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. 2024 Jan 11:10:1247778.
doi: 10.3389/fmed.2023.1247778. eCollection 2023.

Profiling of plasma extracellular vesicles identifies proteins that strongly associate with patient's global assessment of disease activity in rheumatoid arthritis

Onno J Arntz  1 , Rogier M Thurlings  1 , Esmeralda N Blaney Davidson  1 , Pascal W T C Jansen  2 , Michiel Vermeulen  2 , Marije I Koenders  1 , Peter M van der Kraan  1 , Fons A J van de Loo  1
Affiliations

Profiling of plasma extracellular vesicles identifies proteins that strongly associate with patient's global assessment of disease activity in rheumatoid arthritis

Onno J Arntz et al. Front Med (Lausanne). .

Abstract

Background: Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation and cartilage/bone damage. Intercellular messengers such as IL-1 and TNF play a crucial role in the pathophysiology of RA but have limited diagnostic and prognostic values. Therefore, we assessed whether the protein content of the recently discovered extracellular vesicles (EVs), which have gained attention in the pathogenesis of RA, correlates with disease activity parameters in RA patients.

Methods: We identified and quantified proteins in plasma-derived EVs (pEVs), isolated by size exclusion chromatography from 17 RA patients by mass spectrophotometry (MS). Quantified protein levels were correlated with laboratory and clinical parameters and the patient's own global assessment of their disease activity (PGA-VAS). In a second MS run, the pEV proteins of nine other RA patients were quantified and compared to those from nine healthy controls (HC).

Results: No differences were observed in the concentration, size, and protein content of pEVs from RA patients. Proteomics revealed >95% overlapping proteins in RA-pEVs, compared to HC-pEVs (data are available via ProteomeXchange with identifier PXD046058). Remarkably, in both runs, the level of far more RA-pEV proteins correlated positively to PGA-VAS than to either clinical or laboratory parameters. Interestingly, all observed PGA-VAS positively correlated RA-pEV proteins were associated with the actin-cytoskeleton linker proteins, ezrin, and moesin.

Conclusion: Our observation suggests that PGA-VAS (loss of vitality) may have a different underlying pathological mechanism in RA, possibly related to enhanced muscle actin-cytoskeleton activity. Furthermore, our study contributes to the growing awareness and evidence that pEVs contain valuable biomarkers for diseases, with added value for RA patients.

Keywords: PGA-VAS; extracellular vesicles; plasma; proteomics; rheumatoid arthritis; vitality.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Characteristics of EVs isolated from plasma by SEC. As recommended by ISEV, at least two transmembrane or lipid-bound extracellular proteins, cytosolic, intracellular, and extracellular proteins were determined by mass spectrophotometry (A). By western blot, the exosomal markers CD9, Alix, and HSP-70 were detected (B). The pEV density was determined by the separation of pEVs using Optiprep Density Gradient, whereafter EVs were quantified by NTA (C). For the characterization, an equal volume of 17 RA-pEVs (run1) was used.
Figure 2
Figure 2
Most RA-pEV protein levels correlate positively to PGA-VAS. All RA-pEV protein levels, quantified by mass spectrophotometry, were correlated to clinical (TJC; SJC), laboratory (CRP; ESR), and own patient’s own global assessment of their disease activity (PGA-VAS; A). Overlap of some significantly positively correlated RA-pEV protein levels to clinical, laboratory, and patient’s own parameters was observed (B).
Figure 3
Figure 3
Protein profiling of EVs isolated from RA and HC donors and their correlation with disease parameters. Amount of particles (A), mode particle size (B), and protein content per particle (C) from pEVs isolated from 500 μL pfp of HC (n = 9) or RA (n = 9) were determined by NTA and micro-BCA protein assay. After preselection (each protein must be at least detected by two peptides and present in at least four donors) in a PCA plot (D) individual pEV donors are shown (RA in red; HC in green). Venn diagram of all pEVs proteins of HC (gray) or RA (blue) pEV donors, detected by mass spectrophotometry (E). RA-pEV protein levels quantified by mass spectrophotometry were correlated to clinical (TJC; SJC), laboratory (CRP; ESR), and patient’s own global assessment of their disease activity (PGA-VAS; F). All significant RA-pEV proteins (vs. HC) are shown in a heatmap (G). Normalized protein levels highly present are colored black and low, white. Ratio of mean RA vs. HC protein level is added including their statistical p values. For each RA-pEV protein, a significant correlation to PGA-VAS is added from the first and second MS runs. Statistically significant differences (p values) between RA and HC were determined by a two-tailed Mann–Whitney U test.
Figure 4
Figure 4
Biological analysis of enriched RA-pEV proteins. An overview of protein–protein interaction of the significantly enriched and positively correlated RA-pEVs proteins to PGA-VAS using STRING (A). Green nodules are the enriched and positively correlated RA-pEVs proteins to PGA-VAS, and white nodules are interacting proteins. The central proteins are marked as yellow nodules. Intern correlation of the four RA-pEV proteins was calculated (B). In white are correlations of the first run (n = 17) and in gray are correlations of the second run (n = 9). Correlation of the central proteins, ezrin and moesin, to significantly enriched RA-pEV proteins (C). Correlation was calculated by Spearman’s rank correlation coefficient (ρ) and their p values.
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