Vasculitis occurs in many different diseases and syndromes in childhood. It is the predominant manifestation of the disorder in some children, but in others it may be one aspect of a more widespread disease (1). The cause of the majority of the childhood vasculitides is unknown, and it is likely that a complex interaction between inherited determinants and environmental factors, such as infections, trigger the disease. Up-regulated expression of adhesion molecules on endothelial cells and infiltrating inflammatory cells occurs in primary systemic vasculitis, and endothelial dysfunction is likely to be central to the mechanism through which adhesion molecules and cytokines contribute to the pathogenesis of vasculitis (2).
A general feature of all activated cells and cells undergoing apoptosis is a loss of asymmetry of normal cell membrane phospholipids, resulting in an increase in phosphatidylserine on the outer leaflet of the bilipid membrane layer, as well as blebbing of the membrane causing microparticle formation and shedding by a process of exocytic budding (3). Although platelet microparticles (PMPs) have been extensively studied, until recently there has been very little interest in microparticles of endothelial cell origin (EMPs). It is now becoming apparent that EMPs may provide a window to the activated endothelium in a number of disease states in which endothelial injury is central to the disease process, including atherosclerosis (4), acute coronary syndromes (5), antiphospholipid syndrome (6), thrombotic thrombocytopenic purpura (7), and multiple sclerosis (MS) (8).
The functional significance of cellular and platelet phosphatidylserine externalization and microparticle formation has not been fully elucidated, but one important pathophysiologic consequence is a prothrombotic tendency mediated by activation of the extrinsic coagulation pathway (i.e., the tissue factor/factor VII–dependent pathway) (4, 5, 9–11). Recently, it has been suggested that EMPs may be useful diagnostically for the detection of relapses of MS (8), although other investigators have suggested that EMPs may increase in a number of autoimmune states other than MS (12). Moreover, circulating endothelial cells have been found to be increased in small-vessel vasculitis in adults (13) and Kawasaki disease in children (14). Such observations may provide important pathophysiologic insights into the mechanisms involved in vascular injury in these syndromes.
Since endothelial cell activation and injury occur in the vasculitides, the hypothesis that we examined in this study was that circulating EMPs are increased during active vasculitis, providing an opportunity to assess endothelial injury. The aims of this study were, therefore, to examine the profiles of circulating EMPs and PMPs in children with active and inactive vasculitis as compared with healthy control subjects and control patients with childhood septic and autoimmune disease.
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- PATIENTS AND METHODS
This study is the first to show that microparticles are elevated in active childhood systemic vasculitis. This result was independent of immunosuppression per se; the overriding correlation was whether or not disease activity was controlled.
We utilized a number of markers to determine the origin of these microparticles and showed that the endothelium was an important source. CD105 (endoglin) is constitutively expressed by endothelial cells, but is also expressed on some activated monocytes and leukemia cells (22). As such, it is relatively specific for the endothelium. E-selectin is exclusively expressed on activated endothelial cells in vivo (23), although very recently it has been demonstrated to be up-regulated on CD4 T cells in vitro (24). Other potential endothelial markers failed to discriminate between the patient groups. This may be because ICAM-1, constitutively expressed on endothelial cells and up-regulated during endothelial cell activation, has a much wider tissue expression, including expression on activated leukocytes (25). Similarly, although VCAM-1 is predominantly expressed on vascular endothelium, it also has been identified on dendritic cells, macrophages, and non–vascular cell populations within several organs including the joints and kidney (26). Our observation that VCAM-1 microparticles were not increased in active vasculitis (predominantly PAN and Kawasaki disease) is consistent with the findings of Nash et al, who demonstrated that soluble VCAM-1 failed to discriminate patients with active Kawasaki disease from febrile controls (27).
Although our results suggest that these microparticles are of endothelial cell origin, one potential confounder is that soluble E-selectin (increased in many vasculitis syndromes ) could bind phosphatidylserine-rich microparticles released from platelets and other cells. We suggest that this is unlikely, however, because although it is known that L-selectin can bind to negatively charged phospholipids such as phosphatidylserine and cardiolipin, E-selectin has been shown unequivocally not to bind to phospholipids (28).
We also observed an increased number of microparticles of platelet origin expressing CD42a in the active vasculitis group, although the difference between the groups was less dramatic than that observed for EMPs. This finding is not entirely surprising, since most vasculitis syndromes are characterized by high platelet counts and by micro- and macroscopic thrombus formation; indeed, antiplatelet therapy is recommended for most forms of childhood vasculitic illness (29). Interestingly, however, there was no correlation between PMPs and the absolute platelet count in any of the patient groups.
There is an emerging concept that endothelial cell dysfunction is central to the pathogenesis of many diseases including atherosclerosis (30, 31), MS (8), and autoimmune diseases that are typified by thrombosis and vascular injury (7), and SLE with antiphospholipid syndrome (6). In support of this concept, previous studies have been able to demonstrate an elevated number of microparticles of endothelial origin in these disorders, as a consequence of increased endothelial cell activation or apoptosis. However, these studies were performed in adult patients and as such are prone to perturbation from environmental factors such as smoking or other comorbid states such as advanced atherosclerosis. It could be argued that the childhood vasculitides would be the “prototypic” disease states in which to study EMPs, since endothelial dysfunction occurs in most vasculitis syndromes and children do not have the confounding factors that could influence endothelial injury and microparticle generation.
We observed high numbers of E-selectin and CD105 EMPs in the patients with active small-vessel vasculitis (MPA and WG), as well as in those with active medium-sized vessel disease (PAN and Kawasaki disease), although patient numbers in the former group were limited. We described patients with active vasculitis together, irrespective of the size of the artery predominantly affected, because there is a significant degree of polyangiitis overlap in children with vasculitis, perhaps even more so than in adults (21, 32, 33). When considering basic mechanisms of endothelial injury, we would be surprised if molecular indices of endothelial injury fell neatly into the relatively arbitrary categories defined by the Chapel Hill consensus or the American College of Rheumatology classification criteria. Indeed, this point is emphasized by the recent reports with regard to circulating endothelial cells that have now been shown to be increased in ANCA-associated vasculitides (13) and in Kawasaki disease (14).
The functional significance of increased microparticle numbers (of all types) in active vasculitis was not investigated in this study, although it is possible that microparticles themselves could contribute to the pathogenesis of vasculitis. The prothrombotic potential of cellular and platelet microparticles is well established and has been demonstrated in acute coronary syndromes, atherosclerosis, antiphospholipid syndrome, and meningococcal sepsis. This occurs mainly as a result of the rich phosphatidylserine content of the microparticles, which acts as one of the essential lipid cofactors for clotting and supports thrombin generation via the tissue factor/factor VII–mediated pathway. Since a prothrombotic tendency occurs in vasculitis (34, 35), it is entirely conceivable that the increased circulating microparticles observed during active vasculitis may contribute to this.
The recent work by Jimenez et al (36) may provide some insights regarding pathophysiology that are relevant to the data presented in our study. By studying the response of renal and brain microvascular endothelial cell lines to activation (TNFα stimulation) or apoptosis (growth factor deprivation), those authors demonstrated that endothelial cells release qualitatively and quantitatively distinct microparticles in activation compared with apoptosis. EMP-expressing inducible markers (predominantly E-selectin) were markedly increased in activation; in contrast, EMP-expressing constitutively expressed markers (CD31 and CD105) or annexin V were more typically found in apoptosis. Our results demonstrated quantitatively greater rises in EMP-expressing E-selectin than EMP-expressing constitutively expressed markers (CD105 and ICAM-1) in children with active vasculitis, suggesting that the endothelium was expressing an activated, rather than apoptotic, phenotype. Activation and apoptosis may not necessarily be mutually exclusive in the vasculitides, however. Indeed, we also observed increased numbers of EMP-expressing annexin V, which may lend credence to the hypothesis that both activation and apoptosis (and, eventually, necrosis as a downstream event) contribute to the vascular injury associated with vasculitis syndromes of the young. In fact, there are surprisingly little published in vivo data examining endothelial apoptosis in primary systemic vasculitides, an area worthy of future study.
EMPs may subsequently prove useful for the diagnosis and monitoring of vasculitic disease activity, and to differentiate sepsis from vasculitis in patients in whom the diagnosis of vasculitis has already been established. Based on our preliminary observations, we summarized the diagnostic test characteristics of EMPs in this context (Table 2 and Figure 6). The use of EMPs as a diagnostic tool in routine clinical practice is not at present warranted. However, in the absence of reliable tools for diagnosing active vasculitis, this area of investigation could prove useful in the future.
In conclusion, we have demonstrated increased levels of circulating endothelial microparticles in children with active vasculitis. We propose that this novel finding may provide a window to the activated endothelium. Further studies are required to determine the full functional and pathophysiologic significance of this observation.