Targeted proteomics reveals promising biomarkers of disease activity and organ involvement in antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

BackgroundTargeted proteomics, which involves quantitative analysis of targeted proteins using selected reaction monitoring (SRM) mass spectrometry, has emerged as a new methodology for discovery of clinical biomarkers. In this study, we used targeted serum proteomics to identify circulating biomarkers for prediction of disease activity and organ involvement in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).MethodsA large-scale SRM assay targeting 135 biomarker candidates was established using a triple-quadrupole mass spectrometer coupled with nanoflow liquid chromatography. Target proteins in serum samples from patients in the active and remission (6 months after treatment) stages were quantified using the established assays. Identified marker candidates were further validated by enzyme-linked immunosorbent assay using serum samples (n = 169) collected in a large-cohort Japanese study (the RemIT-JAV-RPGN study).ResultsOur proteomic analysis identified the following proteins as biomarkers for discriminating patients with highly active AAV from those in remission or healthy control subjects: tenascin C (TNC), C-reactive protein (CRP), tissue inhibitor of metalloproteinase 1 (TIMP1), leucine-rich alpha-2-glycoprotein 1, S100A8/A9, CD93, matrix metalloproteinase 9, and transketolase (TKT). Of these, TIMP1 was the best-performing marker of disease activity, allowing distinction between mildly active AAV and remission. Moreover, in contrast to CRP, serum levels of TIMP1 in patients with active AAV were significantly higher than those in patients with infectious diseases. The serum levels of TKT and CD93 were higher in patients with renal involvement than in those without, and they predicted kidney outcome. The level of circulating TNC was elevated significantly in patients with lung infiltration. AAV severity was associated with markers reflecting organ involvement (TKT, CD93, and TNC) rather than inflammation. The eight markers and myeloperoxidase (MPO)-ANCA were clustered into three groups: MPO-ANCA, renal involvement (TKT and CD93), and inflammation (the other six markers).ConclusionsWe have identified promising biomarkers of disease activity, disease severity, and organ involvement in AAV with a targeted proteomics approach using serum samples obtained from a large-cohort Japanese study. Especially, our analysis demonstrated the effectiveness of TIMP1 as a marker of AAV activity. In addition, we identified TKT and CD93 as novel markers for evaluation of renal involvement and kidney outcome in AAV.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-017-1429-3) contains supplementary material, which is available to authorized users.

Usefulness of tissue inhibitor of metalloproteinase 1 as a predictor of sustained remission in patients with antineutrophil cytoplasmic antibody-associated vasculitis

“…AUC was calculated. The median serum level of TIMP-1 in healthy controls (n = 52) was 133 ng/mL (IQR 122-150 ng/mL) in our previous study [17]. Based on healthy control data and the cut-off point for distinguishing between patients who relapsed and/or had di culty reducing GC and those in sustained remission without di culty reducing GC in the RemIT-JAV-RPGN study, the high TIMP-1 group comprised patients with a serum TIMP-1 level ≥ 150 ng/mL, whereas the low TIMP-1 group was composed of those with a serum TIMP-1 level < 150 ng/mL.…”

Proteomic analysis of neutrophils in ANCA-associated vasculitis reveals a dysregulation in proteinase 3-associated proteins such as annexin-A1 involved in apoptotic cell clearance

“…Despite this, even in remission, proteomic results from GPA neutrophils do not fully resemble HC, indicating that treatment could not overcome all intrinsic defects likely to contribute to the pathogenesis of this disease. Interestingly, our proteomics analysis of neutrophil cytosols under basal conditions has identified S100A8/A9, which constitutes 40% of cytosolic proteins, a disease biomarker recently identified in GPA, 20,31 demonstrating that examining dysregulated cytosolic proteins could represent a valid tool for biomarker discovery. Notably, S100A8/9 proteins complexes that are stabilized and secreted during apoptosis can promote neutrophil survival, thus potentially delaying the resolution of inflammation.…”

“…It is predominantly found as calprotectin (S100A8/A9 heterodimer) and is a promising biomarker in inflammatory diseases, including rheumatoid arthritis 18 and GPA. 19,20 Ingenuity Pathway Analysis was used to visualize the biological connections between proteins identified as dysregulated in GPA (Figure 2a). Importantly, various known PR3 binding partners were differentially regulated during neutrophil apoptosis in GPA compared to HC, including CRT 12 and annexin-A1 (AnxA1).…”

Vasculitis

“…Proteomic analyses have identified TIMP1 as a marker of ANCA-associated vasculitis activity and TKT and CD93 as markers of renal involvement and outcome in ANCA-associated vasculitis [46]. A recent meta-analysis identified 33 genetic variants, supporting a role for alpha-1-antitrypsin, the major histocompatibility complex system, and inflammatory processes in the pathogenesis of ANCA-associated vasculitis [47].…”