Idiopathic inflammatory myopathies (polymyositis (PM); dermatomyositis (DM) and related conditions) are a heterogenous group of autoimmune disorders whose causes and pathogenesis remain unclear. In DM, inflammatory changes occur both in muscle and in skin. Although there has been no direct comparison, the pathological changes in juvenile and adult DM appear to be similar except that all the basic pathological features are more prominent in the childhood form. The main histopathologic alterations in DM are found in relation to the blood vessels of the connective tissue of the muscle, skin and gastrointestinal tract. In DM, the inflammatory exudate is predominantly perivascular and perimysial and to a lesser extent endomysial. In juvenile DM, intramuscular blood vessels also show endothelial hyperplasia, immune complex and complement deposition, and focal loss of capillaries [1]. It is believed that in adult DM, capillary loss precedes other pathological changes in the muscle, and that the capillary endothelium is an early and possibly primary target of immune attack [2,3]. A physiologic reaction to capillary loss would be formation of new blood vessels, but the evidence for such neovascularization in DM patients has not previously been investigated.

The recruitment of leukocytes involves sequential capture on, rolling along and firm adhesion to the microvascular endothelium, followed by transmigration of leukocytes through the vessel wall and further migration in extra-vascular tissue [4]. The steps in the recruitment cascade are orchestrated by the cell adhesion molecules on both leukocytes and endothelial cells; different subsets of cell adhesion molecules are responsible for different steps. Previous studies have shown that adhesion molecules which facilitate leukocyte transmigration are up-regulated in the capillaries of DM muscle tissue, suggesting an active participation in the recruitment of the inflammatory infiltrate into the muscle tissue [5]. Monocyte-derived dendritic cells play a critical role in controlling immunity by activating naive T cells. Monocytes leave the blood stream by endothelial cell transmigration, engulf tissue antigens, differentiate into mature dendritic cells, and finally reverse-transmigrate into lymph nodes to activate naive T-cells. Recent in vitro studies show that PECAM-1 (CD31) and tissue factor (CD142) play a critical role in transmigration and reverse transmigration respectively [6,7].

Angiogenesis is an important component of the inflammatory response, during which new vessels are formed from preexisting ones via sprouting and non sprouting mechanisms. Because the status of angiogenesis in myositis is not known, we have focused our attention on identifying the molecular processes that facilitate angiogenesis and the immune response in DM. Our first aim was to demonstrate whether there are signs of angiogenesis in muscle tissue of patients with juvenile and adult DM. A second aim was to identify molecular pathways that are relevant for angiogenesis by gene expression profiling using muscle biopsies from adult DM patients, and a third aim was to investigate whether dendritic cells and molecules that facilitate their entry and exit are present in vivo in the inflamed muscle tissue of DM patients.

Whether or not angiogenesis occurs in a particular tissue depends on the balance between the relative amounts of pro- and anti-angiogenic factors [19]. Analysis of myositis and control muscle tissues also showed a significant up regulation of anti-angiogenic genes (angiopoietin-2, tryptophenyl-tRNA synthetase, interleukin 10 receptor, and TGF-beta) in DM patients suggesting an active ongoing anti-angiogenic response (Fig. 3).

The endothelial markers originally identified to be associated with leukocyte recruitment are also involved in neovascularization, suggesting a dual role not only in leukocyte-endothelial adhesion but also in angiogenesis [20]. Significant up-regulation of the markers that participate in both leukocyte trafficking and angiogenesis (e.g., CX3CL1, CCR1, CD47, VCAM-1, ICAM-1, PECAM1 and ICAM2) is noted in DM patients (Fig. 3).

It has been previously shown that immune complex and complement deposition occurs in arterioles and capillaries of DM patients [2,21]. Since the complement cascade is intricately associated not only with vessel damage but also with angiogenesis, we have specifically looked for members of the complement cascade that facilitate angiogenesis. We found that both classical and alternate complement cascade members along with regulators (Factor-B, C7, Factor-I, C1S, Factor H, and C4) are highly up regulated in DM patients (Fig. 3).

Immunohistochemical and global gene expression analysis of independent groups of DM muscle samples clearly showed that angiogenesis and genes that influence angiogenesis, leukocyte migration, and complement activation are highly upregulated in DM. Expression profiling also suggested that several dendritic cell markers are up-regulated in DM patients. We have confirmed the presence of DC-LAMP positive dendritic cells that are predominantly localized in perivascular areas of inflammation and in close proximity to blood vessels. Juvenile and adult DM patients also had dendritic cells in close proximity to blood vessels expressing antigens (e.g., PECAM and Tissue factor) that facilitate their transmigration and reverse transmigration.

It is known that skeletal muscle capillarity is very dynamic, for example repeated exercise results in increased capillarity and inactivity results in reduced capillarity [22]. There is little information, however, regarding the molecular pathways that influence capillary formation in inflammatory muscle diseases. Angiogenesis is generally quiescent in adults with the exception of certain tightly controlled physiological situations such as female reproductive functions and tissue regeneration and repair [23]. It is widely accepted that the "angiogenic switch" is 'off' when the effect of pro-angiogenic molecules is balanced by that of anti-angiogenic molecules and is 'on' when the net balance is tipped in favor of angiogenesis [24]. The relative contributions of both pro- and anti-angiogenic factors are likely to change the vascular network in a diseased tissue. Although the exact mechanisms controlling angiogenesis in inflamed muscle are not fully understood, mechanistically, the major stimuli for neo-vascularization are hypoxia and inflammation. Inflammatory signals recruit lymphocytes and macrophages into areas of neovascularization which further act as a source of angiogenic and arteriogenic factors.

It has been previously shown that there is significant capillary loss in the muscle of adult DM patients associated with deposition of the late components of the complement pathway [21]. Our current findings using CD 146 antibodies support and confirm the previous results in adult DM. It was also suggested that similar loss of capillaries occur in juvenile DM patients, but there have been no systematic quantitative studies addressing the capillary loss in juvenile DM patients. Our data in juvenile DM patients in comparison with childhood non-inflammatory myopathic controls suggest that there is no significant net capillary loss despite activated complement system in juvenile DM patients. These results suggest that either capillary loss is effectively compensated by robust new capillary formation or complement mediated damage is modest. In adult DM the number of [alpha]V[beta]3 positive (new) capillaries is not significantly different from that of controls. The modest increase in new capillaries in adult DM also suggests ongoing loss of existing capillaries. Our findings further confirm and extend previously published gene expression data as well as the recent studies showing up-regulation of angiogenesis-related factors (HIF-1beta, alphaV beta3, VEGFR-1) in DM biopsies [25,26]

Although previous work has shown a decrease in capillaries in DM but not PM, our results suggest that there is a significant upregulation of several pathways that initiate angiogenesis in myositis. The new capillaries are highly significantly increased in JDM in comparison with respective controls. Thus it appears that the association between myositis lesions and neovascularization is not merely an epiphenomenon, but represents a compensatory mechanism. In this study all adult DM patients were used for immunohistochemical analysis were treated with Prednisone. The differences in angiogenic responses between juvenile and adult DM patients are likely not due to the medication, because gene expression profiling of untreated adult DM patients also show up-regulated angiogenic genes.

Pathological conditions, such as the inflammatory process, may perturb the resting state of the endothelial cells and promote transendothelial migration of cells and leakage of molecules that are needed at specific sites. Leukocyte interactions with inflamed endothelial cells are mediated by selectins, signaling molecules that include lipids and chemokines, integrins and their ligands, and junctional molecules. Several of these molecules that were initially identified to have a role in leukocyte trafficking have also been found to influence endothelial cell activation, proliferation and differentiation. For example, different complement activation products can perturb the antithrombotic state of quiescent endothelial cells by various mechanisms for example C5a causes the release of heparin sulfate from endothelial cells, and the membrane attack complex is now widely recognized as a potent promoter of coagulation [27]. The membrane attack complex added to endothelial cells in sublytic concentrations induces release of von Willebrand Factor, which in turn favors platelet adherence to the vessel wall [28], and promotes the assembly of prothrombinase by the exposure of phospholipids [29].

Dendritic cells are antigen presenting cells with the unique ability to initiate an immune response. Immature dendritic cells are localized in peripheral tissues where they exert a sentinel function for incoming antigens. After antigen capture and exposure to inflammatory stimuli, dendritic cells undergo maturation and migrate to regional lymph nodes where the presentation of antigenic peptides to T lymphocytes takes place. It is known that some monocytes differentiate into dendritic cells in response to cues that are endogenous to endothelial cells [6,30]. Passage of leukocytes across the endothelial lining into sites of inflammation has been shown to be regulated largely by platelet/endothelial cell adhesion molecule-1 (PECAM/CD31) and the reverse transmigration involves p-glycoprotein and tissue factor expressed on the leukocytes. The reverse transmigrating cells were shown to be dendritic cells [6]. Both tissue factor and PECAM are highly significantly expressed DM patients suggesting an active participation in dendritic cell migration.

In summary, these results suggest that neoangiogenesis is highly activated in DM patients. It appears that significant new blood vessel formation in patients is influenced not only by traditional pro-and anti-angiogenic genes, but also by genes that participate in leukocyte trafficking and complement activation pathways. Further it is likely that close association of monocytes with activated endothelial cells will initiate rapid dendritic cell maturation and initiation of autoimmune response in DM.

The author(s) declare that they have no competing interests.

KN, RR, MM, CF, YC conducted research, KN, LGR, FW, PHP, KP, EHP, IL, provided samples, designed research, analyzed data and wrote paper. KN oversaw research, designed and conducted experiments, analyzed data, and wrote paper.

Dr. Nagaraju is supported by the Vernon Lynch Memorial Fellowship in Arthritis Research, an Arthritis Investigator Award from National Arthritis Foundation, a grant from the Maryland Arthritis Research Centre, and NIH-AR050478. Also supported by grants from the NIH (NHLBI Programs in Genomic Applications [PGA] HOPGENE U01 HL66614-01, and NINDS N01-NS-1-2339). We sincerely thank Dr. Nina Raben for help in the discussion and review of the manuscript.