Targeting synovial neoangiogenesis in rheumatoid arthritis

    Targeting synovial neoangiogenesis in rheumatoid arthritis Agathe Leblond, Yannick Allanore, J´erˆome Avouac PII: DOI: Reference:

S1568-9972(17)30100-3 doi:10.1016/j.autrev.2017.04.005 AUTREV 1997

To appear in:

Autoimmunity Reviews

Received date: Accepted date:

27 February 2017 3 March 2017

Please cite this article as: Leblond Agathe, Allanore Yannick, Avouac J´erˆome, Targeting synovial neoangiogenesis in rheumatoid arthritis, Autoimmunity Reviews (2017), doi:10.1016/j.autrev.2017.04.005

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ACCEPTED MANUSCRIPT Targeting synovial neoangiogenesis in Rheumatoid arthritis. Agathe Leblond1, Yannick Allanore (MD, PhD)1,2, Jérôme Avouac (MD, PhD)1,2 1

Université Paris Descartes, Sorbonne Paris Cité, INSERM U1016 and CNRS UMR8104,

Université Paris Descartes, Sorbonne Paris Cité, Service de Rhumatologie A, Hôpital

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Institut Cochin, Paris, France

Corresponding author: Dr. Jérôme Avouac Service de Rhumatologie A, Hôpital Cochin

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Université Paris Descartes 27 rue du Faubourg Saint-Jacques

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75014 Paris, France Telephone: + 33 1 58.41.25.63

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Cochin, Paris, France

Fax: + 33 1 58.41.26.24

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e-mail: [email protected]

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Total word count: 4389

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Acknowledgement: “Arthritis foundation” and “Société Française de Rhumatologie”

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The authors have no competing interest to declare

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ACCEPTED MANUSCRIPT Abstract: In Rheumatoid arthritis (RA), neoangiogenesis is an early and crucial event to promote the development of the hyperplasic proliferative pathologic synovium. Endothelial cells are critical for the formation of new blood vessels since they highly contribute to

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angiogenesis and vasculogenesis. Current therapies in RA target the inflammatory

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consequences of autoimmune activation and despite major improvements these last years still refractory patients or incomplete responders may be seen raising the point of the need to

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identify complementary additive and innovative therapies. This review resumes the mechanisms of synovial neoangiogenesis in RA, including recent insights on the implication of vasculogenesis, and the regulation of synovial neoangiogenesis by angiogenic and

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inflammatory mediators. In line with the recent development of vascular-targeted therapies used in cancer and beyond, we also discuss possible therapeutic implications in RA, in

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particular the combination of targeted immunotherapies with anti-angiogenic molecules.

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Keywords: Rheumatoid arthritis, synovial angiogenesis, vasculogenesis, endothelial cells.

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Highlights

Neoangiogenesis is a key process in the development of the synovial pannus.

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The formation of new synovial vessels depends on angiogenesis and vasculogenesis

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A positive feedback loop exists between synovial inflammation and neoangiogenesis

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Hypoxia and VEGF are the most potent proangiogenic growth factors in RA synovium

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Modulation of angiogenesis in experimental arthritis has shown promising results

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ACCEPTED MANUSCRIPT Introduction: Rheumatoid arthritis (RA) is the most common cause of chronic inflammatory arthritis with a

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prevalence ranging from 0.5% to 1% of the adult population worldwide (1, 2).

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The synovium is the primary site of the inflammatory process in RA. The synovium becomes

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inflamed, with infiltration of blood-derived inflammatory cells at the interface between cartilage and bone. This invasive and destructive front (termed ‘pannus’) promotes the development of the erosions observed in RA. Progressive destruction of the articular cartilage,

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subchondral bone and periarticular soft tissues results in deformities that characterize long-

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standing RA. These deformities lead to functional deterioration and long term profound irreversible disability.

An important feature of RA is the role of vascular structures in these invasive and destructive

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processes. Indeed, there are increased number and density of synovial blood vessels in RA, which are required to supply the expansion of synovial tissue and to develop the hyperplasic

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and invasive nature of RA synovium. Endothelial cells (ECs) lining the blood vessels also appear to be an active target for the action of cytokines, growth factors, permeability factors,

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and matrix-degrading enzymes. EC response to these factors both maintain and promotes RA. Thus, neoangiogenesis appears to be central to maintaining and promoting RA. It is also possible that a potential method of attenuating the development of the pannus is to interfere with its blood supply. This possibility is supported by several studies in animal models of arthritis which have suggested that blocking angiogenesis during the course of RA might actually be of therapeutic benefit. The aim of this review is to summarize the current understanding of synovial neoangiogenesis in RA and its regulation. We are also giving a focus on the application of angiogenesis inhibitors in animal models of arthritis, and on the potential for development of new vasculartargeted therapies for treatment of RA.

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ACCEPTED MANUSCRIPT 1/ Increased vascular density in RA synovium and the contrast with tissue hypoxia The synovium is normally a physiologically relatively paucicellular structure with a delicate

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intimal lining between macrophage-like and fibroblast-like synoviocytes. This lining region is

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one or two cells deep and is highly vascularized. Synovial blood flow provides oxygen and

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nutrients to the synoviocytes and to the avascular articular cartilage.

RA is first characterized by a transitory pre-vascular highly inflammatory stage, followed by a prominent vascular stage with a strong increase in vessel growth.

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The pre-vascular stage is characterized by a marked hyperplasia of macrophage-like and

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fibroblast-like synoviocytes in the lining layer. In parallel, the sublining layer is infiltrated by CD4+ T cells, B cells and macrophages, leading to the formation of an invasive and destructive front, called the synovial pannus. This pannus acts like a local tumor that invades

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and damages cartilage and bone (2) (Figure 1). Then, the vascular stage rapidly arises and is usually set at the time of RA clinical diagnosis.

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In this stage, capillary density is increased in the RA synovium, with a more deeply distribution as compared with normal tissue (Figure 2) (3, 4). Increased density of sublining

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blood vessels perpetuates synovitis by increasing the delivery of nutrients and oxygen to the proliferating pannus, and allows immune cells to emigrate from the blood into inflamed synovium where they highly produce a network of pro-inflammatory cytokines and chemokines (5). The increased number of blood vessels correlates with synovial hyperplesia, mononuclear cell infiltration and tender/swollen joint counts (6). Vascular proliferation is usually primarily detected in inflamed joints, whereas mononuclear cell infiltration and increased thickness of the synovial lining layer are observed both in inflamed and noninflamed joints (3). Despite increased vessel density related to active endothelial proliferation and increased EC survival, synovium in RA is chronically hypoxic, particulary in the lining layer (3) (Figure 1).

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ACCEPTED MANUSCRIPT The presence of reduced oxygen levels in the RA synovium was demonstrated by direct measurements of the oxygen tension and more indirectly by an increase of hypoxic

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metabolites in the synovium (7). This observation is not unexpected given the raise in

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synovial cell proliferation and the consequent increase in the distance between the

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proliferating cells and the nearest blood vessels. This leads to a growing metabolic demand for oxygen and nutrients resulting in local hypoxia and relative hypoperfusion. Moreover the vasculature of RA synovium is compromised by movement and accumulation of synovial

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fluid, thus exacerbating hypoxia in an already ischemic environment. Such a combination of

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increased metabolic demand and hypoxia is a potent signal for new vessel formation.

2/ Source of new synovial blood vessels

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The recruitement of ECs is required to form new vascular structures. ECs can be recruited locally through angiogenesis, defined by the capillary sprouting of resident ECs. Circulating

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bone marrow-derived endothelial progenitor cells (EPCs or hemangioblasts) are a second potential source of ECs.

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Angiogenesis

Angiogenesis is the growth of new blood vessels from existing ones and is an important aspect of new tissue development, growth and repair. The numerous proangiogenic signals that target ECs derive from cells primed in an abnormal environment, where the proliferation rate exceeds the supply of nutrients and oxygen (8). Angiogenic stimulation is triggered in RA synovium by the proinflammatory and hypoxic miceoenvironment, with production of a large array of growth factors, cytokines, and chemokines. These factors induce the sprouting of ECs from preexisting vessels, their proliferation and migration into inflamed sites, launching the vascular stage of the disease (Figure 1). This sequence is not specific to RA and is observed in other diseases with angiogenic compentent including cancer, diabetes, and other chronic

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ACCEPTED MANUSCRIPT inflammatory conditions. However, specific cells from the synovium are also able to early drive angiogenenis together with ECs. Indeed, local synovial inflammation drives resident

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stromal synovial cells to acquire a pro-angiogenic profile. Synovial fibroblast from RA

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synovium are sufficient under hypoxic conditions to induce angiogenesis, when used in a

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matrigel plug system engrafted in immunodeficient mouse. (9, 10). In addition, these cells express growth factors (VEGF, b FGF, TGFβ), cytokines (IL-6, IL-8), chemokines (CXCL12), adhesion molecules (ICAM-1, VCAM-1) and matrix remodeling enzymes

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(MMP1, 2, 3 and 9) that regulate angiogenesis.

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Infiltrating macrophages are also a major source of pro-angiogenic molecules producing a broad range of mediators including growth factors, chemokines and matrix-remodeling enzymes (11).

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In RA, increased angiogenesis is associated with morphological alterations of new formed vessels. This fraction of neoangiogenic immature, dilated and leak vessels lacks -smooth

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muscle actin (-SMA) positive mural cells. Chronic VEGF overexpression is implicated in this imbalance between EC proliferation and the lack of concomitant development of pericyte

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coverage. These small size vessels are preferentially located in sublining layer and are surrounded by inflamatory infiltrates (12) (Figure 1). Interestingly, disease progression and activity are related to the density of immature vessels, which is the sole vessel fraction to regress in response to anti-TNFα therapy (12). Vasculogenesis EPCs, first described by Asahara and al (13), are a population of bone marrow-derived cells characterized by the presence of surface markers such as CD34, VEGF receptor-2 (VEGFR-2 or kinase-insert domain receptor, KDR) and CD133, able to differentiate into mature endothelial cells and to participate in the formation of new blood vessels (14) (Figure 1).

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ACCEPTED MANUSCRIPT Two studies have reported the presence of endothelial precursor cells in the synovial tissue of RA patients. In a first study (15), a population of cells expressing CD34 on their surface was

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found in the synovial tissue of 18 RA patients. These cells were detected in close proximity to

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CD133+ cells, forming cell clusters in the area under the synovial membrane. CD34+

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precursor cells produced high levels of the chemokine receptor CXCR4, and VEGFR-2 was expressed on CD34+ and CD133+ cells. In the second study (16), CD34+ cells, purified from the bone marrow of 13 patients with active RA and 9 control subjects, were cultured in the

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presence of stem cell factor and GM-CSF. Significantly more von Willebrand factor-positive

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cells (vWF+) and CD31+/vWF+ cells were generated from RA bone marrow-derived CD34+ cells for RA than control samples. The generation of vWF+ cells from bone marrow CD34+ cells was related to the microvessel densities in the synovial tissues. Thus, bone marrow

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CD34+ cells may contribute to synovial neovascularization by supplying endothelial precursor cells and may be important in the pathogenesis of RA.

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In the peripheral blood of RA patients, contradictory results have been reported (17-21), which could be related to several methodological issues or the characteristics of the RA popu-

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lation enrolled. EPC depletion in the peripheral blood has been reported, which could result from their enhanced recruitment from peripheral blood to synovium. On the other hand, our group has reported increased circulating EPC levels in RA patients, with a correlation between EPC counts and RA disease activity (22). These results were obtained in a relatively large number of patients by using a well-characterized definition of late-outgrowth EPCs and fulfilling recent recommendations for the detection of EPCs. These results emphasize a key role for vasculogenesis in RA, and the association with disease activity supports the implication of vasculogenesis in the perpetuation of synovitis.

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ACCEPTED MANUSCRIPT 3/ Regulation of synovial neoangiogenesis in RA Neovascularization in RA is dependent on the balance of proangiogenic and antiangiogenic

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mediators, including growth factors, cytokines, chemokines, cell adhesion molecules and

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matrix metalloproteinases (23).

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Hypoxia and Hypoxia inducible factor 1-alpha (HIF-1α)

RA synovium is characterised by chronic tissue hypoxia, which is partly related to synovial cell hyperplasia and the disorganization of vascular system within RA synovium. Hypoxia

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may be responsible for rendering RA synovial lining pro-angiogenic and pro-invasive, thus

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contributing to synovial and cartilage damage. Indeed, upregulation of metalloproteinases and RA fibroblast migration across collagen have been showed under hypoxic conditions (24). Furthermore, expression of angiogenic stimuli, including vascular endothelial growth factor

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(VEGF), and VEGF/placental growth factor heterodimer, was also increased. And crucially, it has been demonstrated that hypoxia increases the angiogenic drive of RA cells, as

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demonstrated by enhanced blood vessel formation in an in vitro angiogenesis assay (24). A key regulator of angiogenesis is the transcription factor HIF (Hypoxia-Inducible Factor)

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which consists of an inducible oxygen-regulated subunit (HIF-1α) and a  subunit constitutively expressed in the nucleus (HIF-1β). Regulation of HIF-dependent gene expression requires -subunit accumulation in the cytoplasm and translocation into the nucleus, which enables it to dimerize with -subunits of HIF. HIF heterodimers are then recognized by co-activators and bind to the hypoxia response elements in the target gene to initiate transcription In RA, two, closely related HIF- isoforms (HIF-1, HIF-2) are expressed in human RA synovium and also in experimental rat arthritis, suggesting that synovial hypoxia leads to upregulation of HIF in the synovial joint, accumulation of VEGF and induction of synovial angiogenesis (25-27) (Figure 1). The number of HIF1-α positive cells correlated strongly

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ACCEPTED MANUSCRIPT with the number of blood vessels in RA synovial tissue and with inflammatory EC infiltration, cell proliferation and the synovitis score. Some studies showed the beneficial

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effect of compound targeting HIF-1α on the neovascularization in the RA. Benzophenones are

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potent anti-inflammatory compounds and are tested for anti-arthritic and anti-angiogenic

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agents in animals models (28). A benzophenone analogue, 2-benzoyl-phenoxy acetamide BP1, was known to inhibit HIF-1α mediated VEGF expression and angiogenesis (29). It was shown that BP-1 inhibited HIF-1α nuclear accumulation in HUVECS and in the synovium

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tissue of adjuvant induced arthritis (AIA) rats (30). Moreover BP-1 inhibited the hypoxia

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induced increase of VEGF gene expression in HUVECS and also in AIA rats. The treatment with BP-1 in synovium section of AIA rats were characterized by a pronounced decrease in vascular density causing in a reduction of arthritis severity in AIA rats. More recently, a high-

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mobility group box protein 1 (HGMB1), a nonhistone nuclear protein and a cytokine mediator, have been shown to be implicated in the pathogenesis of RA (31). Stimulation with

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HGMB1 increased both HIF-1α mRNA and protein levels in synovial fibroblasts obtained from RA patients. Moreover, HGMB1 induced HIF-1α transcription in RA fibroblasts occurs

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via NF-ΚB activation. The use of anti-HGMB1 neutralizing antibody prominently ameliorated the inflammatory features in collagen-induce arthritis (CIA) mice and also showed a significant reduction in the number of vessels in CIA mice in association with diminished HIF-1α and VEGF levels in the knee joints of CIA mice. VEGF signaling VEGF is the main signaling protein involved in angiogenesis ; it is detected in RA in synovial tissue and fluid, as well as in serum (32). During RA, VEGF165 is the mainly involved isoform (8) and it exerts its effects through tyrosine kinase receptors Flt-1 (fms-like tyrosine kinase receptor; also known as VEGF-R1) and Flk-1/KDR (fetal liver kinase receptor/kinaseinsert-domain-containing receptor; also known as VEGF-R2). The dual activities of VEGF as

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ACCEPTED MANUSCRIPT an endothelial-cell mitogen and a modulator of changes in vascular permeability are of relevance in the pathogenesis of RA (Figure 1). Immunohistochemical and in situ

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hybridization studies of synovial tissues have shown that VEGF is strongly expressed by

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subsynovial macrophages, fibroblast surrounding microvessels, vascular smooth muscle cells,

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and synovial lining cells (33). The VEGF expression level in synovial fluid and tissues correlates with the clinical severity of human RA and with the degree of joint destruction (34, 35). Interestingly serum VEGF concentrations have been found higher in patients with newly

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diagnosed RA than in those with long-standing, treated disease. This observation may

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represent a response to drug treatment, supporting several observations demonstrating reduction in serum VEGF concentrations after therapeutic intervention. In fact patients with early RA responding to disease-modifying antirheumatic drugs showed significant reductions

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in serum VEGF concentrations compared to non esponders to the same treatment, who showed no significant change in serum VEGF (36). Furthermore, blockade of TNF- in RA

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results in early marked reduction, but not normalization, of serum VEGF concentrations in a dose-dependent manner (37). The synthesis of VEGF by inflammatory and synovial cells is

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induced by numerous cytokines (such as TNF-, IL1-, TGF- and platelet derived growth factor, PDGF), by oxidative stress and by hypoxia (34, 35) (Figure 1). Increasing evidence suggests a complex interaction between VEGF and the angiopoietin (Ang)/Tie-2 system. It is demonstrated that Ang/Tie-2 is overexpressed in human RA synovium (38). Tie-2 signaling regulates arthritis-related angiogenesis in vivo and Ang-1 stabilize new blood vessels (Figure 1). Ang-1 is secreted by pericytes or synoviocytes and activation of Tie2 signalling through Akt pathway is responsible for endothelial cell proliferation and survival. Indeed, TNF-α upregulates Tie-2 in ECs through NFκB and increases the expression of Ang-1 in synoviocytes (39, 40). Gene therapy with the used of a soluble Tie-2 receptor inhibits neovascularization and arthritis development and protects

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ACCEPTED MANUSCRIPT against bone destruction in a mouse model of CIA (41). Interestingly, the effect of a bispecific antibody targeting TNF- and Ang-2 in a TNF- transgenic mouse model of arthritis has

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been recently described (42). The authors has showed a dose-dependent reduction in both

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clinical symptoms and histological scores with the use of this antibody. Moreover the

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magnitude of results observed with this bispecific antibody were more important than those observed with adalimumab. Cytokines

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It is known that many of cytokines such as TNF-α, IL6, IL1-β or IL17 are important

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contributors in RA and may even work in part by regulating neovascularization in the arthritic joint.

TNF-α is a potent inducer of new blood vessel growth (43). Exposure of endothelial cells to

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TNF-α has been reported to induce the release of VEGF and FGF-2 (44) (Figure 1). Production by synovial-joint cells of angiogenic cytokines such as VEGF is at least in part

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induced by TNF-α. Moreover, anti-TNF-α therapy was found to reduce the levels of circulating VEGF, the synovial blood flow assessed by power Doppler ultrasound (45), and

(46).

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the synovial vascularity assessed by immunostaining for CD31 and von Willebrand factor

IL-17 is found in RA synovial tissue and fluid, and the percentage of TH-17 cells is significantly higher in RA synovial fluid compared to RA or normal peripheral blood (47, 48). It was shown that the local expression of IL-17 increases vascularity in mouse ankle joints (49). Moreover, IL-17 markedly promotes blood vessels growth using matrigel plugs in C57BL/6 mice. In vitro, IL-17 induces endothelial Human microvascular endohtelial cells (HMVEC) migration and the used of neutralizing antibody to IL-17 suppressed HMVEC migration (49). IL-17 induced HMVEC chemotaxis and tube formation are mediated

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ACCEPTED MANUSCRIPT primarily through IL-17 receptor (R)-C. Neutralization of either IL-17 in RA fluids or IL17R-C on HMVECS significantly reduces the induction of HMVEC migration.

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Chemokines

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In RA, synoviocytes and inflammatory cells produces chemokines which plays a central role

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in atttracting additional leucocytes. Among these chemokines, one has been mainly studied in RA. CXCL12 or stromal cell derived factor-1 (SDF-1) and it’s receptor, CXCR4, are present in joints of RA patients (50, 51). The injection of a CXCR4 antagonist (52), the bicyclam

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derivative AMD3100, in IFN-γR-deficient DBA/1 mice showed a reduction of incidence of

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arthritis, disease onset and clinical scores. Moreover, the histological examination demonstrated the protective effect of AMD3100 in IFN-γR-deficient DBA/1 mice. Indeed, infiltration of mono-and-polymorphonuclear cells, hyperplesia of the synovium and pannus

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formation were dramatically reduced in IFN-γR-deficient DBA/1 mice. More recently, CXCR7 was identified as an alternative receptor for CXCL12. CXCR7 is

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expressed on RA synovial endothelial cells (53). The use of CXCR7 inhibitor, CCX733, inhibited CXCL12-induced angiogenesis, reduced clinical arthrtitis scores and prevented joint

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destruction in mice with CIA (53). Treatment with a humanized antibody against CXCL12, ch30D8, ameliorated arthritis in CIA mice (54). Indeed, this antibody reduced disease progression, clinical score and bone-erosive changes in CIA mice. The chemokine CXCL16 is also overexpressed in RA SF (55). The depletion of CXCL16 in CIA mice reduced clinical arthritis score, infiltration of inflammatory cells and bone destruction in the synovium of mice with CIA (56). The presence of CXCR6 in HMVECs and EPCs may be upregulated by IL-1β (57) and in vitro CXCL16 can induce HMVEC migration and tube formation. In vivo, intragraft with RA SF immunodepleted of CXCL16 in SCID mice showed a significant diminution in EPC recruitment.

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ACCEPTED MANUSCRIPT 4/ Targeting angiogenesis: Therapeutic application in RA Effects of usual RA trearment on angiogenesis

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Disease-modifying antirheumatic drugs (DMARDs) might affect synovial angiogenesis.

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Methotrexate (MTX) has been shown to inhibit vascular endothelial cell proliferation in vitro

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(58). However, MTX failed to decrease VEGF levels in patients with RA and did not significantly reduce vessel growth in a murine in vivo matrigel model for angiogenesis.(59, 60).

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The use of TNF- inhibitors in RA results in early marked reduction, but not normalization,

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of serum VEGF concentrations in a dose-dependent manner (37, 59, 61). Moreover, antiTNF-α therapy was found to reduce the synovial blood flow assessed by power Doppler

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ultrasound (45), and the synovial vascularity assessed by immunostaining for CD31 and von

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Willebrand factor (36).

Regarding other biologics, treatment with abatacept was associated in RA patients with

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reduced serum levels of A disintegrin and metalloprotease 17 (ADAM17), which is recognized as an important player in exacerbation of inflammation related with increased

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activities of angiogenesis (62). A single study has observed reduced microvessel density in synovial tissues of RA patients treated with tocilizumab, as well as degeneration of lining layers (63).

Available preclinical evidence of anti-angiogenic treatments Direct Targeting of the VEGF pathway may be beneficial in treating RA. In the model of transgenic K/BxN mice, treatment with anti-VEGF-RI strongly attenuated the disease clinically and on histological analyses throughout the study period, while anti-VEGF only transiently delayed disease onset (64). Treatment with anti-VEGF-RII had no effect. Treatment with a VEGF-RI tyrosine kinase inhibitor almost abolished the disease. These results show that VEGF is a key factor in pannus development. This has been emphasised by

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ACCEPTED MANUSCRIPT data showing that enhanced expression of PlGF and VEGF-R1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines (65).

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Of the most interest, treatment with Avastin (Bevacizumab), a VEGF humanized monoclonal

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antibody, on collagen induced arthritis in rat model reduces significantly the arthritis index,

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synovial pathological injury index and serum levels of VEGF and TNFα (66). Avastatin exhibits similar effects to Etanercept to relieve RA in rat model Moreover, angiostatin is known to be an endogenous inhibitor of angiogenesis. It has been described that angiostatin

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inhibits primary tumor growth and metastasis in vivo (67) and endothelial cells migration and

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induces endothelial cell-specific apoptosis in vitro (68). The use of a HIV vector containing the murine angiostatin expression unit significantly reduced synovial cell hyperplesia, pannus formation and progression of CIA (69).

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Other anti-angiogenic treatements have been used, which indirectly target the VEGF pathway. TNP-470, an angiogenesis inhibitor derived from fumagillin, has proven its efficacy in

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inhibiting tumor growth and metastasis in various animals models (70-72). TNP-470 has been shown to inhibit angiogenesis and, in particular, VEGF synthesis (71, 72). The preventive

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treatment with TNP-470 (73), at a dosage of 60 mg/kg of body weight, delayed the onset of arthritis and its clinical intensity was rather mild. At a dosage of 90 mg/kg of TNP-470 the appearance of clinical signs was delayed for a longer period of time and disease was almost abolished. Moreover, the treatment with TNP-470 reduced cartilage and bone destruction, as well as circulating VEGF blood levels. Norisoboldine (NOR), an alkaloid coumpound, can significantly suppress synovial angiogenesis by selectively inhibiting endothelial cell migration (74). Indeed, the use of NOR in the adjuvant-induced arthritis rat model caused a significant reduction in the number of blood vessels and inhibited VEGF-induced endothelial cell migration via the CAMP-PKANF-ΚB/Notch 1 signaling.

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ACCEPTED MANUSCRIPT Treatment with a recombinant human (rh) endostatin in the adjuvant-induced arthritis rat model led to decreased VEGF expression in both cartilage and synovial tissue, which was

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accompanied by an attenuation of paw swelling and a reduction in new blood vessels

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formation (75). Moreover, administration of rh endostatin in rat adjuvant arthritis led to the

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downregulation of VEGF, TNF-α and IL-1β production, wich was associated with decreased arthritis severity and bone destruction (76). In vitro, treatment with rh endostatin showed a reduction of osteoclast formation and bone resorbing function (76).

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Levels of resistin, a small cysteine-rich adipose-derived peptide hormone were overexpressed

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in the synovial fluid and tissue from patients with RA and mice with CIA. Resistin promoted the homing of EPCs into the synovium, thereby inducing angiogenesis (77). Resistin also

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and neoangiogenesis in vivo.

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directly induced a marked VEGF expression in EPCs and its inhibition reduced EPC homing

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Conclusion and research agenda

Neoangiogenesis is a dynamic and a key process in the development and the perpetuation of

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RA. New vessel formation depends on two main processes that are angiogenesis and vasculogenesis, which lead to the recruitment of resident and systemic ECs, respectively. A positive feedback loop exists between chronic inflammation and neoangiogenesis. Inflammatory state promotes enhanced synovial neovasvularization and new blood vessel formation facilitates chronic inflammation, leading to bone and cartilage damages. However, the question whether angiogenesis drives RA or whether the enhanced synovial proliferation promotes angiogenesis is still unsolved since angiogenesis, hypoxia and synovial hyperplasia are intricately linked and are regulated by common mediators. Multiple molecules contribute to the tight regulation of the angiogenic processes, especially hypoxia and VEGF, which is one of the most potent pro-angiogenic growth factors identified.

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ACCEPTED MANUSCRIPT VEGF is implicated in orchestrating the generation of synovial neovessels, and persistent VEGF overexpression leads to the development of an altered vasculature characterized by

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serious structural and functional defects.

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The modulation of many pro- and anti-angiogenic molecules in mouse models of

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experimental arthritis has shown promising results. Consequently, targeting angiogenesis might be considered as a new alternative in the treatment of RA. This is supported by the fact that about 30% of patients have a primary or secondary lack of response, regardless the

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inflammatory cytokine or the immune cell targeted by the different available drugs. However,

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several questions need to be addressed prior positionning anti-angiogenic therapies in RA. The first question is the selection of the most accurate angiogenic pathway to target. Based on the data presented in this review, VEGF pathway seems the most appropriate given the

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prominent synovial and systemic overexpression of this growth factor in RA. Moreover, promising results both on efficacy and safety of anti-VEGF therapies in cancer and other

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chronic diseases are available. Combination of anti-VEGF, bevacizumab and chemotherapy in many cancers (neuroblastoma, melanoma, malignant glioma, metastatic colerectal cancer,

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lung cancer and cervical cancer) showed a significant survival benefit in comparaison with chemotherapy alone (78-87). Moreover, the effect of bevacizumab combined with chemotherapy was better when bevacizumab was administered few days (3 days) before chemotherapy. In addition, inhibition of angiogenesis by anti-VEGF therapy (bevacizumab) has now been used in other diseases than e.g age-related macular degeneration (88) or diabetic retinopathy (89). Research agenda proposed to further address this question: further research is still required to definitely validate the choice of VEGF or identify another candidate molecules or pathways that could be targeted. Several proangiogenic or angiostatic factors have not been studied in RA (Table 1). Moreover, unbiaised genomic approaches on ECs isolated from

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ACCEPTED MANUSCRIPT patients with RA are ongoing and could help to identify pertinent candidates to be further validated.

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The second question is the selection of the most appropriate patients who could receive anti-

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angiogenic therapies. It makes sense to primarily focus on patients in the vascular phase of the

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disease. High circulating levels of angiogenic biomarkers might be helpful for this selection, but the precise nature of the biomarkers should be assessed, as well as a routine dosage of these markers. Power Doppler ultrasound might also be useful to select patient with high

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synovial blood flow. Several data support to focus on patiens with newly diagnosed patients

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given the detection of high VEGF serum and synovial fluid levels, compared to patients with long lasting disease. Another important point to consider is the high cardiovascular risk of RA

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upon antiangiogenic therapies.

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patients, given the demonstration of reduced collateral vessel formation following ischaemia

Research agenda proposed to further address this question: Further studies assessing the

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link between angiogenic markers, clinical/biological signs of disease activity and synovial blood flow on doppler are required to better define the population that could benefit of anti-

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angiogenic therapies.

The third question is the administration of angiogenic therapies alone or in combination. The combination of an anti-angiogenic theray with a drug targeting the inflammation process seems more appropriate, by analogy with cancer therapy, in which of cytotoxic treatments are combined with anti-angiogenic molecules (90). Treatment targeting inflammation has already a variable anti-angiogenic effect, but which is in all cases incomplete. The addition of an antiangiogenic treatment could help to reach a complete effect on the pathological angiogenic process.

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ACCEPTED MANUSCRIPT Research agenda proposed to further address this question: New preclinical studies testing this combination therapy is required before positionning anti-angiogenic drugs with

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human proof of concept studies.

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Criscione LG, E.W. SC. Tumor necrosis factor-alpha for the treatment of rheumatic

Firestein GS. Starving the synovium: angiogenesis and inflammation in rheumatoid

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arthritis. J Clin Invest. 1999;103:3-4.

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Chemokines

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Angiogenin ANG-1

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IL-8 (CXCL8)

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Other mediators

Anti-angiogenic molecules

TGF-

TNF- IL-1

Cryptic proteins

Proangiogenic molecules

IL-12

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Cytokines

VEGF FGF-1, FGF-2, HGF TGF- PDGF PlGF G-CSF

D

Growth factors

Anti-angiogenic molecules

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Proangiogenic molecules

Molecules suspected to contribute to synovial neoangiogenesis

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Molecules known to contribute to synovial neoangiogenesis

Endostatin Angiostatin

Fibronectin fragment Kringle-5 Vasostatin Midkine, Pleiotrophin Ephrin-B2 Ephrin B4

Cartilage-derived inhibitor Interferon-inducible protein (IP-10) TIMPs Plasminogen activator inhibitor Platelet factor-4 Thrombospondin-1

VEGF: Vascular Endothelial Growth Factor, FGF: Fibroblast Growth Factor, HGF: Hepatocyte Growth Factor, TGF-β: Transforming Growth Factor beta, PDGF: Plateletderived Growth Factor, PIGF: Phosphatidylinositol-glycan biosynthesis class F protein, GCSF: Granulocyte colony-stimulating factor, TNF-α: Tumor Necrosis Factor α, IL-1β: Interleukin-1β, IL-12: Interleukin-12, IL-8: Interleukin-8, ANG-1: Angiopoietin-1, TIMPs: Tissue Inhibitor of metalloproteinase.

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ACCEPTED MANUSCRIPT Figure legends

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Figure 1: Mechanisms of synovial neovasculariation in rheumatoid arthritis A, Rheumatoid Arthritis (RA) is characterized by inflammatory polyarthritis involving particularly metacarpophalangeal and proximal interphalangeal joints. B, The synovium (in red) is the primary site of inflammation in RA leading to bone erosions and cartilage destruction. C-F: Neoangiogenesis is a crucial factor for the development of the synovial pannus. The lining layer becomes hypertrophic due to the marked hyperplasia of fibroblast-like synoviocytes. This cell hyperplasia promotes chronic tissue hypoxia with activation of the transcription factor HIF (Hypoxia inducible factor), leading to VEGF overexpression in the sublining layer of the synovium, the synovial fluid and the serum. The synovial sublining layer is characterized by increased vessel density, which perpetuates synovitis by allowing inflammatory cells to emigrate from the blood to inflamed synovium. These cells secrete matrix metalloproteinases and activate the RANK/RANKL pathway to induce bone and cartilage damages. Increased vessel density contrasts with hypoxic areas favored by synovial cell proliferation and formation of morphologically abnormal, immature and non-functional vessels secondary to chronic VEGF overexpression. VEGF is the main signaling protein involved in synovial angiogenesis. Hypoxia of the lining and sublining layers and inflammatory cytokines are the major source of VEGF production. VEGF induces the proliferation and the migration of endothelial cells (ECs) to form new blood vessels (D). The proinflammatory cytokine TNF- stimulates the production of VEGF through the NFKB pathway in ECs (E) and fibroblast-like synoviocytes (F). Angiopoietin-1 synthetized by pericytes stimulates vessel stabilization (D) through the activation of the tie2/AKT pathway (E). The overexpression of joint and systemic VEGF stimulates vasculogenesis with the recruitment of endothelial progenitor cells from the bone marrow (EPCs) (C). Circulating EPCs are recruited in the inflamed synovial to stimulate synovial neovascularization (D).

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Figure 2: Sections of synovial tissue stained for CD31 from a patient with osteoarthritis (OA) (A) and rheumatoid arthritis (RA). Increased vessel density is observed in the syovial tissue of the patient with RA compared to the patient with OA (black arrows). Perivascular inflammatory infiltrates are also visible in the patient with RA.

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