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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Objective

Ankylosing spondylitis (AS) has been considered a seronegative rheumatic disease based on absent or low levels of antibodies against citrullinated proteins. The present study was undertaken to evaluate whether a citrullinated and matrix metalloproteinase–degraded fragment of vimentin (VICM) could be a prognostic biomarker in AS.

Methods

VICM was measured in serum samples from healthy controls (n = 35), control patients with rheumatoid arthritis (RA) (n = 47), and patients with AS (n = 201). The optimal cutoff for diagnostic sensitivity and specificity was determined by receiver operating characteristic curve analysis. Baseline and 2-year spine radiographs were available from 118 AS patients, and were scored using the modified Stoke AS Spine Score (mSASSS). We assessed correlations with patient demographic characteristics (age, disease duration), disease activity (Bath AS Disease Activity Index [BASDAI], C-reactive protein level), and disease severity (mSASSS) using Spearman's rho. The independent association of VICM with 2-year radiographic progression, defined as a change of >0 in the mSASSS or the development of a new syndesmophyte, was analyzed by multivariate regression.

Results

Levels of degraded VICM were significantly higher in both RA patients and AS patients than in healthy controls (both P < 0.001). AS patients with the highest levels of VICM had the largest burden of disease (P < 0.01), i.e., highest mSASSS score and BASDAI. VICM levels were significantly and independently associated with radiographic progression after 2 years (β = 0.69, P = 0.0005). Patients with both a high VICM level and a high baseline mSASSS had the highest risk of radiographic progression (odds ratio 13 for mSASSS change, 32 for new syndesmophytes), with progression occurring in 67% of these patients.

Conclusion

The present findings show that serum VICM may be of prognostic value in AS. The data also suggest that citrullination may be relevant in AS pathogenesis.

In rheumatic diseases, inflammation, wear and tear, and trauma lead to abnormal or excessive tissue remodeling and turnover, with common and distinct characteristics dependent on the specific disease. The consequence of this remodeling process in connective tissue is the release of a range of degradation products of extracellular and intracellular proteins generated by the proteases expressed locally in the pathologically affected area. Such products constitute fingerprint changes to joint tissue and can provide information about the pathogenesis of disease and serve as target biomarkers (1, 2). An example of such a protein fingerprint biomarker is C-telopeptide of type I collagen, which is a marker of bone resorption measured by the release of crosslinked and cathepsin K–generated type I collagen C-terminal telopeptide (3–5). This and other similar biomarkers have provided diagnostic and prognostic information with regard to rheumatic diseases (6). Using protein fingerprint technology, we recently developed a degradation biomarker assay that measures a circulating citrullinated and matrix metalloproteinase (MMP)–degraded fragment of vimentin (VICM) (7). Thus, the biomarker assay only measures a vimentin peptide fragment with a free C-terminal citrulline (7).

Vimentin is a type III intermediate filament protein that is expressed by various cells as an important part of the cytoskeleton. It has been linked to cell migration, adhesion, and signaling (8), but also to the differentiation of monocytes and polykaryons such as osteoclasts, and, crucially, to inflammation and apoptosis (9, 10). In addition, vimentin has been shown to be secreted by activated macrophages, which accounts for its presence in the extracellular matrix and potentially in the circulation (11). Vimentin is also a well-established target for intracellular citrullination (12). This is a process, mainly driven by peptidylarginine deiminase (PAD) enzymes, that mediates a posttranslational modification of the amino acid arginine into citrulline (13). Citrulline modification may irreversibly disrupt protein structure and function (14, 15). The citrulline may mark the protein for destruction, which can lead to processing of the protein through major histocompatibility complex presentation and (auto)antibody generation (16, 17), as well as to cellular apoptosis, which in turn leads to the release of the citrullinated protein fragments. Vimentin is one of the proteins that are modified as part of the cellular processing and secretion (18).

The presence of antibodies directed against citrullinated proteins is well described in rheumatoid arthritis (RA) (12, 19), and these antibodies are used as diagnostic biomarkers. However, citrullination as such is not found in RA only, and has been described in joint tissue of patients with osteoarthritis and spondyloarthritis (SpA) (20). The relevance of this to the development of SpA has not been assessed, however. Tilleman et al (21) identified the presence of citrullinated vimentin fragments in synovial tissue from both RA and SpA patients, although it was predominantly found in RA patients. Increased levels of metalloproteinases have been found in both RA and ankylosing spondylitis (AS) and have been linked to adverse prognosis in these diseases (22, 23). We recently demonstrated that the level of circulating VICM was elevated in patients with liver fibrosis (7), which indicates that VICM could be a marker of local inflammation. The aim of the current study was to evaluate the presence of MMP-derived fragments of VICM in patients with AS and to determine whether this might be of prognostic significance.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Patients.

Serum samples were collected from 201 patients with AS. Of the 201 patients, 155 had radiographs obtained at baseline and 118 had 2-year followup radiographs. Twenty-seven of the 201 patients had 1 or more swollen peripheral joints, 168 had no swollen peripheral joints, and data regarding the number of swollen joints were not available on 6 patients. All patients had an established diagnosis of AS according to the modified New York criteria (24). Patients had received standard care, including physical therapy and treatment with a nonsteroidal antiinflammatory drug (NSAID), but have not received biologic agents at baseline. The Bath AS Disease Activity Index (BASDAI) (25) and modified Stoke AS Spine Score (mSASSS) (26) were recorded for each AS patient. For progression analysis, patients were divided into 2 groups based on the presence of a new syndesmophyte at 2-year followup (yes/no) or having an increase in the mSASSS score (change of >0) at 2-year followup (yes/no).

Forty-seven control patients with RA were included in the study. Serum samples from these patients were obtained prior to the start of treatment with biologic agents.

In addition, sera from 35 healthy controls were studied. The healthy control serum samples had been obtained for 2 earlier studies (27, 28). Healthy control subjects were all lean (body mass index <25 kg/m2), between 21 and 72 years of age, and had no history of rheumatic or arthritic disease or treatment for such. All healthy subjects reported that they felt well and had no pain or symptoms of any disease.

Written informed consent was obtained from all of the study subjects.

Reagents.

All materials and chemicals not from the specific sources noted below were purchased from Sigma-Aldrich or VWR.

Biomarker assay.

The identification and development of the serum biomarker VICM have been described in detail by our group (7). Briefly, human cartilage tissue was digested with a range of proteases, and the proteolyzed peptide products were identified by mass spectrometry as previously described (29). Several peptide sequences of interest, which contained a C-terminal arginine, were identified. These arginines are sites for citrullination. Monoclonal antibodies against the C-terminal end of synthetic peptides that included the VICM peptide RLRSSVPGV-citrulline were raised in 6-week-old BALB/c mice (30). After careful screening of primary clones and 3 rounds of subclones, a specific competitive enzyme-linked immunosorbent assay (ELISA) was developed using the clone NB202-1C5. The assay was optimized for specificity against the citrullinated free C-terminal end; thus, the antibody did not recognize uncleaved or noncitrullinated peptide sequences. In addition, the ELISA was tested for sensitivity in human serum, affinity, accuracy, and precision (including interference test) using native material, such as purified protein cleaved with MMPs, samples from various patients, animal samples, and synthetic peptides (specific, noncitrullinated, nonsense).

In this specific VICM ELISA, samples were prediluted 4 times in incubation buffer (50 mM phosphate buffered saline [PBS]/bovine serum albumin–Tween–Bronidox). Biotin–RLRSSVPGV–citrulline (1 ng/ml, 100 μl) (American Peptide) was coated onto streptavidin-coated 96-well Nunc plates (Roche Diagnostics) for 30 minutes at 20°C and shaken at 300 revolutions per minute. The plate was washed 5 times with standard washing buffer (PBS–10% Tween 20). Calibrators, controls, and prediluted serum samples (20 μl) were added, followed by addition of 100 horseradish peroxidase–conjugated monoclonal antibody 1C5 and incubation overnight at 4°C on a shaker set at 300 rpm. After sample/calibrator incubation, the wells were washed 5 times and incubated with 100 μl 3,3′,5,5′-tetramethylbenzidine (Kem-En-Tec) for 15 minutes at 20°C with shaking at 300 rpm, followed by addition of 100 μl stop solution (sulfuric acid) to each well. The colorimetric reaction was measured at 450 nmoles/liter (with reference at 650 nmoles/liter) using SoftMax Pro software, version 5 (Molecular Devices).

The technical properties of the final ELISA were evaluated according to internal standard operating procedures developed under Good Laboratory Practice/Good Manufacturing Practice. These include, but are not limited to, tests of specificity for the degraded and citrullinated vimentin fragment, intra- and interassay variation, detection limits, analyte recovery (e.g., dilution recovery, spiking recovery), analyte stability (e.g., freeze–thaw cycle, stress tests, sample type), kit stability (e.g., stress tests, prolonged storage), and inter–kit lot variation. Briefly, the specificity of the VICM assay was tested by its ability to bind the specific peptide RLRSSVPGV-citrulline, the elongated peptide RLRSSVPGV-citrulline–L, and the related arginine peptide RLRSSVPGVR. There was no cross-reactivity with the elongated peptide (<0.3%) or with the arginine peptide, suggesting that the VICM assay is highly specific for the neoepitope. The assay had good intra- and interassay reproducibility (coefficient of variation <3% and <7%, respectively), sample recovery (>98%), and lot–lot recovery (>94%). The analyte was stable even after 10 runs of freeze–thaw cycles. The technical performance test showed that the assay was specific and robust. There was no relationship between serum VICM levels and sex or age.

Statistical analysis.

Statistical analyses of correlations and logistic regression were performed using MedCalc, version 12 and GraphPad Prism, version 5. VICM levels were compared between patients and controls using descriptive statistics (mean ± SD) and the Mann-Whitney test. Correlations with patient demographic characteristics (age, disease duration), disease activity (BASDAI, C-reactive protein [CRP] level), and disease severity (mSASSS) were determined using Spearman's rho. The Mann-Whitney test was used to compare geometric mean VICM levels in the healthy control and AS groups, as well as the healthy control and RA groups. The optimal cutoff for patients versus controls was determined by plotting the receiver operating characteristic curve (ROC) and discrimination was assessed by area under the curve (AUC) analysis and peak likelihood plotting, as proposed by Landewe and van der Heijde (31). In a preliminary analysis, AS patients were divided into tertiles according to their baseline mSASSS and the difference between groups evaluated by Kruskal-Wallis one-way analysis of variance. Discrimination between low and high tertiles was determined using odds ratios (ORs). VICM values were Z score transformed and analyzed for associations with disease progression, with age, sex, disease duration, and baseline mSASSS entered as independent variables in a multivariate linear regression model. AS patients who had 2-year followup data available were dichotomized as progressors or nonprogressors (new syndesmophyte yes/no or mSASSS change >0 yes/no) and the OR for unadjusted and adjusted VICM as predictor was investigated by logistic regression. We also determined the optimal cutoff level of VICM for progression/no progression by ROC analysis and then calculated odds and maximum likelihood ratios for progression in patients with low or high baseline mSASSS, defined as an mSASSS of >10.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Baseline demographic and clinical characteristics.

Table 1 depicts the baseline characteristics of each of the groups. AS patients and healthy controls were of similar age, while the RA patients were, on average, slightly more than 10 years older. The proportion of men was similar in the AS and RA groups (74.5% and 78.6%, respectively); the control group consisted of a nearly equal number of men and women. A high percentage of the AS patients (∼80%) and approximately one-fourth of the RA patients were taking NSAIDs. The mean level of serum CRP was 10 times higher in RA patients compared to controls (P = 0.0002). In the AS group, the mean serum CRP level was 4 times higher than that in the control group (P = 0.004) and ∼3 times lower than that in the RA group (P < 0.0001). The erythrocyte sedimentation rate (ESR) was significantly higher in the RA patients compared to the AS patients (P < 0.0001).

Table 1. Demographic and clinical characteristics of the groups studied*
 Healthy controls (n = 35)RA controls (n = 47)AS patients (n = 201)
  • *

    Except where indicated otherwise, values are the mean ± SD. AS = ankylosing spondylitis; NSAIDs = nonsteroidal antiinflammatory drugs; CRP = C-reactive protein; ESR = erythrocyte sedimentation rate; DAS = Disease Activity Score; mSASSS = modified Stoke AS Spine Score; BASDAI = Bath AS Disease Activity Index; VICM = citrullinated and matrix metalloproteinase–degraded fragment of vimentin.

  • P < 0.001 versus controls.

  • P < 0.01 versus controls; P < 0.001 versus rheumatoid arthritis (RA) patients.

  • §

    P < 0.01 versus controls.

  • P < 0.001 versus controls; P < 0.01 versus RA patients.

Age, years42.5 ± 8.9555.3 ± 12.443.4 ± 12.8
Male, %48.674.578.6
Disease duration, years14.4 ± 10.719.1 ± 12.2
Patients taking NSAIDs, %023.179.9
Baseline CRP, mg/dl3.02 ± 4.4433.5 ± 45.112.6 ± 19.2
Baseline ESR, mm/hour39.7 ± 29.221.4 ± 19.2§
Baseline DAS6.63 ± 1.19
Baseline mSASSS16.1 ± 19.5
Patients with baseline mSASSS >0, %84.0
Baseline BASDAI5.68 ± 2.24
Change (>0) in mSASSS at 2 years1.60 ± 2.64
Patients with worsening of mSASSS at 2 years, %42
Patients with new syndesmophytes at 2 years, %48
Patients with ≥1 swollen peripheral joint at baseline, %013.8
Baseline serum VICM, nmoles/liter6.60 ± 7.1388.9 ± 17516.4 ± 16.8
Difference in baseline serum VICM level between men and women, mean ± SD nmoles/liter−2.04 ± 4.74−18.0 ± 1113.59 ± 4.33
Peripheral joint affected yes/no, mean ± SD difference−0.99 ± 0.81

Baseline serum levels of VICM in the healthy controls, RA patients, and AS patients.

The mean level of serum VICM at baseline was highest in the RA group (88.9 nmoles/liter [range 0.12–543]), followed by the AS group (16.4 nmoles/liter [range 0.12–123]), and was lowest in the control group (6.6 nmoles/liter [range 0.43–36.4]) (Table 1). The level of VICM was significantly higher in the RA group and the AS group as compared to controls (both P < 0.001), and the level was significantly higher in the RA group compared to the AS group (P < 0.01). There were no significant differences between female and male subjects in any of the groups. The mean VICM level also did not differ between patients with swollen peripheral joints (17.2 nmoles/liter [range 1.6–93]) and those without swollen peripheral joints (16.2 nmoles/liter [range 0.12–123]). In the ROC analysis, the AUC was 0.75 (Figure 1A). The highest positive likelihood ratio was calculated to be 9.8 at an optimal cutoff of 20.7 nmoles/liter (Figure 1B). At this level, 28% of AS patients and 97% of controls were correctly identified (Figure 1C). The tradeoff of increasing the sensitivity to 40%, 60%, and 80% was investigated and resulted in a reduction in the corresponding specificity to 91%, 77%, and 54%. The optimal cutoff of 20.7 nmoles/liter was used in further analyses outlined below to determine associations of VICM level with disease burden and prognosis.

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Figure 1. Serum levels of citrullinated and matrix metalloproteinase–degraded fragment of vimentin (VICM) in ankylosing spondylitis (AS) patients and healthy controls, and association with burden of disease (radiographic severity, assessed with the modified Stoke AS Spine Score [mSASSS] and disease activity, assessed with the Bath AS Disease Activity Index [BASDAI]). A, Receiver operating characteristic curve (ROC) analysis comparing controls (n = 35) and AS patients (n = 201). B, Identification of the optimal cutoff serum VICM level (>20.7 nmoles/liter) by identifying the peak likelihood ratio (9.80). C, Serum VICM cutoff levels, specificities, and likelihood ratios corresponding to sensitivities of 80%, 60%, and 40%, compared with the values obtained with the 28% sensitivity at a VICM cutoff level of 20.7 nmoles/liter. Ninety-five percent confidence intervals (95% CIs) are shown in brackets. D, Geometric mean levels of serum VICM in controls (C.) (n = 35), in the group of all AS patients (A.) (n = 201), and in the AS patients divided into tertiles according to baseline mSASSS (n = 155 with available data). T1 = lowest tertile (mSASSS 0–3.6) (n = 52); T2 = middle tertile (mSASSS 3.7–15) (n = 52); T3 = highest tertile (mSASSS 15.1–72) (n = 51). Vertical lines show the 95% CIs. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus controls. E, Odds of being in the different tertiles of disease burden according to the mSASSS (definitions and n values shown in D) and disease activity according to the BASDAI at baseline, among patients with VICM levels above the cutoff that had the highest likelihood ratio, i.e., 20.7 nmoles/liter. For the BASDAI, T1 = lowest tertile (BASDAI 0–4) (n = 66); T2 = middle tertile (BASDAI 4.1–5.3) (n = 65); T3 = highest tertile (BASDAI 5.4–10) (n = 66). ∗ = P < 0.05; ∗∗ = P < 0.01 versus lowest tertile.

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Association between baseline serum levels of VICM and disease burden (BASDAI, mSASSS).

The association between serum VICM level and disease burden (radiographic severity and activity) was assessed in the AS patients. Patients were divided into tertiles based on their baseline mSASSS and compared to controls and to the group of AS patients overall (Figure 1D). The geometric mean serum VICM levels in the healthy controls and the overall group of AS patients, respectively, were 3.91 nmoles/liter (95% confidence interval [95% CI] 2.65–5.78) and 11.8 nmoles/liter (95% CI 9.9–14.1) (P < 0.001). In the mSASSS tertile groups, the mean levels of VICM were as follows: lowest tertile (n = 52) 10.5 nmoles/liter (95% CI 8.1–13.6), middle tertile (n = 52) 12.2 nmoles/liter (95% CI 9.5–16.2), highest tertile (n = 51) 14.5 nmoles/liter (95% CI 10.8–19.4) (Figure 1D). The level of VICM in each of the 3 AS subgroups was significantly higher compared with controls (P < 0.01). In addition, the VICM level was significantly higher in patients in the highest versus the lowest mSASSS tertile (P < 0.01). When AS patients were divided into tertiles according to scores on the BASDAI, the geometric mean serum levels of VICM in the lowest tertile (n = 66), middle tertile (n = 66), and highest tertile (n = 65), respectively, were 9.14 nmoles/liter (95% CI 6.85–12.2), 12.4 nmoles/liter (95% CI 9.63–16.0), and 13.9 nmoles/liter (95% CI 10.8–17.7). There was a significant difference between each of the tertiles and the healthy controls, but not between the tertiles.

The odds ratios for association with disease burden as assessed using the optimal discriminatory cutoff for serum VICM of 20.7 nmoles/liter are shown in Figure 1E. Patients with VICM levels above the 20.7 nmoles/liter cutoff had significantly increased odds of having a moderate disease burden according to the baseline mSASSS (mSASSS tertile 2) (OR 7.5 [95% CI 1.6–35]) or a severe disease burden (mSASSS tertile 3) (OR 8.7 [95% CI 1.9–40]) compared to controls (P < 0.01) and compared to patients whose baseline mSASSS was in the lowest tertile (P < 0.01). A similar pattern was seen for disease activity assessed according to the BASDAI: patients with VICM levels of >20.7 nmoles/liter had significantly increased odds of having moderate disease activity (BASDAI tertile 2) (OR 7.7 [95% CI 1.7–35], P < 0.01) or high disease activity (BASDAI tertile 3) (OR 7.0 [95% CI 1.5–32], P < 0.05). Thus, at the optimal cutoff level of 20.7 nmoles/liter, VICM was associated with a higher burden of disease.

Association between baseline serum levels of VICM and radiographic progression.

The correlation between baseline VICM level, clinical characteristics, baseline mSASSS, and change in mSASSS was investigated in a univariate analysis (Table 2). Serum VICM levels were not correlated with age or disease duration, but were significantly correlated with the acute-phase reactants CRP and ESR (P = 0.0005 and P = 0.0017, respectively). There was no significant correlation between baseline BASDAI and serum VICM level. In contrast, baseline mSASSS correlated significantly with VICM level (P = 0.022), as well as with age (P < 0.0001), disease duration (P < 0.0001), CRP level (P = 0.0001), and ESR (P = 0.0009). The 2-year change in mSASSS was also significantly correlated with VICM level (P = 0.032) and with age and disease duration, but was not correlated with the BASDAI or acute-phase reactant levels. There was a strong correlation between baseline mSASSS and 2-year change in the mSASSS (P < 0.0001).

Table 2. Univariate correlation between clinical parameters and serum VICM levels in the patients with AS*
 Baseline VICMBaseline mSASSS2-year change in mSASSS
nSpearman's rhoPnSpearman's rhoPnSpearman's rhoP
  • *

    See Table 1 for definitions.

Age201−0.030.661550.60<0.00011180.270.003
Disease duration1960.040.611500.57<0.00011150.200.034
Serum CRP1950.250.00051510.310.00011150.130.16
ESR1990.220.00171540.270.00091180.130.16
Baseline BASDAI1930.130.0651500.100.231150.060.51
Baseline mSASSS1550.180.022  1180.42<0.0001
Baseline VICM    1180.200.032

Multivariate analysis for disease progression.

In a multivariate linear regression model that included all of the significant predictors as continuous variables, VICM was significantly associated with mSASSS progression after 2 years (β = 0.69, P = 0.0005) (Table 3). There was limited change in the effect size when the covariates were included in the model (β = 0.66–0.69), even when baseline mSASSS was entered into the model as an independent variable.

Table 3. Multivariate linear regression analysis of baseline VICM level as an independent predictor of radiographic progression in AS (change in mSASSS) over 2 years*
 nβSEMP
  • *

    See Table 1 for definitions.

VICM alone1180.660.200.0011
VICM adjusted for age, sex, and disease duration1150.680.200.0007
VICM adjusted for age, sex, disease duration, and baseline mSASSS1150.690.190.0005

We further investigated whether we could identify patients with any radiographic progression over 2 years by analysis of the baseline predictors. For this purpose, VICM levels were dichotomized using ROC analysis to identify the best cutoff level for any progression (change of >0 in the mSASSS [yes/no] or new syndesmophytes [yes/no]). This analysis revealed that a baseline VICM cutoff level of 7.2 nmoles/liter had the highest combination of sensitivity (82% [95% CI 69–91]) and specificity (40% [95% CI 28–52]) for mSASSS progression, at an AUC of 0.59. The positive and negative likelihood ratios were 1.4 and 0.45, respectively. Using the same cutoff of 7.2 nmoles/liter for the analysis of new syndesmophyte development, the sensitivity was 84% [95% CI 69–94] and the specificity was 39% [95% CI 28–50]. In a logistic regression model that included this VICM level of 7.2 nmoles/liter as a dichotomous independent variable, VICM was a significant predictor of 2-year progression in the mSASSS after adjustment for age, sex, disease duration, and baseline mSASSS (Table 4).

Table 4. Logistic regression analysis of baseline VICM level as a predictor of radiographic progression in AS over 2 years*
 nOR (95% CI)P
  • *

    A VICM cutoff level of 7.2 nmoles/liter was derived from receiver operating characteristic curve analysis assessing radiographic progressors over 2 years (change of >0 in the mSASSS or new syndesmophytes) and tested as a dichotomous variable. OR = odds ratio; 95% CI = 95% confidence interval (see Table 1 for other definitions).

  • R2 for the model was 18% with inclusion of VICM and 13% without.

  • R2 for the model was 20% with inclusion of VICM and 17% without.

Increase in mSASSS   
 VICM unadjusted1182.82 (1.18–6.74)0.020
 VICM adjusted for age, sex, and disease duration1152.97 (1.14–7.70)0.025
 VICM adjusted for age, sex, disease duration, and baseline mSASSS1152.94 (1.12–7.66)0.028
New syndesmophytes   
 VICM unadjusted1183.03 (1.13–8.11)0.027
 VICM adjusted for age, sex, and disease duration1142.64 (0.93–7.47)0.068
 VICM adjusted for age, sex, disease duration, and baseline mSASSS1142.57 (0.88–7.50)0.083

We next investigated whether a combination of baseline VICM level and baseline mSASSS could be an even stronger predictor of progression, using a previously reported methodology (23). Patients were divided into 2 groups: low baseline radiographic damage (mSASSS <10) and high baseline radiographic damage (mSASSS ≥10), and the prognostic ability of VICM at the 7.2 nmoles/liter cutoff derived from ROC analysis was further tested within these 2 groups. Patients with both high VICM (>7.2 nmoles/liter) and a high baseline mSASSS had significantly higher odds of developing radiographic progression (OR 13 [95% CI 3.4–52], P < 0.0001 for mSASSS change; OR 32 [95% CI 3.9-257], P < 0.0001 for new syndesmophytes) compared to patients with both low baseline mSASSS and low VICM levels (Figures 2A and B). Of the patients with both high VICM and high mSASSS, 67% had radiographic progression after 2 years, compared to only 13% of those with low VICM levels but high baseline mSASSS and 9% of those with low baseline mSASSS but high VICM levels (Figure 2C).

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Figure 2. Odds ratios for 2-year radiographic progression in AS patients grouped according to various combinations of high/low VICM level and high/low baseline mSASSS. A and B, Radiographic progression at 2 years, assessed as a change of >0 in the mSASSS (A) and as development of new syndesmophytes (B). Odds ratios are versus the group with low VICM level and low baseline mSASSS (set at 1). C, Cumulative probability plot for radiographic progression at 2 years, assessed as a change of >0 in the mSASSS. See Figure 1 for definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

In the present analysis of VICM as a biomarker in AS, it was demonstrated that 1) levels of VICM in AS patients were increased compared to those in healthy controls but significantly lower than those in patients with RA; 2) the AS patients with the highest levels of VICM had the largest burden of disease, i.e., radiographic severity as assessed with the mSASSS and disease activity as assessed with the BASDAI; and 3) the AS patients with the highest levels of VICM had the highest odds of radiographic progression, and the prognostic value of VICM was particularly evident in those patients who already had some degree of radiographic progression. These findings suggest that serum VICM may be a novel biomarker associated with AS disease burden and progression.

The VICM epitope was identified, by mass spectrometry, as a unique neoepitope, from in vitro proteolytic cleavage of different tissues, including human articular cartilage, with MMPs (7). Bang et al identified several citrullinated and mutated fragments in synovial fluid and cultured fibroblasts from RA patients (18), whereas we searched for fragments that were both generated by MMPs and were citrullinated. Several lines of evidence support the notion that MMPs have a role in AS. In particular, a previous study demonstrated that MMP-3 was elevated in patients with AS and was associated with syndesmophyte formation (23). Recently, we also reported that levels of MMP-generated type I, II, III, V, and VI collagen neoepitopes were highly elevated in AS (27). MMPs are expressed by synovial fibroblasts and chondrocytes, among other joint cells (32). The role of fibroblasts in the pathogenesis of AS, and especially in disease progression, has not been well studied, but mesenchymal cells likely contribute to the structural disease progression characterized by ankylosis. Moreover, mesenchymal cells could be target cells in the enthesis, the anatomic zone proposed as the primary disease location in AS.

Although citrullination is not a specific process and can be found in the synovial tissue in different forms of arthritis and has also been associated with other remodeling diseases, we believe this is the first report of the detection of circulating citrullinated degradation fragments in patients with AS. The specific development of autoantibodies against citrullinated proteins appears to be a relatively unique feature of RA. As has been extensively discussed elsewhere (12, 13, 16), this disease process–contributing feature is associated with specific antigen presentation in the context of the shared epitope HLA–DR genes, and with polymorphisms in the PAD-4 gene. The contribution of citrullination to AS pathogenesis is yet to be fully explored, and the detection of VICM may represent a secondary phenomenon. AS has been associated with smoking in terms of both disease activity and severity (33). It is unclear whether this reflects any involvement of citrullination in the pathogenesis of the disease. Our data suggest that further study of this process in AS is warranted.

A limitation of our study is that we investigated the presence of only one citrullinated sequence, which was selected from a protein that has been well described in autoimmunity in RA. Indeed, we observed highly elevated levels of VICM in the RA controls, which confirms the presence of citrullinated antigens. However, while this sequence may be present, it does not necessarily imply any role of citrullination in the pathogenesis of disease. Of note, a peptide analysis of target sequences for anticitrullinated vimentin in RA patients did not include the fragment we described (18). Further research is needed to understand the sequences that may be part of the disease or consequent to the disease.

We recently demonstrated that citrullinated peptides, including VICM, may also be found in other disorders, such as liver fibrosis, of which low-grade inflammation is a well-described consequence (7). This suggests that citrullination and citrullination-directed tissue degradation may be a central event in other diseases besides AS and RA. Further analysis is needed to investigate the common denominator in diseases with high levels of citrullinated peptides and why the presence of anti–citrullinated protein antibody appears limited to RA.

Other investigators have demonstrated that mutated vimentin and antibodies to mutated vimentin have diagnostic ability in RA (12,18). In a similar context, autoantibodies to citrullinated sequences, including citrullinated telopeptides of type I and type II collagen, have also been shown to have diagnostic potential in RA (34). Whether these and other citrullinated peptide autoantibodies would also be biomarkers in seronegative diseases such as AS remains in question.

These important findings highlight the notion that posttranslational modifications are becoming increasingly important for the understanding of the pathogenesis of disease, and that some of these modifications may reflect both the course as well as the consequence of disease (2). Most importantly, they emphasize that advanced clinical chemistry investigations should focus on protein fingerprinting technologies directed at proteins known to be relevant to disease, rather than just on measurement of proteins in their healthy conformation (1). In summary, we have presented data suggesting that serum VICM has potential as a biomarker in AS. With further validation, the use of VICM as a biomarker alone or in combination with other serologic and/or radiographic markers may be found to have clinical utility.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Bay-Jensen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Bay-Jensen, Karsdal, Vassiliadis, Lories, Christiansen, Maksymowych.

Acquisition of data. Bay-Jensen, Karsdal, Vassiliadis, Wichuk, Marcher-Mikkelsen, Maksymowych.

Analysis and interpretation of data. Bay-Jensen, Karsdal, Vassiliadis, Lories, Christiansen, Maksymowych.

ADDITIONAL DISCLOSURES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

Authors Bay-Jensen, Karsdal, Vassiliadis, and Marcher-Mikkelsen are employees of Nordic Bioscience. Dr. Christiansen is the founder of CCBR Group ALS and chairman of the board of Nordic Bioscience.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES

We would like to acknowledge our skilled technicians Sedi Tavallaee, Maibritt Andersen, and Trine Overgaard, who all contributed to the development of the ELISAs.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. ADDITIONAL DISCLOSURES
  8. Acknowledgements
  9. REFERENCES
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