Why Is the Microvasculature Important in RA

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As the lining of the vasculature, endothelium has the capacity to integrate many different signals and responses, and as a consequence is involved in the pathogenesis of many diseases. The involvement of endothelium in the pathogenesis of RA can be inferred from observations that RA is associated with vascular and hematological abnormalities. For example, the excess mortality in RA is predominantly due to coronary artery atherosclerosis [2]. A recent study showed that the adjusted relative risks of myocardial infarction and stroke in women with RA were 2.0 and 1.48, respectively, when compared to women without RA. Many studies have reported an association between RA and traditional cardiovascular risk factors such as cholesterol and low-density lipoprotein levels. The acute phase response marker C-reactive protein (CRP) is a novel risk factor for atherosclerosis and predicts future risk of coronary artery disease in initially healthy individuals. Since CRP levels are markedly elevated in RA, as part of the ongoing systemic inflammatory processes, such an augmented inflammatory burden may account for the increased cardiovascular risk. However, in addition to these somewhat circumstantial observations, the vasculature is also implicated in RA by the fact that many of the events known to occur in RA—such as leukocyte extravasation—involve the participation of endothelial cells.

Effector Role of Vascular Endothelium in RA

It was recognized more than 30 years ago that endothe-lial cells in RA synovium acquire the characteristic appearance of lymphatic endothelium, which controls lymphocyte emigration. Considerable evidence has now converged, documenting the responsiveness of endothelium to cytokines expressed in RA, the presence on endothelial cells of receptors for these cytokines, and expression by endothelium of adhesion molecules and chemoattractants. Endothelium can also contribute to RA pathogenesis through the formation of new blood vessels.

Recruitment and Activation of Leukocytes in RA

Adhesion of leukocytes to vascular endothelium in vivo must overcome the normal vascular mobility of circulating cells and result in a localized arrest of leukocytes at relevant sites [3]. Over the past few years there have been a number of studies using immunohistochemical analyses of RA tissue, documenting alterations in the pattern of expression of adhesion molecules. It has been shown, for example, that antibody to E-selectin stained endothelium in RA synovium. In addition, RA synovial endothelial cells were also found to express increased levels of vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1. As discussed previously, the typical RA synovial infiltrate is rich in memory CD45RO+ T cells. Synovial membrane and synovial fluid T cells display an enhanced capacity to interact with purified E-selectin and VCAM-1, relative to peripheral blood lymphocytes from either the same patients or from healthy donors, due to increased levels of VLA-4a, the counterligand for VCAM-1. In addition, synovial fluid lymphocytes show higher expression of other integrins such as CD29 (b1), VLA-1a, VLA-5a, and VLA-6a. Accumulation of memory T cells in RA synovium thus appears to result from elevated expression of adhesion receptors on synovial microvascular endothelium, leading to the selective emigration of memory T lymphocytes, which may bear enhanced levels of ligands for these adhesion molecules as a result of a previous activation step.

Moreover, endothelial cells are a source of a range of proinflammatory cytokines, including interleukin (IL)-1, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF). Many of the features of the rheumatoid synovial environment suggest possible roles for chemoattractant cytokines, in that the large number of infiltrating leukocytes, especially the selective accumulation of memory T cells, could in part be a response to the elaboration of chemokines. Endothelial cells secrete and present on cell surface proteo-glycans chemokines of both C-C and C-X-C subsets, in particular IL-8, monocyte chemoattractant protein (MCP)-1, RANTES, and Groa. The ability of endothelium to capture chemokines may be of particular significance in RA, in that mediators such as RANTES or MIP-1a, secreted by other participating cells and anchored on the endothelial cells surface, would ensure a relatively high concentration of chemoattractants close to the blood vessel wall, and hence temporally and spatially restricted activation of circulating cells.

Angiogenesis in RA

A consequence of the synovial hyperplasia, which occurs in RA, is an increase in the distance between the proliferating cells and the nearest blood vessels, leading to local hypoxia and hypoperfusion. It has been reported, for example, that synovial oxygen tension is low in aspirated synovial fluid samples taken from human RA knee joints. In general, the in vivo response to hypoxia is to form new blood vessels, to restore perfusion and oxygenation to the compromised area. Endothelial cells lining blood vessels within RA synovium express cell cycle-associated antigens, and indices of endothelial turnover are increased in synovia from patients with RA compared with noninflamed controls. A morphometric study also suggested that capillaries are distributed more deeply in RA synovium. Many of the cytokines and growth factors expressed in RA have the potential to stimulate angiogenesis [4]. For example, serum levels of vascular endothelial growth factor (VEGF) are markedly elevated in RA, relative to either patients with OA or normal controls, and correlate with levels of CRP. Expression of VEGF by RA lining layer cells has been reported, and microvascular endothelial cells in the vicinity of VEGF-positive cells express VEGF receptors. Since VEGF is upregulated by hypoxia, it is likely that this factor is central to the regulation of angiogenesis in RA. Other proangiogenic molecules expressed in RA include fibroblast growth factor (FGF)-1 and FGF-2, which stimulate proliferation of a variety of cell types, including endothelial cells. Members of the FGF family (FGF-1 and FGF-2) have been detected in human RA synovium. However, in RA the role of the FGFs, and other mitogens expressed in RA, such as platelet-derived growth factor, hepatocyte growth factor, and transforming growth factor-b, is unclear.

In summary, the invasive synovium in RA is highly vascularized, and numerous growth factors are expressed, which might promote new blood vessel formation.

Target Role of Vascular Endothelium in RA

As the interface between the blood and tissues, endothelium can respond to a range of cytokines and growth factors. In RA, many studies have focused on the identity of media-tor(s) involved in disease pathogenesis, but a key milestone was the realization that intercellular messengers, now termed cytokines, may play a central role. Both TNFa and IL-1 have the capacity to activate cells in the synovium, as well as regulating cartilage turnover, stimulating bone resorption and inhibiting proteoglycan synthesis. In the context of RA pathogenesis, TNFa is produced in large quantities by enzymatically dissociated synovial cells from RA patients, and immunohistochemical analyses have demonstrated the presence in RA synovium of TNFa and its receptors. Addition of anti-TNFa antibodies to RA synovial cell cultures resulted in downregulated expression of IL-1, and reduced expression of other cytokines and angiogenic factors [5]. These studies were instrumental in the formation of a key hypothesis by Marc Feldmann and Ravinder N. Maini and their researchers—namely, that TNFa plays a vital role in the pathogenesis of RA.

In terms of vascular endothelium, TNFa is a fundamental inducer of endothelial cell responses. For TNFa to transmit a signal and exert an effect on endothelial cells, it must initially bind to specific cell surface receptors. The TNF receptor family has been studied extensively, in our own and other laboratories, and two receptors for TNFa, CD 120a or TNF-R1 and 75-kDa CD 120b or TNF-R2, have been cloned. Both receptors have been detected on the surface of cultured endothelium. We and others have observed that selective stimulation of endothelial cells through TNF-R1 induced responses comparable to those observed for TNFa, actions not mimicked by TNF-R2 agonists. It is likely that the presence of the higher affinity TNF-R2 on the cell surface serves to "pass" ligand to the lower affinity TNF-R1, leading to cell activation. Studies from our laboratory using sections of RA synovium have also localized TNFa and its receptors to endothelium.

TNFa has the potential capacity to regulate many of the events occurring in the RA microvasculature—leukocyte extravasation, chemotaxis, and angiogenesis [6]. E-selectin, which is not expressed on unstimulated endothelial cells, is induced in response to TNFa and IL-1b, with maximal expression observed after approximately 4 to 6 hours. Induction of ICAM-1 and VCAM-1 by TNFa and IL-10 occurs over a slower time-course, peaking at 16 to 24 hours. TNFa also induces production by endothelial cells of IL-6, GM-CSF, IL-8, monocyte chemoattractant protein-1 (MCP-1), RANTES, and Groa. The effects of TNFa on the angio-genic process are both stimulatory and inhibitory, depending on the experimental system. TNFa inhibits basal and FGF-2-stimulated endothelial cell proliferation and migration in vitro, but stimulates neovascularization in the rabbit cornea. TNFa treatment of endothelial cells has been reported to enhance urokinase-type plasminogen activator activity, which could contribute to the proangiogenic effects of TNFa. Brief exposure to TNFa induced release from endothelial cells of VEGF and FGF-2, whereas prolonged exposure of microvascular cells to TNFa inhibited capillary-like structure formation in vitro, suggesting that the net effect of TNFa on angiogenesis may reflect a balance of pro- and anti-angiogenic responses.

Another key endothelial cell activator is VEGF. Several members of the VEGF family have been described, including VEGF-A, -B, -C, and -D, which bind differentially to tyrosine kinase receptors VEGF-R1 or Flt (fms-like tyrosine kinase)-1, VEGF-R2 (kinase insert domain containing receptor or KDR/Flk-1) and VEGF-R3 (Flt-4). Signaling through VEGF-R1 appears to be important in controlling the number of endothelial cells during vessel formation, as well as in the organization of these cells during angiogenesis. Activation of VEGF-R2 enables the differentiation of endothelial cells and blood vessel formation [7]. As well as being essential for vasculogenesis and angiogenesis, VEGF is also capable of inducing procoagulant tissue factor and expression of cytokines and adhesion molecules. For example, VEGF was shown to induce expression of MCP-1 and IL-8, as well as ICAM-1, VCAM-1, and E-selectin. Essential to its proangiogenic function, VEGF can inhibit apoptosis, or programmed cell death. It has been shown, for example, that VEGF withdrawal results in obliteration of immature blood vessels. The antiapoptotic effects of VEGF are mediated via the induction of members of the Bcl family and inhibitors of apoptosis (IAP), that in turn inhibit terminal effector caspases-3 and -9. VEGF induces Bcl-2, as well as the IAPs survivin and XIAP. Thus the continued expression of VEGF in RA, as well as promoting angiogen-esis, is likely to sustain the vasculature, thus further augmenting the inflammatory process.

Finally, in the context of an ongoing inflammatory response, it is likely that under conditions of flow, cytokines such as TNFa may be less important as activators of vascular endothelium than cell-bound stimuli. Although activation of endothelial cells via soluble factors has been extensively studied, cell-cell interactions can also modulate the responses of the target cells, and could thus potentially further perpetuate inflammation. T cell adhesion to the endothelial lining of blood vessels is an early event in inflammation, and cell contact-mediated signaling to endo-thelial cells by stimulated T-cells could be an important endogenous mechanism of endothelial activation [8]. We recently described the ability of stimulated, but not resting, human T lymphocytes to modulate endothelial cell activation by direct cell-cell contact. T cells, either activated through the T cell receptor (TCR) with immobilized anti-CD3 monoclonal antibody or incubated in the presence of a combination of TNFa, IL-6, and IL-2, were able to induce endothelial cell production of chemokines and cytokines (MCP-1, IL-8, and IL-6), although the relative amounts of different cytokines were dependent on the method of T cell stimulation. For example, TCR-activated T cells induced relatively greater amounts of endothelial cell MCP-1 than did cytokine-activated T cells. In contrast, production of IL-6 was upregulated to a greater extent in the presence of cytokine-activated T cells. Furthermore, addition of anti-CD154 (anti-CD40 ligand) and anti-TNFa antibodies reduced release of IL-6, IL-8, and MCP-1 from endothelial cells cocultured with T cells. Such direct cell-cell contact between stimulated T lymphocytes and endothelium represents a novel pathogenic mechanism in inflammatory diseases such as RA. For example, overexpression of MCP-1 induced by TCR-activated lymphocyte-endothelial cell contact interactions might preferentially regulate the influx of monocytes into the RA lesions. Cytokine-activated lymphocyte-endothelial cells contact interactions might also contribute to the systemic inflammatory response in RA patients, for example, elevated expression of IL-6.

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