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.
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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.
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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.