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Abstract

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

Objective

To evaluate the effect of duration of untreated disease on vascular cell adhesion molecule 1 (VCAM-1) and microRNA (miRNA) expression in muscle biopsy samples from children with juvenile dermatomyositis (DM) as well as its effect on soluble VCAM-1 (sVCAM-1) and tumor necrosis factor α (TNFα) concentrations in sera from these children.

Methods

We enrolled 28 untreated children with juvenile DM and 8 pediatric controls. Eleven children with juvenile DM had short duration of untreated disease (symptoms for ≤2 months before muscle biopsy), and 17 had long duration of untreated disease (symptoms for >2 months before muscle biopsy). Vascular structures, characterized by immunofluorescence using antibodies against von Willebrand factor, VCAM-1, and α-smooth muscle actin, were measured for total area and intensity. Circulating sVCAM-1 and TNFα levels were determined in patients with short duration of untreated disease, patients with long duration of untreated disease, and controls. Differential expression of microRNA-126 (miR-126) in muscle biopsy samples from the 2 patient groups and the control group was detected by miRNA expression profiling and confirmed by quantitative reverse transcription–polymerase chain reaction in muscle biopsy samples from the 3 groups.

Results

Juvenile DM patients with short duration of untreated disease had significantly higher total positive area and intensity/high power field of VCAM-1 expression than did juvenile DM patients with long duration of untreated disease (P = 0.043 and P = 0.015, respectively) or controls (P = 0.004 and P = 0.001, respectively). Von Willebrand factor antigen–positive vasculature displayed greater VCAM-1 intensity in patients with short duration of untreated disease than in patients with long duration of untreated disease (P = 0.001). Circulating levels of sVCAM-1 and TNFα were significantly higher in patients with short duration of untreated disease than in controls (P = 0.013 and P = 0.048, respectively). The miRNA miR-126, a negative regulator of VCAM-1 expression, was significantly decreased (3.39-fold; P < 0.006) in patients with short duration of untreated disease compared to controls, while miR-126 expression in patients with long duration of untreated disease did not differ significantly compared to controls.

Conclusion

In patients with short duration of untreated disease, miR-126 down-regulation is associated with increased VCAM-1 in both muscle and blood, suggesting that VCAM-1 plays a critical role early in juvenile DM disease pathophysiology, augmented by TNFα.

Although a rare disease, with an incidence of 3.2 per million children per year (1), juvenile dermatomyositis (DM) is one of the more easily recognized pediatric rheumatic conditions. The patient, who is usually age ∼6–7 years or younger (1), develops the characteristic rash (erythema of eyelids, malar area, extensor joint surfaces, and, if severe, the trunk) with progressive, symmetric proximal muscle weakness (2). The systemic vasculopathy is documented by deformation and loss of microvascular structures, reflected both in the muscle histology (3, 4) and in the loss of nailfold capillary end row loops (5, 6), which is associated with decreased gastrointestinal absorption (7). It is well documented that endothelial cell activation and neovascularization are major components of the disease pathophysiology (8), but the effect of untreated chronic inflammation on muscle vasculature in juvenile DM is unknown.

Recent epidemiologic studies established that disease chronicity had a previously unrecognized effect on the disease's phenotype (9, 10) as well as a direct association with loss of end row capillary loops determined by nailfold capillaroscopy and impaired capacity for microvascular regeneration (5). Gene expression profile studies of muscle and peripheral blood of untreated children with juvenile DM demonstrated a florid up-regulation of type I interferon (IFN)–induced genes related to disease severity (11, 12) and, after ≥2 months of illness, a dysregulation of genes associated with vascular remodeling (13). This molecular evidence is supplemented by careful studies of the physical structures in muscle which suggest that capillary abnormalities precede other structural changes (14). Despite this observation, there is no information about the effect of either disease duration or microRNA (miRNA)–126 (miR-126) levels on the expression of vascular-associated adhesion molecules in the muscle of children with untreated juvenile DM.

Expression of adhesion molecules, specifically intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), has been inconsistently identified in muscle from patients with DM (15–20). VCAM-1 is expressed on differentiating skeletal muscle (21) but not on adult skeletal muscle fibers (22) and on activated but not quiescent endothelial cells, dendritic cells, macrophages, and epithelium, and is involved in the recruitment of leukocytes from the blood into almost all tissues (15–25). The ligand for VCAM-1, very late activation antigen 4, has been identified on a wide range of cells including leukocytes, hematopoietic progenitors, and stem cells as well as on developing myotubes (22). VCAM-1 is released from activated endothelial cells, resulting in soluble VCAM-1 (sVCAM-1) (25). The increase in VCAM-1 expression and associated endothelial activation contribute to the promotion of inflammation and tissue damage, which is augmented by tumor necrosis factor α (TNFα), a proinflammatory cytokine (21) found to be elevated in children with juvenile DM (26). Because VCAM-1 plays an integral role in the inflammatory process, it is a key factor in the pathophysiology of several different autoimmune diseases, such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and scleroderma (23). VCAM-1 participates in systemic disease activity in SLE (24) as well as in the evolution of heart disease, in which this adhesion molecule plays a dominant role in the initial phases of the development of atherosclerosis (27).

The potential role of miRNAs as regulators of VCAM-1 in the juvenile DM inflammatory cascade is unknown. Modulation of key miRNA levels can affect several physiologic and pathologic functions, offering a new prototype for therapeutic intervention (28). MicroRNAs are noncoding RNAs usually 18–25 bp in length that regulate several messenger RNAs simultaneously by mechanisms such as incomplete base pairing and posttranscriptional gene silencing (29). They control a network of genes involved in endothelial cell function, vascular disease, and angiogenesis. The purpose of this study was to evaluate the effect of the duration of untreated disease in children with definite/probable juvenile DM on VCAM-1 and miRNA expression in diagnostic muscle biopsy samples and on concentrations of sVCAM-1 and TNFα in the serum.

SUBJECTS AND METHODS

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

Juvenile DM patients and pediatric controls.

Muscle biopsy samples (6 μm thick) from 28 patients (24 girls and 4 boys, mean ± SD age 7.07 ± 3.75 years) with definite/probable juvenile DM according to the Bohan and Peter criteria (2) were obtained before the start of therapy after their families gave their age-appropriate, informed consent (Institutional Review Board approval no. 10778). Muscle biopsy samples were derived from areas of inflammation as determined by magnetic resonance imaging (MRI) (T2-weighted, fat suppressed), usually the vastus lateralis. Of the juvenile DM patients, 17 were characterized as having a long duration of untreated disease (>2 months), and 11 were characterized as having a short duration of untreated disease (≤2 months). The duration of untreated disease was defined as the amount of time from the first symptom (rash and/or weakness) until muscle biopsy. Three to four muscle pieces were obtained from the MRI–directed biopsy. One of the samples was wrapped, placed in a sterile container, and transported on wet ice to the Neuromuscular Laboratory at Northwestern University Medical School for diagnosis. The remaining muscle pieces were snap-frozen in liquid nitrogen and transferred to a −180°C freezer until molecular studies (miRNA and quantitative reverse transcription–polymerase chain reaction [qRT-PCR]) were performed.

Fourteen of the children with juvenile DM who had the TNFα –308 A allele (AA/AG) were matched for age and duration of untreated disease with 14 children with juvenile DM who were TNFα −308 GG positive. Eight control muscle biopsy samples (from trunk and proximal muscles, areas affected in inflammatory myopathies) were obtained from healthy pediatric patients undergoing orthopedic surgery; all 8 controls were positive for TNFα –308 GG, the most common allelic representation, and had no evidence of inflammation in their biopsy samples. For the miRNA expression profiling, we tested muscle biopsy samples obtained from 6 girls (mean ± SD age 5.8 ± 2.13 years) with untreated definite/probable juvenile DM; 3 had short duration of untreated disease (here defined as ≤2.4 months) and 3 had long duration of untreated disease (here defined as >7 months). The controls were 2 healthy age- and race-matched females. The miRNA data were validated by testing with qRT-PCR in an independent group of age-matched muscle biopsy samples from 5 girls with short duration of untreated juvenile DM, 5 girls with long duration of untreated juvenile DM, and 5 healthy girls.

VCAM-1 determined by triple immunofluorescence staining.

Frozen muscle slides were fixed with PIPES buffer and 3.7% formaldehyde in a 2.1:1 ratio. The slides were blocked with 10% donkey serum and incubated overnight in a 4°C humidified chamber with 2 primary antibodies: mouse anti–VCAM-1 (BBA5, diluted 1:100; R&D Systems) and rabbit anti–α-smooth muscle actin (anti–α-SMA) (ab5694, diluted 1:50; Abcam). After using 1× phosphate buffered saline with 0.1% saponin as the wash, the slides were incubated with secondary antibodies (Cy3-conjugated anti-mouse Ig and Cy5-conjugated anti-rabbit Ig [715-165-152 and 711-175-152, respectively, both diluted 1:100; Jackson ImmunoResearch]) for 1 hour at room temperature. After washing and a second round of fixation and blocking, the Zenon technique was used to couple a mouse monoclonal antibody against von Willebrand factor (vWF) (ab20435, diluted 1:100; Abcam) with Fab conjugated with Alexa Fluor 488 (Z-25002; Invitrogen) according to the manufacturers' instructions. The slides were washed, mounted with FluorSave (Calbiochem), and coverslipped.

Image capturing and analysis.

Images of the triple-stained tissue sections were acquired within 48 hours, using Openlab computer software 4.04 (Improvision) and a Leica DMR-HC microscope coupled to a Photometric Cool Snap CCD camera. SlideBook 4.2 was used to measure the positive fluorescence of the area (μ2) and intensity (pixels) of VCAM-1, α-SMA, and vWF. VCAM-1 expression on arterioles was defined as α-SMA strong positive, vWF positive, and VCAM-1 positive. VCAM-1 expression on venules was defined as minimally α-SMA positive or negative, vWF positive, and VCAM-1 positive. Capillaries were defined as α-SMA negative, vWF positive, and VCAM-1 negative.

Determination of TNFα −308 polymorphism.

A single basepair substitution of an A for the more common G at the TNFα −308 promoter site was analyzed using PCR as previously described (26). Digestion of amplified product with Nco I restriction enzyme confirmed genotype of the juvenile DM samples as GG, GA, or AA (26).

Measurement of serum levels of sVCAM-1, TNFα, interleukin-1β (IL-1β), IL-6, and IFNγ.

Five of the children with long duration of untreated disease had a corresponding fasting serum sample obtained at the time of the muscle biopsy. We tested an additional 3 serum samples from patients with long duration of untreated juvenile DM (total of 8), 6 serum samples from patients with short duration of untreated juvenile DM, and 6 serum samples from pediatric controls. An MSD Multi-Spot 96-well 4 Spot Human VCAM-1 plate (K151EQC-1; Meso Scale Discovery) and an MSD Multi-Spot 96-well Human Proinflammatory I 4-plex Assay (K15009C-1; Meso Scale Discovery) were used to measure concentrations of sVCAM-1, TNFα, IL-1β, IL-6, and IFNγ in sera. The manufacturer's protocol was followed, and data were analyzed using MSD Workbench (Meso Scale Discovery). To test for a potential inhibitor of IL-1β, plasma from 10 children with juvenile DM (5 with a short duration of untreated disease and 5 with a long duration of untreated disease) and 5 healthy children was spiked with 2 concentrations of IL-1β (62.5 pg/ml and 15.6 pg/ml) and assessed with both negative and positive controls using Meso Scale Discovery technology. There was no evidence of an IL-1β inhibitor in any of the children's plasma, either from juvenile DM patients or from controls.

MicroRNA expression profiling.

Total RNA was extracted using a miRCURY RNA Isolation Kit (Exiqon). RNA quality control was performed using a 2100 Bioanalyzer (Agilent). The 2100 Bioanalyzer Expert software was used to generate an RNA integrity number that provides a numerical assessment of the integrity of RNA. Following quality control, RNA samples were labeled using a miRCURY LNA microRNA Power Labeling Kit according to Exiqon's protocol. Array experiments were conducted as double-channel Hy3/Hy5 experiments in triplicates on Exiqon's miRCURY LNA microRNA Array, version 11.0. Hybridization of labeled RNA to the array was performed on Tecan HS4800 Pro automated hybridization stations and scanned with Agilent G2505B Microarray Scanners. The obtained microarray images were analyzed using GenepixPro 4.0 software.

MicroRNA-126 qRT-PCR.

Total RNA was extracted from the muscle samples using a miRCURY RNA Isolation Kit. The first-strand complementary DNA (cDNA) synthesis and real-time PCR for microRNA were conducted using a miRCURY LNA Universal RT miRNA PCR kit (Exiqon). Briefly, for first-strand cDNA synthesis, 20 ng of total RNA was used in 20 μl of reaction mixture. The reaction was carried out for 60 minutes at 42°C followed by heat-inactivation of reverse transcriptase for 5 minutes at 95°C. For real-time PCR amplification, cDNA was diluted 80 times; 4 μl of diluted cDNA was used for the real-time PCR. The miR-126 primer set and SYBR Green Master Mix were purchased from Exiqon, and the 2-step PCR program was performed in an ABI 7500 Fast thermal cycler with the following protocol: 95°C for 10 minutes to initially denature DNA, followed by 40 cycles of 95°C for 10 seconds, and ending with 60°C for 1 minute to achieve DNA amplification.

Statistical analysis.

The total area and total intensity/high-power field (hpf) of VCAM-1 were compared among diagnostic muscle biopsy samples from juvenile DM patients with short duration of untreated disease, juvenile DM patients with long duration of untreated disease, and pediatric controls, by one-way analysis of variance with Tukey's test (SPSS software, version 18.0). The same method was used to compare sera from these 2 juvenile DM patient groups with sera from controls for levels of sVCAM-1 and the cytokines listed, including TNFα. For miRNA profiling, RNAs from muscle samples were hybridized and analyzed against a common reference pool of all 8 samples. For these samples the quantified signals (background corrected) were normalized using the global lowess regression algorithm. The microRNA expression in juvenile DM patients with short duration of untreated disease was compared with that in juvenile DM patients with long duration of untreated disease and with that in pediatric controls, by Student's t-test. Fold change was calculated, and miRNAs were selected that fulfilled both criteria of P ≤ 0.05 and fold change ≥1.5 or fold change ≤−1.5. Ingenuity Pathways Analysis software (Ingenuity Systems) and PubMed were used to search and identify target protein-coding genes potentially regulated by the top differentially expressed miRNAs.

RESULTS

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

Expression of VCAM-1 in muscle biopsy samples from untreated children.

Figure 1 presents representative images of the vascular system in the muscle from a patient with short duration of untreated juvenile DM, a patient with long duration of untreated juvenile DM, an age- and sex-matched pediatric control, and an isotype-negative control (IgG from the same species of nonimmunized animal donor, not directed against any known antigen). In the juvenile DM patients with short duration of untreated disease, the capillaries were positive for vWF antigen and negative for both VCAM-1 and α-SMA, which identifies the arterioles and some venules, compared with the negative controls.

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Figure 1. Immunohistochemistry of muscle and vasculature from patients with untreated juvenile dermatomyositis (DM) and from a pediatric control, showing vascular cell adhesion molecule 1 (red), α-smooth muscle actin (blue), and von Willebrand factor antigen (green). Shown are triple immunofluorescence–stained images of muscle biopsy samples from a child with short duration of untreated juvenile DM (≤2 months) (a), a child with long duration of untreated juvenile DM (>2 months) (b), an age- and sex-matched control child (c), and an isotype-negative control (d). Original magnification × 20.

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The analysis of the juvenile DM diagnostic muscle biopsy tissue as a whole documented that children with a short duration of untreated disease displayed a significantly higher total area of VCAM-1 expression compared to children with a long duration of untreated disease (P = 0.043) and compared to controls (P = 0.004) (Figure 2a). The intensity of VCAM-1 expression/hpf was also increased in juvenile DM patients with untreated disease of short duration compared to juvenile DM patients with untreated disease of long duration (P = 0.015) and compared to controls (P = 0.001) (Figure 2b). Analysis of VCAM-1 expression in vWF-positive blood vessels similarly showed a significant increase in total area/hpf in juvenile DM patients with short duration of untreated disease compared to juvenile DM patients with long duration of untreated disease (P = 0.022) (Figure 3a), while intensity/hpf was also higher in vWF-positive blood vessels in muscle biopsy samples from juvenile DM patients with short duration of untreated disease compared to those from juvenile DM patients with long duration of untreated disease (P = 0.001) and compared to those from controls (P = 0.003) (Figure 3b).

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Figure 2. Increased total area and intensity of vascular cell adhesion molecule 1 (VCAM-1)–positive staining in diagnostic muscle biopsy samples from children with short duration of untreated juvenile dermatomyositis (DM). Total area (a) and total intensity (b) of VCAM-1–positive expression in muscle biopsy samples were increased in juvenile DM patients with short duration of untreated disease (DUD) (≤2 months) (n = 11) compared with those in juvenile DM patients with long duration of untreated disease (>2 months) (n = 17) and compared with those in age- and sex-matched controls (n = 8). Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the horizontal lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. hpf = high-power field.

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Figure 3. Increased VCAM-1–positive areas and intensity are localized to von Willebrand factor (vWF) antigen–positive vasculature in muscle biopsy samples from patients with short duration of untreated juvenile DM. Total area (a) and total intensity (b) of VCAM-1–positive and vWF antigen–positive expression in blood vessels from muscle biopsy samples from patients with short duration of untreated juvenile DM (n = 11), patients with long duration of untreated juvenile DM (n = 17), and age- and sex-matched controls (n = 8) are shown. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the horizontal lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. See Figure 2 for other definitions.

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Serologic findings.

A similar pattern was observed with sVCAM-1 levels in sera from juvenile DM patients compared to sera from pediatric controls. Juvenile DM patients with short duration of untreated disease had significantly higher sVCAM-1 levels compared to those in controls (P = 0.013) (Figure 4a). In parallel, the levels of TNFα in juvenile DM patients with short duration of untreated disease were higher than those in controls (P = 0.048) (Figure 4b). Levels of the other proinflammatory cytokines (IL-1β, IL-6, and IFNγ) did not differ significantly among the 3 groups. Although the levels of IL-1β were below the level of detection (data not shown), the controlled experiments using the addition of 2 concentrations (62.5 pg/ml and 15.6 pg/ml) of IL-1β did not demonstrate the presence of an inhibitor.

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Figure 4. Elevated circulating levels of soluble VCAM-1 (sVCAM-1) and tumor necrosis factor α (TNFα) in children with short duration of untreated juvenile DM compared with those in controls. Soluble VCAM-1 levels (a) and TNFα levels (b) in sera from patients with short duration of untreated juvenile DM (n = 6), patients with long duration of untreated juvenile DM (n = 8), and age- and sex-matched controls (n = 5) are shown. Data are presented as box plots, where the boxes represent the 25th to 75th percentiles, the horizontal lines within the boxes represent the median, and the whiskers represent the minimum and maximum values. conc = concentration (see Figure 2 for other definitions).

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MicroRNA expression profiles and qRT-PCR.

Of 841 human miRNAs represented in the array, 195 were expressed across all the samples. The most down-regulated miRNA in muscle biopsy samples from patients was miR-126, which was decreased 1.92-fold in patients with short duration of untreated disease compared to patients with long duration of untreated disease (P = 0.014) (Figure 5a). To confirm these findings, miR-126 qRT-PCR was performed in an independent set of untreated subjects (5 juvenile DM patients with short duration of untreated disease, 5 juvenile DM patients with long duration of untreated disease, and 5 healthy controls). Expression of miR-126 was 3.39-fold lower in patients with short duration of untreated disease compared to controls (P < 0.006), while miR-126 expression in controls and in patients with long duration of untreated disease did not differ significantly (0.145-fold; P = 0.548) (Figure 5b). These data provide confirmatory evidence that expression of miRNA-126 is lower in muscle from juvenile DM patients with short duration of untreated disease than in muscle from juvenile DM patients with long duration of untreated disease and in muscle from pediatric controls.

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Figure 5. a, Intensity plot of the expression levels of miRNA-126 (miR-126) in muscle biopsy samples from girls with short duration of untreated juvenile DM (≤2.4 months) (n = 3), girls with long duration of untreated juvenile DM (>7.0 months) (n = 3), and controls (n = 2). The microRNA miR-126 was down-regulated in samples from girls with short duration of untreated juvenile DM compared with that in samples from girls with long duration of untreated juvenile DM (fold change −1.92; P = 0.014). b, Quantitative reverse transcription–polymerase chain reaction data from a different set of muscle biopsy samples from girls with short duration of untreated juvenile DM, girls with long duration of untreated juvenile DM, and healthy controls. Values are the mean ± SD. These data confirm that miR-126 was down-regulated 3.39-fold in samples from juvenile DM patients with short duration of disease compared with samples from juvenile DM patients with long duration of disease. Expression of miR-126 in controls and in patients with long duration of untreated disease did not differ significantly (0.145-fold; P = 0.548). See Figure 2 for definitions.

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DISCUSSION

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

We believe this is the first investigation to evaluate the intensity and localization of VCAM-1 expression in the muscle of untreated children with juvenile DM, classified by the duration of untreated disease in conjunction with assessment of miRNA expression. Our results show an increase in VCAM-1 protein in muscle and vasculature from patients with juvenile DM who had a short duration of untreated disease compared to children with juvenile DM who had a long duration of untreated disease and compared to pediatric controls. In addition, our results implicate miR-126 as an important early regulating factor of VCAM-1 expression, suggesting that this miRNA may play a critical role in developing juvenile DM pathophysiology.

Past studies have provided conflicting results concerning VCAM-1 expression in muscle of patients with DM, but neither the age of the donor nor the duration of untreated disease at the time of biopsy has been consistently considered. It is important to remember that normal endothelium, as in our pediatric controls, expresses little or no VCAM-1. Sallum et al did not find a difference in VCAM-1 expression when 27 juvenile DM and control muscle biopsy samples were compared, but the mean duration of untreated disease in their juvenile DM patients was 8 months (range 1–64 months) (18). Subsequently, the same group reported that muscle biopsy samples from patients with juvenile DM differed from brachial muscle biopsy samples from adults with DM, polymyositis, or inclusion body myositis with respect to variations in expression of ICAM-1, but not of VCAM-1, leading to their conclusion that VCAM-1 did not play a major role in juvenile DM; little to no expression of VCAM-1 was identified in adult DM biopsy samples (19). In contrast, Tews and Goebel, in a study of patients with inflammatory myopathy (age and duration of untreated disease not stated), documented an increase in VCAM-1 expression in blood vessels and described detectable VCAM-1 expression in areas of the muscle without an obvious inflammatory infiltrate (20), while also recognizing inflammatory cell–associated VCAM-1 staining. Cid et al reported that VCAM-1 was increased on vWF antigen–positive microvasculature in DM patients (average age 57 years) compared to controls (15).

There are conflicting data concerning VCAM-1 expression on muscle fibers. Our study of young untreated children documented significantly higher expression of VCAM-1 in vWF antigen–positive, α-SMA–negative blood vessels as well as in the muscle fibers themselves. These findings are consistent with the data reported by Iademarco et al, who showed that VCAM-1 was present in the basal lamina of muscle cells, prominent in regenerating muscle cells, and appeared to be constitutively expressed, but not usually induced by cytokines, thus differing from the obligatory cytokine-induced VCAM-1 expression on endothelial cells (21). This observation could explain the varied reports of VCAM-1 expression on muscle fibers found in samples from adult DM patients and provides evidence for the importance of not only controlling for duration of untreated disease and the age of the patients studied, but also identifying the specific location of VCAM-1 expression. Our study solidifies the data pertaining to the effect of time on the untreated inflammatory process in the muscle of children with juvenile DM. Furthermore, these data document the impressive differences in VCAM-1 expression when a defined length of time was established between the onset of symptoms and the date that the muscle biopsy sample was obtained.

Soluble adhesion molecules have been studied in other related rheumatic, autoimmune diseases, such as SLE, RA, and localized scleroderma (30–33). In treated adult RA patients, sVCAM-1 levels were significantly higher compared to those in controls (30). Soluble VCAM-1 levels were also higher in SLE patients compared to those in controls, and these levels were correlated with SLE Disease Activity Index (34) scores and inversely related to C3 levels (31). Bloom et al compared soluble adhesion molecules in sera from children with SLE, mixed connective tissue disease (MCTD), vasculitis, and juvenile DM. They found that the children with juvenile DM and MCTD had elevated levels of sVCAM-1 compared to the children with the other rheumatic diseases and compared to the controls (33). In the present study, the significantly higher concentration of sVCAM-1 in the sera of children with juvenile DM and short duration of untreated disease was paralleled by the increased VCAM-1 expression in the muscle of these same children, confirming that circulating sVCAM-1 could be used as an accessible indicator of the inflammatory process within the muscle.

We hypothesized that elevated levels of TNFα and IL-1β, both known to participate in the juvenile DM inflammatory response (26, 35, 36) and to be potent inducers of VCAM-1 expression, might result in differences of VCAM-1 expression in patients with short versus long duration of untreated disease. Examination of our data confirms only part of this conjecture. Significant differences in both TNFα and sVCAM-1 expression were found only when patients with short duration of untreated disease were compared to controls; when juvenile DM patients with short duration of untreated disease were compared to those with long duration of untreated disease, there were no significant differences in the TNFα level. In addition, circulating IL-1β was below detection levels, similar to findings in other serum studies (35, 36), and despite specific testing for a possible circulating inhibitor, none was identified. These data suggest that TNFα may contribute to vascular bed damage in juvenile DM early in the disease course by enhancing the activation of VCAM-1.

Recent studies have shown the importance of miRNA in regulation of endothelial cell activation (37, 38). Specifically, miR-126 exerts an inhibitory regulation on VCAM-1 expression (39). The significant down- regulation of miR-126 early in the course of untreated disease in children with juvenile DM exemplifies the central role that miRNAs appear to play in the regulation of these critical inflammatory pathways (37–39). The miRNA miR-126 inhibits ischemia-induced neovascularization (40), also present in juvenile DM, and also targets insulin receptor substrate 1, which is involved in the development of insulin resistance associated with mitochondrial dysfunction (41). Mitochondrial dysfunction has been previously described in the muscle of children with juvenile DM (4, 12), as has the development of insulin resistance in older patients with juvenile DM (42), which may be associated with observed premature cardiovascular damage (43). The effect of miR-126 on both VCAM-1 and endothelial cell structure and function adds another layer of complexity to the pathophysiology of juvenile DM and the evolution of the inflammatory process as defined by the duration of untreated disease.

In summary, we have demonstrated that VCAM-1 expression is increased in diagnostic muscle biopsy samples, both in the inflammatory muscle tissue and in the vasculature, from children with juvenile DM who have a short duration of untreated disease (≤2 months) compared to those from children with juvenile DM who have a long duration of untreated disease (>2 months). This finding is mirrored by elevated levels of sVCAM-1 and TNFα in the sera of children with juvenile DM who have a short duration of untreated disease, and it is accompanied by consistent down-regulation of miR-126 in those same children. Based on these data, we conclude that VCAM-1 expression, augmented by TNFα and regulated by miR-126, may play a critical role in early juvenile DM pathophysiology, thus possibly opening a new avenue of therapeutic intervention.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. SUBJECTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. 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. Pachman 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. Kim, Cook-Mills, Sredni, Pachman.

Acquisition of data. Kim, Cook-Mills, Morgan, Sredni, Pachman.

Analysis and interpretation of data. Kim, Cook-Mills, Sredni, Pachman.

Acknowledgements

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

The authors acknowledge with great appreciation Dr. John Sarwark and Theresa Phillip for obtaining healthy muscle, the skilled review of Dong Xu, MD, and the technical expertise of Akadia Kachaochana and Jan Caliendo.

REFERENCES

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