | |||
| CK, U/L | 1,507.85 ± 3,275.67 | 1,121.16 ± 3,447.8 | 2,547.06 ± 2,573.78 |
| CK‐MB, U/L | 79.4 ± 157.65 | 64.48 ± 149.53 | 120.44 ± 176.66 |
| LDH, U/L | 494.05 ± 367.45 | 466.75 ± 377.63 | 569.13 ± 337.77 |
| Liver function‐related indicators | |||
| ALT, U/L | 65.67 ± 80.86 | 65.91 ± 91.18 | 65 ± 43.43 |
| AST, U/L | 90.9 ± 135.22 | 92.98 ± 155.16 | 85.19 ± 53.51 |
| ALP, U/L | 82.08 ± 39.96 | 86.09 ± 43.34 | 71.06 ± 26.82 |
| γ‐GT, U/L | 67.63 ± 95.67 | 74.14 ± 102.91 | 49.75 ± 71.95 |
| TP, g/L | 68.86 ± 7.77 | 67.69 ± 7.25 | 72.08 ± 8.48 |
| ALB, g/L | 37.88 ± 5.16 | 37.12 ± 4.5 | 39.99 ± 6.35 |
| GLO, g/L | 30.98 ± 5.99 | 30.57 ± 5.8 | 32.1 ± 6.53 |
| A/G | 1.27 ± 0.29 | 1.26 ± 0.29 | 1.29 ± 0.31 |
| TBIL, μmol/L | 12.7 ± 5.5 | 12.51 ± 5.25 | 13.23 ± 6.28 |
| DBIL, μmol/L | 2.45 ± 1.32 | 2.63 ± 1.24 | 1.96 ± 1.43 |
| IBIL, μmol/L | 10.24 ± 5.02 | 9.86 ± 4.78 | 11.27 ± 5.67 |
| Indicators related to renal function and metabolism | |||
| Urea, mmol/L | 5.4 ± 1.65 | 5.25 ± 1.47 | 5.82 ± 2.06 |
| Crea, μmol/L | 49.64 ± 17.86 | 50.86 ± 15.75 | 46.28 ± 22.98 |
| UA, μmol/L | 322.71 ± 111.97 | 320.03 ± 114.53 | 330.08 ± 107.86 |
| GLU, mmol/L | 5.52 ± 3.02 | 5.69 ± 3.42 | 5.07 ± 1.44 |
Patients were compared to 20 healthy controls (7 male and 13 female; age ± SD, 41.95 ± 10.42 years). γ‐GT, γ‐glutamyl transpeptidase; ++/+/±/−, strong positive or positive or weak positive or negative; A/G, albumin‐to‐globulin ratio; ALB, albumin; ALT, alanine aminotransferase; ALP, alkaline phosphatase; AMA‐M2, antimitochondrial antibody M2 type; ANA, antinuclear antibody; AST, aspartate aminotransferase; CENP B, anticentromere protein B antibody; CK, creatine kinase; CK‐MB, creatine kinase–MB isoenzyme; Crea, creatinine; CRP, C‐reactive protein; DBIL, direct bilirubin; DM, dermatomyositis; ESR, erythrocyte sedimentation rate; GLO, globulin; GLU, glucose; HGB, hemoglobin; IBIL, indirect bilirubin; IIM, idiopathic inflammatory myopathy; IL‐6, interleukin‐6; Jo‐1, anti–Jo‐1 antibody; LDH, lactate dehydrogenase; LY, lymphocyte; MO, monocyte; NE, neutrophil; nPNP/Sm, anti‐nPNP/Sm antibody; PCNA, anti–proliferating cell nuclear antigen antibody; PCT, procalcitonin; PLT, platelet count; PM, polymyositis; PM‐scl, anti–PM‐Scl antibody; RBC, red blood cell; Rib.P, anti–ribosomal P protein antibody; Ro‐52, anti–Ro‐52 antibody; Scl‐70, anti–Scl‐70 antibody; SF, serum ferritin; Sm, anti‐Sm antibody; SSA, anti‐SSA antibody; SSB, anti‐SSB antibody; TBIL, total bilirubin; TP, total protein; UA, uric acid; WBC, white blood cell.
Elevated serum ET‐1 and cfDNA levels in patients with IIM
Serum ET‐1 levels in patients with IIM were significantly elevated compared to healthy controls (10.89 ± 3.49 pg/mL vs 2.09 ± 0.56 pg/mL, P < 0.0001), representing a 5.2‐fold increase (Figure 1A). Similarly, serum cfDNA levels were markedly higher in patients with IIM (252.4 ± 67.06 ng/mL) than in controls (145.0 ± 25.89 ng/mL, P < 0.0001; Figure 1C). Subgroup analysis revealed no significant differences in ET‐1 or cfDNA levels between patients with DM (ET‐1 10.71 ± 3.33 pg/mL; cfDNA 252.1 ± 67.54 ng/mL) and PM (ET‐1 11.41 ± 3.97 pg/mL; cfDNA 253.3 ± 67.90 ng/mL; P > 0.05 for both comparisons; Figure 1B and D). Pearson correlation analysis revealed a significant positive correlation between serum ET‐1 levels and corresponding CT scores in patients with ILD (r = 0.7677, P < 0.0001; Figure 1E), as well as between serum ET‐1 and cfDNA levels (r = 0.3744, P = 0.0032; Figure 1F).
Figure 1.
(A) Serum endothelin‐1 (ET‐1) levels were significantly elevated in patients with idiopathic inflammatory myopathy (IIM) compared to healthy controls (HC). (B) No significant differences in serum ET‐1 levels were observed between patients with dermatomyositis (DM) and polymyositis (PM). (C) Serum cell‐free DNA (cfDNA) levels were markedly higher in patients with IIM than in HCs. (D) No significant differences in serum cfDNA levels were observed between patients with DM and PM. (E) Serum ET‐1 levels showed a significant positive correlation with corresponding computed tomography (CT) scores in patients with IIM‐associated interstitial lung disease. (F) A significant positive correlation was observed between serum ET‐1 and cfDNA levels in patients with IIM. Bar graphs represent mean ± SD. Statistical significance was defined as P < 0.05.
Significantly elevated serum ET‐1 levels in patients with IIM‐ILD compared to patients with IIM‐non‐ILD
Patients with IIM‐ILD exhibited significantly elevated serum ET‐1 levels compared to patients with IIM‐non‐ILD (12.17 ± 3.67 pg/mL vs 9.34 ± 2.53 pg/mL, P = 0.0012), representing a 36.3% increase (Figure 2A). Stratified analysis by IIM subtypes showed that patients with DM‐ILD had higher ET‐1 levels than patients with DM‐non‐ILD (11.94 ± 3.46 pg/mL vs 8.92 ± 2.16 pg/mL, P = 0.0021; Figure 2B). In patients with PM, ET‐1 levels were 27.9% higher in the subgroup with ILD (13.01 ± 4.59 pg/mL) compared to the subgroup without ILD (10.17 ± 3.12 pg/mL), though this difference did not reach statistical significance (P = 0.1629; Figure 2C). No significant differences in cfDNA levels were observed between the subgroup with ILD and without ILD (Figure 2D–F).
Figure 2.
Differences in serum endothelin‐1 (ET‐1) and cell‐free DNA (cfDNA) levels between patients with idiopathic inflammatory myopathy (IIM) with and without ILD. (A) Serum ET‐1 levels in patients with IIM‐associated ILD (IIM‐ILD) versus IIM without ILD (IIM‐non‐ILD). (B) Serum ET‐1 levels in patients with dermatomyositis (DM) with ILD versus DM‐non‐ILD. (C) Serum ET‐1 levels in patients with polymyositis (PM) with ILD versus PM‐non‐ILD. (D) Serum cfDNA levels in patients with IIM with ILD versus IIM‐non‐ILD. (E) Serum cfDNA levels in patients with DM with ILD versus DM‐non‐ILD. (F) Serum cfDNA levels in patients with PM with ILD versus PM‐non‐ILD. Bar graphs represent mean ± SD. Statistical significance was defined as P < 0.05.
Risk factors for ILD in patients with IIM: Regression analysis
Spearman correlation analysis identified serum ET‐1 levels (r = 0.4208, P = 0.0008), Ro‐52 antibody positivity (r = 0.4169, P = 0.0009), elevated IgA (r = 0.3637, P = 0.0043), IgG (r = 0.383, P = 0.0025), and decreased complement C4 (r = −0.2857, P = 0.0269) as significantly associated with ILD comorbidity (Table 2). Multivariate logistic regression confirmed ET‐1 as an independent risk factor for ILD (odds ratio [OR] 1.442, 95% confidence interval [CI] 1.082–1.923, P = 0.013). Conversely, albumin‐to‐globulin ratio (A/G; OR 0.018, P = 0.009), lactate dehydrogenase (LDH; OR 0.996, P = 0.021), and complement C4 (OR 0.000, P = 0.008) were identified as protective factors (Table 3).
Table 2.
Spearman correlation analysis among serum ET‐1 levels, clinical parameters, and ILD comorbidity in patients with IIM*
| Characteristic | Spearman | |
|---|---|---|
| r | P | |
| ET‐1 | 0.4208 | 0.0008 |
| Ro‐52 | 0.4169 | 0.0009 |
| IgA | 0.3637 | 0.0043 |
| IgG | 0.383 | 0.0025 |
| Complement C4 | −0.2857 | 0.0269 |
| CK | −0.3198 | 0.0136 |
| CK‐MB | −0.2656 | 0.0403 |
| LDH | −0.2911 | 0.024 |
| ALT | −0.2863 | 0.0266 |
| AST | −0.2719 | 0.0356 |
| A/G | −0.3115 | 0.0154 |
A/G, albumin‐to‐globulin ratio; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; CK‐MB, creatine kinase–MB isoenzyme; ET‐1, endothelin‐1; IIM, idiopathic inflammatory myopathy; ILD, interstitial lung disease; LDH, lactate dehydrogenase; Ro‐52, anti–Ro‐52 antibody.
Table 3.
Multivariate logistic regression analysis of risk factors for ILD in patients with IIM*
| Characteristic | B value | SE | Wald score | P value | OR (95% CI) |
|---|---|---|---|---|---|
| ET‐1 | 0.366 | 0.147 | 6.219 | 0.013 | 1.442 (1.082 ~ 1.923) |
| A/G | −3.992 | 1.519 | 6.908 | 0.009 | 0.018 (0.001 ~ 0.362) |
| LDH | −0.004 | 0.002 | 5.303 | 0.021 | 0.996 (0.993 ~ 0.999) |
| Complement C4 | −12.171 | 4.581 | 7.058 | 0.008 | 0.000 (0.000 ~ 0.041) |
| Constant | 7.258 | 3.243 | 5.008 | 0.025 | – |
A/G, albumin‐to‐globulin ratio; CI, confidence interval; ET‐1, endothelin‐1; IIM, idiopathic inflammatory myopathy; ILD, interstitial lung disease; LDH, lactate dehydrogenase; OR, odds ratio.
Dose–response relationship between serum ET‐1 levels and ILD risk
For dose–response analysis, patients were stratified into four quartiles based on serum ET‐1 levels. The Cochran–Armitage trend test revealed a significant linear increase in the risk of ILD with ascending ET‐1 quartiles (P = 0.002). Compared with the lowest quartile (quartile 1, ILD prevalence 26.7%) for ILD, ORs were as follows: 2.41 (95% CI 0.52–11.10) in quartile 2, 6.05 (95% CI 1.28–28.73) in quartile 3, and 10.08 (95% CI 1.81–56.00) in the highest quartile (quartile 4, ILD prevalence 78.6%), demonstrating a clear dose–response relationship between serum ET‐1 levels and the risk of ILD in patients with IIM.
Diagnostic performance of biomarkers for IIM‐ILD
ROC curve analysis demonstrated that a combined model incorporating ET‐1, A/G, LDH, and complement C4 achieved superior diagnostic accuracy for IIM‐ILD, with an optimal cutoff value of 0.53 determined by the Youden index (area under the ROC curve [AUC] 0.888, 95% CI 0.802–0.975; sensitivity 78.8% and specificity 88.9%), compared to individual biomarkers (Figure 3). Among single biomarkers, ET‐1 exhibited moderate diagnostic value (AUC 0.744, 95% CI 0.617–0.871; sensitivity 84.85% and specificity 66.67%), whereas other parameters (A/G, LDH, and complement C4) showed lower discriminatory power (AUC < 0.700).
Figure 3.
Receiver operating characteristic curves for the combined model and individual biomarkers in diagnosing idiopathic inflammatory myopathy–interstitial lung disease. A/G, albumin‐to‐globulin ratio; ET‐1, endothelin‐1; LDH, lactate dehydrogenase.
DISCUSSION
The present study revealed that serum ET‐1 levels were significantly elevated in patients with IIM, including those with DM and PM, compared to healthy controls. This finding suggests that ET‐1 may play a role in the pathogenesis of IIM. As a potent vasoactive peptide, ET‐1 has been implicated in promoting inflammatory responses and tissue damage through multiple pathways in various diseases, including systemic lupus erythematosus. 19 , 20 Under inflammatory conditions, endothelial cells and fibroblasts are stimulated to synthesize and release ET‐1, which subsequently binds to its receptors to activate signaling pathways such as PLC‐PKC, MAPK, and NF‐κB, thereby inducing tissue remodeling. In fibrotic processes, ET‐1 promotes fibroblast proliferation, enhances collagen synthesis, reduces collagen degradation, and drives EMT, leading to extracellular matrix alterations and fibrosis progression. In inflammatory responses, ET‐1 activates transcription factors such as NF‐κB, triggering the production of cytokine‐like interleukin‐6, which exacerbates inflammation. 21 Given that IIM is characterized by inflammatory cell infiltration and muscle fiber necrosis in skeletal muscles, with potential involvement of interstitial lung tissue, the abnormal elevation of ET‐1 may contribute to its pathophysiology by aggravating local tissue ischemia, promoting inflammatory cell recruitment, and accelerating fibrosis. This study further revealed a significant positive correlation between serum ET‐1 levels and CT scores, based on the Ichikado scoring system, in patients with IIM‐ILD (r = 0.7677, P < 0.0001). This finding suggests that elevated ET‐1 levels may directly reflect the radiographic severity of pulmonary fibrosis or inflammation, providing critical evidence to support ET‐1 as a potential biomarker for disease activity in IIM‐ILD.
Notably, no significant differences in serum ET‐1 or cfDNA levels were observed between patients with DM and PM. Although DM and PM differ in clinical manifestations (eg, skin involvement or muscle inflammation patterns) and histopathological features, both subtypes share core pathophysiologic mechanisms, such as skeletal muscle inflammation and immune dysregulation. 22 These shared mechanisms may similarly influence ET‐1 and cfDNA levels. Bioactive substances released during muscle injury, such as myoglobin and creatine kinase, could directly or indirectly affect endothelial cell function, modulating ET‐1 synthesis and release. Although studies on the relationship between muscle injury and serum ET‐1 levels remain limited, previous research has suggested a link between skeletal muscle ischemia‐reperfusion injury and endothelial dysfunction. 23
Subgroup analysis demonstrated that patients with IIM with ILD exhibited significantly higher serum ET‐1 levels than those with IIM‐non‐ILD. This observation highlights the potential role of ET‐1 in the pathogenesis and progression of IIM‐ILD. ILD, a common and severe complication of IIM, involves intricate mechanisms encompassing inflammation, immune dysregulation, and fibrosis. ET‐1 may drive pulmonary fibrosis by promoting fibroblast proliferation, enhancing TGFβ1 expression and inducing alveolar epithelial cell apoptosis. Furthermore, ET‐1 enhances the adhesion, migration, and activation of inflammatory cells in lung tissue, amplifying pulmonary inflammation and contributing to ILD progression.
Additionally, this study identified a positive correlation between serum cfDNA and ET‐1 levels in patients with IIM (r = 0.374, P = 0.003). This correlation implies that cfDNA, primarily derived from NETs, may mediate endothelial injury or inflammatory responses, thereby stimulating ET‐1 synthesis and release. NETs have been shown to activate the TLR9 signaling pathway, which promotes fibroblast activity and collagen deposition 9 while also driving EMT via the cGAS–STING axis. 12 Notably, NETs formation and TLR9 activation not only directly participate in pulmonary fibrosis but may also up‐regulate endothelial ET‐1 expression, forming a “NETs–cfDNA–ET‐1” positive feedback loop that exacerbates inflammation and fibrosis in ILD. Future studies should explore the precise interplay between cfDNA and ET‐1—for instance, whether cfDNA directly regulates ET‐1 expression or activity—to provide a theoretical foundation for targeted interventions. Elevated cfDNA levels, reflecting tissue injury or apoptosis, may indicate the extent of inflammation or fibrosis and promote ET‐1 release via endothelial damage. Conversely, ET‐1 may aggravate cellular injury through vasoconstriction, inflammation, and fibrosis, further increasing cfDNA release. This bidirectional relationship offers novel insights into the pathogenesis of IIM‐ILD.
ROC curve analysis revealed that a combined model incorporating ET‐1, A/G, LDH, and complement C4 achieved superior diagnostic accuracy for IIM‐ILD (AUC = 0.8883) compared to individual biomarkers. This finding underscores the clinical utility of multiparameter models in early identification of ILD among patients with IIM, enabling timely intervention to improve prognosis. However, the relatively small sample size may limit the robustness of these diagnostic thresholds. Larger‐scale studies are warranted to optimize cutoff values and validate diagnostic performance. Furthermore, the complex pathogenesis of IIM‐ILD likely involves additional factors beyond those examined here. Future investigations should identify novel biomarkers to enhance diagnostic precision. In particular, correlation with specific myositis autoantibodies (such as anti‐MDA‐5) and established fibrosis markers (eg, KL‐6) represents a critical direction for future research and will be incorporated into our planned multicenter validation studies.
This study has several limitations. First, its single‐center design and modest sample size may introduce selection bias, necessitating validation through multicenter, large‐scale cohorts. Second, although serum ET‐1 levels were measured, tissue‐specific expression patterns in skeletal muscle or lung were not explored, leaving the local mechanistic role of ET‐1 unresolved. As an exploratory clinical study, this work is inherently correlative and does not establish causality. However, it provides the essential clinical rationale and serum biomarker foundation for subsequent interventional studies aimed at elucidating the mechanistic role of ET‐1 in IIM‐ILD, which are currently underway in preclinical models. Third, longitudinal data on ET‐1 levels before and after treatment were lacking, precluding assessment of ET‐1 as a therapeutic monitoring biomarker. Addressing these limitations in future research will deepen our understanding of the role of ET‐1 in IIM and its clinical implications.
This study demonstrates that serum ET‐1 levels are significantly elevated in patients with IIM, particularly in those complicated by ILD. These findings suggest that ET‐1 may serve as a potential biomarker implicated in the pathogenesis and progression of IIM. The observed positive correlation between ET‐1 and cfDNA levels further underscores a potential synergistic role of these mediators in driving inflammation and fibrosis, particularly in IIM‐ILD. The combined diagnostic model incorporating ET‐1, A/G, LDH, and complement C4 exhibited superior performance in identifying ILD among patients with IIM, highlighting its clinical utility for early detection and risk stratification, and providing a rationale for further investigation into ET‐1 as a therapeutic target in IIM‐ILD.
AUTHOR CONTRIBUTIONS
All authors contributed to at least one of the following manuscript preparation roles: conceptualization AND/OR methodology, software, investigation, formal analysis, data curation, visualization, and validation AND drafting or reviewing/editing the final draft. As corresponding author, Dr Zhang confirms that all authors have provided the final approval of the version to be published, and takes responsibility for the affirmations regarding article submission (eg, not under consideration by another journal), the integrity of the data presented, and the statements regarding compliance with institutional review board/Declaration of Helsinki requirements. All authors contributed to the investigation, formal analysis of the data, and writing, reviewing, and editing of the manuscript.
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ACKNOWLEDGMENTS
We sincerely thank the healthy volunteers who donated blood for experiments and the patients who provided lung tissue samples for the experiment, and also thank the Gansu clinical medical research center for rheumatic and immunologic diseases, and the Cuiying biomedical research center and the department of rheumatology of Lanzhou University Second Hospital for their support in this study.
Supported by the National Natural Science Foundation of China (grants 82060302 and 82260325), Natural Science Foundation of Gansu Province (grants 24JRRA923 and 25YFFA055), Fundamental Research Funds for the Central Universities of Lanzhou University (grant lzujbky‐2024‐oy03), Gansu Province Traditional Chinese Medicine Research Project (grant GZKZ‐2024‐29), Cuiying Scientific and Technological Innovation Program of Lanzhou University Second Hospital (grants CY2024‐MS‐A03 and CY2023‐QN‐B06), and Cuiying Student Research Cultivation Program Project of Lanzhou University Second Hospital (grant CYXZPT2025‐25).
Author disclosures and graphical abstract are available at https://onlinelibrary.wiley.com/doi/10.1002/acr2.90044.
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