The potential role of angiogenic factors in rheumatoid arthritis.

International Journal of Rheumatic Diseases 2014; 17: 369–383

REVIEW ARTICLE

The potential role of angiogenic factors in rheumatoid arthritis Gholamreza AZIZI,1 Roobina BOGHOZIAN2 and Abbas MIRSHAFIEY2 1 Imam Hassan Mojtaba Hospital, Alborz University of Medical Sciences, Karaj, and 2Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

Abstract Angiogenesis is an important phenomenon in the pathogenesis of some diseases, such as numerous types of tumors and autoimmunity, and also a number of soluble and cell-bound factors may stimulate neovascularization in inflammatory reaction processes. Here, by highlighting the significance of angiogenesis reaction in rheumatoid arthritis (RA), we will mainly focus on the role of various growth factors, cytokines, enzymes, cells, hypoxic conditions and transcription factors in the angiogenic process and we will then explain some therapeutic strategies based on blockage of angiogenesis and modification of the vascular pathology in RA. Key words: angiogenesis, cytokines, hypoxia, RA, VEGF.

ANGIOGENESIS AND ANGIOGENIC FACTORS Angiogenesis is the growth of new capillaries from preexisting vasculature and is classically defined as the process of development and formation of new blood vessels that occurs during the growth and development of tissues. It can also play a key role in several physiological events, including embryonic development, reproduction, tissue repair and normal wound healing.1,2 There are two separate pathological angiogenesis: 1 Excessive angiogenesis; in some diseases the excessive angiogenesis plays an essential role in pathological processes, such as diabetic retinopathy, rheumatoid arthritis (RA), osteoarthritis, psoriasis, atherosclerosis and neoplasms.3 In many cancers, following tumor growth, neovascularization could be a negative prognostic indicator signifying aggressive disease and increased metastasis.4

Correspondence: Professor Abbas Mirshafiey, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Box 6446, Tehran-14155, Iran. Email: [email protected]

2 Insufficient angiogenesis; delayed wound healing, and also the lack of angiogenesis, may lead to cardiac failure and limb ischemia as well as stroke.1,2 Angiogenesis is a complex multistep process requiring stimulation of proliferation and migration of endothelial cells (ECs). It involves a series of coordinated events: activation of ECs, disruption of vascular basement membrane and extra-cellular surrounding matrix, migration of the ECs to distal sites, proliferation of ECs, differentiation of ECs, and subsequent formation and maturation of new blood vessels.5 Blood vessels are composed of two interacting cell types. ECs form the inner lining of the vessel wall, and pericytes (mural cells or vascular smooth muscle cells) envelop the surface of the vascular tube. In the past decades, investigations of blood vessels had focused mainly on the ECs component, while the interest in pericytes had lagged behind. Recently, pericytes have acquired new consideration as critical contributors to angiogenesis and potential therapeutic targets for antiangiogenic treatment.6 Furthermore, the heterotypic interactions of pericytes and ECs and extracellular matrix (ECM) components, such as the neural cell adhesion molecule (NCAM) are critical for congregation, stability and maturation of

© 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd

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blood vessels.7 It is demonstrated that an unstable EC and pericyte interaction and vessel survival deficit are related to NCAM deficiency.8 This process is dependent on cell survival signals, which may affect nuclear instability, including telomere length shortening induced by high levels of oxidative stress.9,10 Angiogenesis phenomenon is self-restricted and tightly controlled by proangiogenic stimulators and antiangiogenic inhibitors. These factors comprise several cell types and mediators, which are found both in peripheral blood and in affected tissues.11 These elements can be divided into two overall groups: one group is composed of factors affecting EC proliferation and differentiation and the other group is composed of the factors affecting only EC differentiation but not their proliferation.12 Humeral factors and cells, which interact to establish a specific microenvironment suitable for new vessel formation, are vascular endothelial growth factor (VEGF), angiopoietin (Ang), placenta growth factor (PLGF), platelet-derived growth factor (PDGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor (TGF)-b, cytokines (tumor necrosis factor [TNF]-a, interferon [IFN]-c, interleukin [IL]-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, IL-18 and IL-19), chemokines (C-C motif ligand 2 [CCL2], C-X-C motif ligand 1 [CXCL1], CXCL2, CXCL4, CXCL8 and stromal cell-derived factor 1 [SDF-1]), enzyme (galectins and matrix metalloproteinases [MMPs]), neutrophils, monocytes, macrophages and lymphocytes (Table 1).1,13 These mediators affect EC function in the angiogenesis process. However, some of them promote angiogenesis while others have angiostatic properties. Moreover, differential interactions between some of them, including VEGF, Ang/ Tie-2 system and PLGF, PDGF or TGF-b, are critically important for determining blood vessel maturity, stability and survival.14,15

cells and humoral factors, such as cytokines (IL-1b, IL6, TNF-a and IL-18), chemokines (CXCL-8, CXCL-10, monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1 [MIP-1] and RANTES [regulated upon activation, normal T cell expressed and secreted]), cell adhesion molecules (intracellular [I] CAM-1, vascular [V]CAM-1, P- and E-selectin), growth factors (VEGF, PLGF, IGF and FGF), and MMP-1, -2, -3, -9 and -13, metabolic proteins (cylco-oxygenase [Cox]1, Cox-2 and inducible nitric oxide synthase [iNOS]) as well as genetic susceptibility to environmental factors, have also been postulated pathogeneses of RA.18,19 The joints of patients with RA are characterized by an infiltration of immune cells into the synovium, leading to chronic inflammation, pannus formation and subsequent irreversible joint and cartilage damage.20 The RA synovium comprises largely of macrophages (30–40%), T cells (30%) and synovial fibroblasts and also of B cells, dendritic cells, other immune cells and synovial cells, such as endothelium.20,21 Recognition of Th17 cells led to breaking the dichotomy of the Th1/Th2 axis in the immunopathogenesis of RA. Th17 cells produce cytokines, including IL-17, IL-6, IL-21, IL-22 and TNF-a, with pro-inflammatory effects, which appear to have a role in immunopathogenesis of RA. Regarding the wide range of production of cytokines and chemokines by Th17 cells, it is expected that Th17 cells could be a potent pathogenic factor in disease immunopathophysiology.22 Regarding the role of autoreactive T cells (Th1 and Th17 cells) in pathophysiology of RA, it might be assumed that the regulatory T cells (Tregs) will be able to control the initiation and progression of disease. Recently, the frequency, function and properties of various subsets of Tregs, including natural Tregs (nTregs), IL-10 producing type 1 Tregs (Tr1 cells), TGF-b producing Th3 cells, CD8+ Tregs, and also defects in Tregs function or their reduced numbers, have been investigated in several human autoimmune diseases, including RA and juvenile idiopathic arthritis.23,24

RHEUMATOID ARTHRITIS Rheumatoid arthritis is a chronic inflammatory and autoimmune disorder characterized by dysfunctional cellular and humoral immunity, enhanced migration and attachment of peripheral macrophages and inflammatory leukocytes to the synovium and articular cartilage of diarthrodial joints. It can lead to a severely debilitating form with pulmonary, renal and cardiovascular involvement.16,17 Although the etiology of RA has not been clearly identified, the immune system, various

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ANGIOGENESIS IN RA Rheumatoid arthritis is a chronic inflammatory disease, and synovial angiogenesis is considered to be a notable stage in its pathogenesis.25 However, the molecular mechanisms that promote angiogenesis in RA have not been clearly identified.26 Angiogenesis has been suggested to be a pivotal mechanism involved both in inflammation/immune activation and in joint damage. During RA, angiogenesis contributes to


Potential role of angiogenic factors in RA

disease progression at multiple levels, including synovial growth, leukocyte recruitment and tissue remodeling.27 During RA, the most important role of vascularization is an increased capacity to sustain the metabolic and nutritional requirements for synovium hyperproliferation.28 However, it has been found that neoangiogenesis by itself is not entirely sufficient to mitigate the intra-articular hypoxia associated with RA.29 Indeed, the result of synovial hyperplasia and augmented proliferation of the synovial cells is increased distance from the nearest blood vessels and also increase demand for nutrients and oxygen. The effects of hypoxia and hypoperfusion, quickly imposes an additional demand on the vasculature, further promoting hypoxia. During RA, hypoxia drives angiogenesis and maintains the chronic inflammatory state by transporting inflammatory cells to the site of inflammation and supplying nutrients and oxygen for proliferating the inflamed tissue to bring about hyperplasia.30 Like many other diseases, various components of immune responses are involved in angiogenesis through T cell subsets, B cells, macrophages, fibroblasts and many growth factors, cytokines and chemokines.31 Moreover, synovial mesenchymal cells are thought to play significant roles in the pathogenesis of rheumatoid joint demolition through antigen presentation and the elaboration of the inflammatory cytokines.32 In RA, disregulation in immune responses through different immune cells and mediator’s results in a multistep complex process in angiogenesis reactions.25 Neoangiogenesis, and the subsequent increased vascular headstock content, can increase leukocyte recruitment into the synovial tissue. The activated immune cells in RA can produce angiogenic mediators; however, they also cause local microvascular blockage and damage. Moreover, increased EC injury occurs directly through the release of reactive oxygen species (ROS) and proteolytic enzymes in extreme values.33 However, in recent studies the prevailing hypothesis that ROS provoke inflammation was challenged when polymorphisms in neutrophil cytosolic factor 1 (Ncf1) that diminish oxidative bursts were shown to increase disease severity in animal models. It has been shown that oxygen radicals might also have a significant role in controlling disease severity and reducing connective tissue damage and joint inflammation.34 On the other hand, local microvascular injury by ROS and proteolytic enzymes will subsequently stimulate a reparative angiogenic response from joined and adjacent vessels.29


In RA joints, it has been shown that synovial fluids promote EC proliferation and migration, to induce vessel formation, which reflects an active, pro-angiogenic phenotype of the arthritic synovium.35,36 Moreover, the increased endothelial surface area creates a capacity for the production of cytokines, chemokines, adhesion molecules and other inflammatory stimuli. Simultaneously, the development of new blood vessels in the synovial membrane allows the invasion of this tissue supporting the active infiltration of synovial cells into cartilage and resulting in erosions and damage of the cartilage.30 Overall, during RA an imbalance in synovial tissue between the immune cells and the main cytokine system, including VEGF, IL-1, IL-6, TNF-a, IL-15, IL-17, IL-18 and so on, occurs which can lead to angiogenesis as one of the inflammatory reactions.31 Also, angiogenesis was recognized as a key event in the formation and growth of the synovial pannus in RA.37 Rheumatoid pannus, a vascular and inflamed, granulated sheet of tissue arising from the perichondral synovial membrane, spreads into cartilage surfaces with a layer of morphologically quiescent fibroblastic mesenchymal cells. Pannus subsequently starts invasion into cartilage matrix with the advent of macrophage-like cells and causing considerable destruction as it invades the subchondral bone.32 Indeed, the invasive growth and spread of pannus tissue in RA have been compared to neoplastic tumors, and it has been considered that the pannus may be indicated as a form of benign tumor.38 The increased synovial volume and its mass effects have scarcely been reviewed in the particular. Although synovial swelling is clinically evident, obstructive effects on movement of the joint or synovial fluid may not be of great consequence. Intervention of expanded, innervated synovium between articulating surfaces may contribute to pain on movement. In addition, the expanded synovium and pannus formation is identified as an abnormal tissue that has acquired novel activities, such as cytokine and antibody production, adhesion, and invasion of articular cartilage and bone.39 Therefore, angiogenesis as well as pannus formation within the joint, could play an important role in the erosion of articular cartilage and bone in the pathological process of RA.37 Briefly, angiogenesis is essential for maintaining RA progression because the formation of new blood vessels provides a supply for nutrients and oxygen to the augmented inflammatory cells and conducting inflammatory cells and mediators inside the joints for progression of RA.40

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Hypoxia The lack of an adequate blood supply and increasing distances from blood vessels lead to formation of hypoxic regions. In RA the vascular network in joints is dysfunctional, thus the synovium remains an hypoxic environment which in turn leads to the generation of ROS and joint damage. Other findings suggest that hypoxia is an important factor in aggravating inflammatory lesions in RA, through increased production of Cox-2-derived nociceptive eicosanoids and increased release of tissue-damaging MMPs. Hypoxia can also induce the production of some angiogenic cytokines and chemokines in the joints from macrophages, ECs and peripheral blood mononuclear cells.41–43 In confirmation of these data, Murdoch et al.42 in 2005 suggested that macrophages in hypoxic conditions secrete angiogenic cytokines (IL-1, IL-6) and enzymes such as MMP-7 that stimulate EC migration during angiogenesis. As mentioned earlier in RA joints hypoxic status is seen and hypoxia-inducible factor-1 alpha (HIF-1a) as a transcription factor is a major regulator in the cellular response to hypoxic conditions. HIF-1a induces cell migration, angiogenesis and cartilage destruction, inhibits the apoptosis of synovial and inflammatory cells and initiates glycolysis for energy supply by up-regulating specific protein levels. HIF-1a expression is strongest in the sub-lining layer of RA synovium and is related to both angiogenesis and inflammation in synovium from RA patients. Moreover macrophages expressing the pro-angiogenic transcription factor HIF-1a have been demonstrated in the synovial tissue of RA. Generally the number of HIF-1a-positive cells is strongly correlated with the number of blood vessels in RA synovial tissue and with inflammatory EC infiltration.44,45 Some data demonstrate that HIF-1a causes a noticeable reduction in the ability of smooth muscle cells to migrate and adhere to extracellular matrices. Moreover, findings by Kennedy et al. in 2010 indicate the presence of unstable vessels in inflamed joints is correlated with hypoxia, insufficient ECs/pericyte interactions, and increased DNA damage. These changes may contribute to persistent hypoxia in the inflamed joint to further manage this unstable microenvironment.10 In fibroblast-like synoviocytes (FLS), hypoxiainduced MMP-3 expression is exclusively regulated by HIF-1a, while hypoxia-induced MMP-1 or IL-8 expression appears to have salvage pathways other than the HIF-1a pathway.46 This demonstrated that migration

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and invasion of FLSs are critical in the pathogenesis of RA. Li and colleagues in their current study observed that RA-FLSs exposed to hypoxic conditions experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion. In this study hypoxia-induced EMT was accompanied by increased HIF-1a expression and activation of Akt. Therefore activation of the PI3K/Akt/HIF-1a pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia status.47 As we know, the combination of hypoxia and IL-17A factor promote the migration and invasion of FLSs, which are critical for the pathogenesis of RA. However, the biochemical pathways regulating IL-17A combined with hypoxia are not well defined, but recent observations suggest a synergetic effect of IL-17A and hypoxia that might contribute to the migration and invasion of RA-FLSs by up-regulating the expression of MMP-2 and MMP-9 by activation of the NF-jB/HIF-1a pathway.48 Alternatively, hypoxia is thought to drive an increase in the synovial angiogenesis process that occurs in RA, through expression of a number of angiogenic factors, including VEGF, Ang, HGF and FGF-2. Here, HIF-1a and HIF-2a are also essential in regulating transcription of the VEGF gene and finally increased vascularity in the inflammation region. This process promotes further infiltration of inflammatory cells and production of inflammatory mediators, perpetuating synovitis.36,44,49 Notch signaling pathways are crucial for angiogenesis and EC fate. In a recent study, the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis synovial tissue was examined. The results indicate that Notch-1 is expressed in synovial tissue and that increased Notch-1 intracellular domain (NICD) expression is associated with low in vivo tissue oxygen levels. Furthermore, Notch-1/HIF-1a interactions via VEGF/Ang-2, mediate hypoxia-induced angiogenesis and invasion in inflammatory arthritis.50 In another study Liu et al. revealed that Ang-2 expression is significantly reduced in the absence of Notch-3. In addition, in vitro experiments represented that Notch-3 is sufficient for Ang-2 induction, and this expression is additionally enhanced in the presence of HIF-1a. These data prepare compelling evidence that Notch-3 is important for the investment of pericytes and is a critical regulator of blood vessel formation.7 Here, it is necessary to note Intergrin/Rho guanosine triphosphatases (GTPases) coordination, so that this complex along with the Notch signaling pathway can



determine blood vessel sprouting, shape, morphology and ability to branch, which influence O2 perfusion, thus leading back to hypoxia again. The Rho family of small GTP-binding proteins comprises a group of signaling molecules (Rho, Rac and Cdc42) which significantly impact angiogenesis. Intracellular signaling molecules phosphatidylinositol 3-kinase (PI3-K), protein kinase B (PKB), Akt, p38 MAPK (mitogen-activated protein kinase), focal adhesion kinase (FAK), and Rho-associated-kinase (ROCK) all provide molecular linkages among VEGF receptor-2 (VEGFR-2) mediated Rho GTPase signal transduction pathways in EC migration.51 Integrins are the main adhesion receptors used by ECs to interact with their extracellular microenvironment. Variations in the repertoire and/or activity of integrins, and also the availability and structural nature of their ligands, regulate the vascular cell during blood vessel growth or repair.52 Integrin avb3 has also been the focal point of intensive research because of its major role in several distinct processes, particularly a critical part in activated macrophage-dependent inflammation, osteoclast development, migration, and bone resorption, and pathological angiogenesis, which show their important relation with RA.53 Interestingly, Rho family GTPase and integrin functions coordinate to mediate cell adhesion-dependent incidents. Recently, it has been revealed that Rho GTPases are able to regulate integrins. Therefore, GTPases and integrins might be organized into complex signaling cascades that regulate EC function.54 In addition, ECs in rheumatoid synovium are subject to continuous production of angiogenic stimuli, including TNF-a and VEGF, resulting in the expression of avb3 on sprouting EC buds and new blood vessel development in pathological neovascularization.55

Growth factor Vascular endothelial growth factor is an endotheliumspecific mitogen and one of the most important proangiogenic mediators related to inflammation-associated synovial angiogenesis. VEGF is originally identified as an EC-specific growth factor to prevent the apoptosis of endothelial cells which is induced by serum starvation. Studies show that the serum level of VEGF elevates throughout the course of RA and this elevation is correlated with disease activity. Moreover, in experimental models, the administration of pro-angiogenic factors, such as VEGF or FGF, has been shown to increase the severity of the disease.26,56,57 Emerging evidence suggests that fluid and serum VEGF levels not only are elevated in RA patients with hypoxic conditions but


also by pro-inflammatory cytokines IL-1 and TNF-a.58 Currently, VEGF and its receptors are the best characterized system in the angiogenesis regulation of rheumatoid joints. The VEGFRs on EC membranes consist of the tyrosine kinases VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/ KDR) and VEGFR-3 (Flt-4). KDR is a main mediator of angiogenic, mitogenic and permeability-enhancing effects of VEGF. Moreover, KDR is up-regulated in response to hypoxia, a main inducer of VEGF gene transcription.59 It is demonstrated that hypoxia stimulated VEGF-A (the most important member of the VEGF family) and VEGFR-1 expression decrease VEGFR-2 levels in ECs. During hypoxic conditions, plasma membrane VEGFR1 levels are elevated, while VEGFR-2 levels are depleted. One functional consequence of hypoxia is a decrease in VEGF-A-stimulated and VEGFR-2-regulated intracellular signaling along with lowered EC NOS activation. In addition, the capillary, arterial and venous ECs subjected to hypoxia display a decreased cell migration in response to VEGF-A. A mechanistic elucidation is that VEGFR-1/VEGFR-2 ratio is substantially increased during hypoxia to obstruct VEGF-A-stimulated and VEGFR-2 regulated endothelial responses to magnify cell recovery and viability.60 In another study, Eubank et al. in 2011 showed that hypoxia can selectively stimulate anti-angiogenic molecule expression in mononuclear phagocytes in a granulocyte macrophage colony-stimulating factor (GM-CSF) enriched environment. The soluble VEGFR-1 (sVEGFR-1) is one of these molecules that act as a negative regulator for VEGF activity through VEGFR-2. Therefore, anti-angiogenic molecules can effect proliferation, migration and survival of ECs.61 Placenta growth factor is another angiogenic factor and highly homologous with VEGF. PLGF can exert its angiogenic effect by synergizing with VEGF. However, it does not have an effect on lymphatic vessel functionality.62,63 Furthermore, PLGF can promote the production of VEGF from monocytes and macrophages.64 It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines, including TNF-a and IL-6.65 Oncostatin M (OSM) belongs to the IL-6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of RA patients and it may rise the inflammatory responses in joints and eventually lead to bone erosion.65 OSM promotes angiogenesis and EC migration, and potentiates the effects of IL-1b in promoting extracellular matrix turnover and human cartilage degradation.66 It was also

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demonstrated that OSM increased messenger RNA (mRNA) and protein levels of PLGF in a time- and concentration-dependent manner in RA synovial fibroblasts.65 Other angiogenic mediators, such as PDGF, FGF-2, EGF, CTGF, IGF, HGF, tissue factor (TF), endothelin-1 (ET-1), Ang, cytokines and chemokines and TGF-b, play important roles in RA angiogenesis. In addition, thrombospondin-1 and -2 as angiostatic mediators in RA and also endogenous angiostatic factors, such as angiostatin, endostatin, IL-4, IL-13, IFNs and some angiostatic chemokines, are also produced within the rheumatoid synovium.37,67–69

Immune cells T cells Rheumatoid T cells promote VEGF, TNF-a and chemokine production in the synovium. VEGF is secreted by T cells following the stimulation by specific antigens or by IL-2 and by hypoxia; thus, activated T cells might enhance angiogenesis. Hypoxia also induces the VEGFR-2 expression in T cells, suggesting that T cells might respond to VEGF. Indeed, VEGF augments IFN-c and inhibits IL-10 secretion by T cells responding to mitogen or antigen. Thus, T cells can play a role in angiogenesis by delivering VEGF to inflammatory sites, and VEGF can augment pro-inflammatory T cell differentiation and enhance Th1 phenotype expansion.70,71 Macrophage Macrophages are differentiated from peripheral-blood monocytes. Both monocytes and synovial macrophages are key players in RA. These cells are involved in the initiation and perpetuation of inflammation, leukocyte adhesion and migration, matrix degradation and angiogenesis. Macrophages express adhesion molecules, chemokine receptors and other surface antigens. Activated macrophages produce many molecules, such as IL1, IL-6, TNF-a, TGF-b and MMPs, thus they can promote the re-epithelization.72 Macrophages are the main cell type which releases TGF-b cytokine. TGF-b stimulates neovascularization through attracting macrophages and increasing the production of many growth factors that act on ECs.56 In addition several proteinases, including cathepsin G, are produced by macrophages during RA-associated inflammatory and angiogenic events.73

Cytokines and chemokines Angiogenesis is an early and critical event in the pathogenesis of RA. Monocytes, macrophages and T lymphocytes fully participate in the angiogenesis process via

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their different cytokines, which play an essential role in angiogenesis and can control this complex process. Proinflammatory cytokines, such as TNF-a and IL-1 stimulate synovial fibroblasts and other cells to release VEGF; also other cytokines, including IL-6, IL-15, IL-17 and IL18 act indirectly on angiogenesis by promoting VEGF production.74 TNF-a promotes neovascularization and it may also regulate capillary formation via VEGF, Ang-1 and -2 and their receptors, Tie-2.75 TNF-a induces HUVECs (human umbilical vein endothelial cells) to proliferate and form new blood vessels. Thus, TNF-induced neovascularization plays a critical role in rheumatic disease pathogenesis. However, the molecular mechanism that underlies TNF-induced angiogenesis is largely unknown.76 IL-6 can act synergistically with TNF-a and IL-1 to induce the production of VEGF. More particularly, IL-6 promotes TNF-induced angiogenesis by inducing NF-jB (nuclear factor kappa-light-chain-enhancer of activated B cells) and IL-8, which are strong cell growth factors.76 IL-6 also promotes increased production of MMPs.77 Neovascularization is dependent on EC activation, migration and proliferation. Pickens et al. introduced a novel function for IL-17 as an angiogenic mediator in RA.78 IL-17 synergizes with TNF-a in stimulating VEGF, EGF, HGF and KGF production by synovial fibroblasts. IL-17 also acts through CXCR2-dependent pathways. IL-18 is a pro-inflammatory cytokine that is elevated in synovial fluids and synovial tissues in patients with RA. In the RA joint, IL-18 can contribute to the inflammatory process by inducing leukocyte extravasation through up-regulation of EC adhesion molecules, the release of chemokines from RA synovial fibroblasts, and directly as monocytes, lymphocytes and neutrophil chemoattractants. IL-18 up-regulates the production of key regulators of osteoclastogenesis (RANKL [receptor activator of nuclear factor kappa-B ligand], M-CSF, GMCSF and osteoprotegerin) from FLS and also induces the serum amyloid A protein synthesis from rheumatoid synovial cells in a dose-dependent manner.79,80 IL-18 can also help maintain and develop the inflammatory pannus by inducing EC migration and angiogenesis. IL-18 does this function directly by binding and activating ECs and indirectly by inducing RA synovial fibroblasts to produce angiogenic chemokines and VEGF.81 These data support the notion that IL-18 has a unique role in inducing the secretion of angiogenic elements by synovial fibroblasts in RA.82 IL-18 is present in RA synovial fluid in high levels, where it functions as



a leukocyte chemoattractant and angiogenic mediator to effect angiogenesis by inducing the secretion of angiogenic factors such as SDF-1a/CXCL12, MCP-1/ CCL2, and VEGF.82,83 In conclusion, synovial fluid IL-18 levels in RA patients are good indicators of disease activity. IL-10 potentially inhibits angiogenesis through preventing the production of angiogenic mediators such as IL-8. It can also inhibit the proliferation of ECs, which is mediated by VEGF and FGF2. Also the release of angiogenic cytokies IL-1, IL-6 and TNF-a can be inhibited by IL-10. However, IL-4 activities in angiogenesis are controversial.41 IL-4 can modulate neovascularization through pro-angiogenic and angiostatic mediators. It can also down-regulate IL-12R expression.84 Angiostatin can inhibit angiogenesis in collageninduced arthritis (CIA). Albini et al. presented evidence that IL-12 with potent anti-angiogenic activity is the mediator of angiostatin activity. Also it is demonstrated that angiostatin induces IL-12 mRNA synthesis by macrophages, suggesting that these cells produce IL-12 upon angiostatin stimulation.85 IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-c and angiostatic CXCR3 chemokine ligands. IFN-c is a strong anti-proliferative cytokine that is produced by T lymphocytes and natural killer (NK) cells. IFN-c inhibits EC growth as well as the expression of MMP-2 and MMP-9.41 It can also induce expression of anti-angiogenic chemokines, such as CXCL10 and CXCL11 and down-regulate expression of pro-angiogenic CXCL12 chemokine.1 In RA, other chemokines, such as CCL21, fractalkine and MIF mediate the synovial angiogenesis and migration of inflammatory leukocytes into the synovium.86,87 MIF has drawn significant attention recently. This chemokine is involved in macrophage activation and cytokine production.73 MIF is primarily produced by synovial macrophages and is involved in macrophage-derived synovial angiogenesis.73,88 MIF acts via the induction of VEGF and IL-8/ CXCL8 release by RA synovial fibroblasts.89 Moreover, IL-8 is an angiogenic factor. This cytokine seems to be an important factor in which synovial tissue macrophages derive chemotactic activity in ECs, so that angiogenesis could be significantly decreased if IL-8 is immunodepleted.90

Proteinase A disintegrin and metalloproteinases (ADAMs) comprise a new family of metalloproteinases, responsible


for liberating a variety of cell surface expressed proteins. ADAMs has been implicated in several inflammatory reactions as RA.91 Several recent studies have demonstrated the effect of cytokines, such as IL-1b, TNF-a and TGF-b, on the expression of ADAMs with thrombospondin motifs 4 (ADAMTS-4) and ADAMTS-5 in FLS. Mimata and colleagues suggest that IL-6 may participate in cartilage destruction in RA as an inducer of ADAMTS-4 expression from FLS.92 Furthermore, ADAMTS-12 has been observed in the cartilage, synovial fluid and serum of arthritic patients, which may play an important role in the pathogenesis of arthritis. Nah et al. suggest that ADAMTS12 may be a susceptible gene for RA development.93 In particular, ADAM10 has been shown to cleave various inflammatory and angiogenic mediators from the cell surface, including CXCL16 and CX3CL1. Soluble CXCL16 plays an important role in T cell accumulation and stimulation in RA synovium, and ADAM10 was identified as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant for T cells.94 Also, ADAM10 is involved in the constitutive cleavage of CX3CL1 and thereby may regulate the recruitment of monocytic cells to CX3CL1-expressing cell layers.95 Recently, Isozaki et al. show that ADAM10 is overexpressed in RA and suggest that ADAM10 may play a role in RA angiogenesis.96 Moreover, other studies have shown that ADAM15 and ADAM17 are active in RA.97 Komiya et al. in 2005 indicated that among the ADAMs, ADAM15 mRNA was more frequently expressed in the RA patients and also it was expressed in the synovial lining cells, ECs of blood vessels and macrophage-like cells in the sublining layer of RA synovium. Therefore, these data demonstrate that ADAM15 is overexpressed in RA synovium and its expression is up-regulated by the action of VEGF through VEGFR-2, and suggest the possibility that ADAM15 is involved in angiogenesis in RA synovium.98

ANTI-ANGIOGENIC THERAPY During RA, chemical mediators in inflamed tissue invade and destroy cartilage and bone. The tissue pathological expansion, invasion and expression of growth factors, cytokines and hypoxic microenvironment which are a feature of RA have resulted in the hypothesis that angiogenesis inhibition may be useful in the treatment of RA.99 Disruption of the new vessel formation would not only prevent delivery of nutrients to the

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Table 1 The most important angiogenic elements and mediators in RA Angiogenic elements

Mediators

Growth factors

Stimulator: VEGF, FGF, HGF, HIF-1, HIF-2, PDGF, EGF, KGF, IGF-1, TGF-b, PLGF, PGF

Cytokines

Stimulator: TNF-a, IL-1, IL-6, IL-8, IL-15, IL-17, IL-18, G-CSF, GM-CSF, oncostatin M Inhibitor: IFN-a, IFN-c, IL-4, IL-12, IL-13, LIF, IP-10 Stimulator: CXCL8, CXCL5, CXCL1, CXCL6, CXCL12, CCL2, CX3CL1, MIF CXCR2, CXCR4, CCR2 Inhibitor: CXCL4, CXCL9, CXCL10, CCL21, CXCR3 avb3 integrin, E-selectin, VCAM-1, ICAM-2, PECAM-1, CD34, MUC18, endoglin, JAM-A, JAM-C Stimulator: MMPs, Plasminogen activators, ADAM10, ADAM15 Inhibitor: TIMPs, PAIs Stimulator: HIF-1a and HIF-2a, MMPs, COX-2, Angiogenic Cytokines and Chemokines, VEGF, Angiopoietins, HGF and FGF-2 Inhibitor: sVEGFR1 Stimulator: Ang 1/Tie-2, Angiotropin, Angiogenin, COX/Prostaglandin E2, PAF, NO, ET-1, Serum Amyloid A, Histamine, Substance P Inhibitor: Angiostatin, Endostatin, Kallistatin, Paclitaxel, 2-Methoxyestradiol, Osteonectin, Opioids, Troponin I, Chondromodulin, Kringle 5, Prolactin, Vasostatin, Thrombospondin-1,-2, Cartilage-derived angiogenesis inhibitor

Chemokines/ Receptors

Cell adhesion molecules enzymes

Hypoxia

Others

Source

Functions

Synovial tissue, MO, MQ and endothelium MO, MQ, T, NK and ECs

Angiogenic (direct effects on endothelium)

MO, MQ, T and ECs

Induction of VEGF and chemotactic activity for inflammatory leukocytes and ECs into the synovium

EC and ECM

Permit EC migration

Synovial, ECs and MQ

Cleave various inflammatory and angiogenic mediators

Vascular ECs, FLS, MQ and PBMCs

Cell migration, angiogenesis, cartilage destruction and initiates glycolysis, transcriptional activator of VEGF, iNOS

Angiogenesis by ECs migration and stimulation of synovial fibroblasts and other cells to release growth factors and MMPs

Several cells

ET, Endothelin; MO, Monocyte; MQ, Macrophage; EC, Endothelial cell; IL, Interleukin; MMP, Matrix metalloproteinase; VEGF, Vascular endothelial growth factor; LIF, Leukemia inhibitory factor; IP-10, Interferon gamma-induced protein 10; HIF, hypoxia-inducible transcription factor; Ang, angiopoietin; NO, Nitric oxide; COX, Cyclooxygenase.

inflammatory site, but could also result in vessel regression and possibly reversal of disease. There are several specific and non-specific angiogenesis inhibitors that have been FDA-approved or are currently being assessed in clinical trials which are safe for humans usage; however, their effects on RA remain untested.100 The first line of angiostatic agents includes antagonists of VEGF, Ang and avb3 integrin and also non-specific angiogenesis inhibitors, including traditional disease-modifying anti rheumatic drugs (DMARDs), anti-TNF biologics, endostatin, angiostatin, fumagillin analogues or thalidomide.101 However, their adverse effects (other than anti-TNF therapy) such as increased aminotransferase levels, hypertension,

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congestive heart failure, gastro-intestinal perforation, neutropenia, increased risk of serious infections, variations in the gammaglobulin profile and high cost are major concerns.16,102

VEGF associated drugs These drugs include anti-VEGF neutralizing monoclonal antibodies (bevacizumab), anti-sense VEGF cDNA, chimeric proteins consisting of the extracellular domain of VEGFR-1 and VEGFR-2 joined to the Fc portion of IgG, soluble VEGFRs, adenoviral expression of soluble VEGFRs and molecules that act through the inhibition of VEGF signaling.103–106 In tumor research, VEGF signaling inhibitors stop angiogenesis and destroy or



change tumor vessels. Inhibition of VEGF and its receptors causes the loss of endothelial fenestrations, regression of tumor vessels and decrease in diameter, permeability and inflection of tumor vessels.107,108 Considering the important role of VEGF/VEGFR in regulating vascular function in RA, inhibitors of VEGF signaling could be beneficial. Norisoboldine (NOR), administered orally, significantly reduced the number of blood vessels and expression of growth factors in the synovium of adjuvant-induced arthritis (AIA) rats. NOR is able to stop synovial angiogenesis, which could be a supposedly new mechanism responsible for its antirheumatoid effect. The anti-angiogenesis activity of NOR was possibly achieved by decreasing the Notch-1 pathway-related endothelial tip cell phenotype with potential action of Notch-1 transcription complex.109 In a recent study, Wei et al. suggest that NOR can also alleviate joint destruction in AIA rats by reducing RANKL, IL-6, PGE2, and MMP-13 expression via the p38/ERK/AKT/AP-1 pathway.110 Pseudolarix acid B (PAB) is a traditional pregnancyterminating agent, which has previously been shown to reduce tumor growth and angiogenesis. PAB inhibits VEGF-mediated anti-apoptotic effects on ECs and also inhibit phosphorilation by the VEGFRs.2,111 It was demonstrated that PAB in combination with 5-fluorouracil (5-Fu) could act in angiogenesis by down-regulation of VEGF, HIF-1a and cyclin E expression.112 Anti-VEGF antibody, bevacizumab, which is already being used as an anti-tumor agent, was approved in 2004 for colorectal cancer, and has since been approved for other cancers which may play a significant role in longstanding RA. However, its adverse side effects, such as ischemic heart disease, gastro-intestinal perforation, hypertension and the high cost of bevacizumab are major problems.16,99,113 Endostatin is an endogenous inhibitor of angiogenesis and findings indicate that recombinant endostatin (rhEndostatin) has a therapeutic effect on RA. In an animal model rhEndostatin reduced the expression of VEGF in both cartilage and synovial tissue. These indicate that rhEndostatin as a VEGF expression inhibitor contributes to the regression of rat adjuvant arthritis.114 Furthermore, rhEndostatin has anti-angiogenic effects by inducing FLS apoptosis, which is firmly associated with increased expression of Fas, c-jun and caspase-3, but not NF-jB.115 Moreover, recent data propose that rhEndostatin inhibits adjuvant arthritis by down-regulating VEGF expression and suppression of inflammatory cytokine production such as TNF-a, IL-1b.116


Another molecule which can be the target of angiogenesis blockade is FGF following the use of compound-1 and compound-2 of stibene glycosides which are derived from some medicinal plants.31,117,118

avb3 integrin antagonist Recent advances in anti-angiogenic therapies in oncology, including the recognition of integrin avb3 as a crucial effector of angiogenesis, indicate a means to assess the role of angiogenesis in RA.27 It should be noted that the cells that express the highest levels of avb3 such as ECs, which are involved in pathological angiogenesis, activated macrophages, are involved in producing pro-inflammatory cytokines and osteoclasts, which mediate inflammatory osteolysis. Macrophagedependent activities, angiogenesis and inflammatory osteolysis are clearly involved in the pathobiology of RA.53 Previous experiments in animal arthritis models have shown benefit after using the broad spectrum avb3 integrin antagonists. However, formal evaluation of integrin-targeted anti-angiogenic activity is now underway.27 Vitaxin, also known as MEDI-522 is a humanized monoclonal IgG1 antibody that specifically binds to a conformational epitope formed by both the integrin av and b3 subunits. It blocks the interaction of avb3 with diverse ligands such as osteopontin and vitronectin.53 In animal models of arthritis, vitaxin inhibited synovial angiogenesis; however, in a phase II human RA trial vitaxin displayed a limited efficacy.119 Cilengitide (cyclic peptidic avb3 antagonist) and small interfering RNA (siRNA) that specifically silence integrin avb3, as well as angiostatin and endostatin as endogenous inhibitors that act via aVb3 integrin-dependent mechanisms, have great therapeutic potential.120,121 Also, selective mast cell silencing with either salbutamol or cromolyn can prevent avb3 integrin activation, angiogenesis and joint destruction.122 Moreover, it is suggested that IL-4 can modulate neovascularization in part through avb3 integrin. In rat AIA, IL-4 reduces synovial tissue vascularization through angiostatic effects. IL-4 mediates angiogenesis inhibition by pro- and anti-angiogenic cytokine alteration, and may also inhibit VEGF-mediated angiogenesis. These data about the specific angiostatic effects of IL-4 may help optimize target-oriented treatment of inflammatory RA.84

Cytokine blockers Cytokine blockade may modify vascular pathology in RA, and can significantly reduce clinical progression of

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atherosclerosis. Inhibition of some cytokines such as IL-1 and TNF-a can reduce the production of VEGF.123 Golimumab and infliximab (TNF-a-blocking monoclonal antibodies), certolizumab (a fragment of a monoclonal antibody to human TNF-a), etanercept (recombinant human soluble TNF-a receptor fusion protein), adalimumab (a human recombinant antibody which binds to TNF-a and blocks the interaction of TNF-a with its receptors), tocilizumab (IL-6 receptorinhibiting monoclonal antibody), canakinumab (human IL-1b monoclonal antibody) and aurothiomalateare (reduced COX-2, MMP-3 and IL-6 expression in human RA cartilage) are some useful cytokine blocker agents for reduction of inflammation, bone destruction and angiogenesis.124–129 Emerging evidence suggests that TNF-a blockade may modify vascular pathology in RA, as it is revealed that anti-TNF therapy in RA patients reduces Ang-1/Tie-2 and survivin, whereas it stimulates Ang-2 expression.75 Administration of infliximab down-regulates mucosal angiogenesis in patients with Crohn’s disease and restrains VEGF-A production by mucosal fibroblasts. It is suggested that this alleviates inflammation-driven angiogenesis in the gut mucosa and contributes to the therapeutic efficacy of TNF-a blockage.130 In another study, Shu et al. in 2012 investigated the effects of certolizumab on endothelial cell function and angiogenesis. Their findings support the hypothesis that certolizumab inhibits TNF-a-dependent leukocyte adhesion and angiogenesis, maybe via inhibition of angiogenic adhesion molecules (E-selectin, ICAM-1 and VCAM-1) expression, and angiogenic chemokine secretion.131 Moreover, it has been reported that the use of combined cytokine blockers could be more effective in controlling collagen degradation than using TNF-a blockers alone. In RA, infliximab therapy in combination with methotrexate (MTX) inhibited systemic and synovial VEGF release, resulting in attenuated synovial vascularization.132 The Shono study demonstrated that golimumab plus MTX effectively reduced the signs and symptoms of RA and was generally well tolerated in patients with an inadequate response to MTX and other biological agents.133 In another study, Kanbe et al. demonstrated that in RA patients, golimumab may involve the inhibition of cell proliferation, with decrease in macrophages, B cells, T cells, b-1 integrin, RANKL and c-Jun N-terminal kinase (JNK) in the synovium, compared with MTX therapy.134 The inhibitory function of atorvastatin (used for lowering blood cholesterol), Qubi Zhentong Recipe (Chinese medical formula) and genistein (soy-derived

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isoflavone and phytoestrogen with antineoplastic activity) on VEGF, TGF-b, IL-1b and TNF-a as main components of inflammatory angiogenesis was revealed.135–137 The hypoxia/HIF pathway may also be a therapeutic target using non-specific inhibitor compounds. For instance, anti-angiogenic YC-1, a superoxide-sensitive stimulator of soluble guanylyl cyclase is also a HIF-1a inhibitor. 2-methoxyestradiol and paclitaxel, on one side destabilize the intracellular cytoskeleton and on the other side block HIF-1a expression and activity.119,138 Inhibition of HIF-1a expression or activation, by blocking signal transduction pathways, results in HIF-1a induction through inhibiting the HIF-1a protein accumulation, and represents a new strategy which is of interest for the treatment of RA.139 However, in the treatment process the predominance of the differential interactions between VEGF, Ang/Tie-2 system and PDGF/TGF-b for determining blood vessel maturity, stability and survival as well as ECs/pericyte alignment which can influence the hypoxic environment, has been observed.

CONCLUSION Various studies have shown that the different immune components such as cells, cytokines, chemokines, integrins, growth and transcription factors, as well as the hypoxic microenvironment, are involved in the inflammatory and angiogenic events of RA. Angiogenesis has a key role in pannus formation and also in infiltration of inflammatory cells into the joints. Some specific components of the immune system are suitable targets for immunomodulatory therapies that may stop joint destruction and disease progression. As a result, a better understanding of this process can help in reduction of disease progression and promote the efficacy of new recommended treatments. Particularly as the latest strategy, HIF-1a, avb3 integrin and ADAM10 may be considered as potential therapeutic targets in RA which is known as an inflammatory and angiogenic disease.96

CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

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Interleukin-34 in rheumatoid arthritis: potential role in clinical therapy.

Angiogenic T cells are decreased in rheumatoid arthritis patients.

Anti-angiogenic effect of triptolide in rheumatoid arthritis by targeting angiogenic cascade.

Rheumatoid factors and the disease state in rheumatoid arthritis.

Rheumatoid factors specific for active rheumatoid arthritis.

Genetics of neuroendocrine factors in rheumatoid arthritis.

Adipokines as potential biomarkers in rheumatoid arthritis.

The role of rheumatologists vis-à-vis assessment of traditional cardiovascular risk factors in rheumatoid arthritis.

Redox-sensitive transcription factors play a significant role in the development of rheumatoid arthritis.

[Factors associated with infection in rheumatoid arthritis].

The pathogenic potential of autoreactive antibodies in rheumatoid arthritis.

The role of leptin in the pathophysiology of rheumatoid arthritis.

The role of angiogenic factors in endometrial cancer.

Role of cysteine‑rich angiogenic inducer 61 in fibroblast‑like synovial cell proliferation and invasion in rheumatoid arthritis.

Contribution of inherited factors to rheumatoid arthritis.

The role of leukotrienes in immunopathogenesis of rheumatoid arthritis.

Role of ineffective erythropoiesis in the anaemia of rheumatoid arthritis.

The pathogenic role of angiogenesis in rheumatoid arthritis.

The possible role of Epstein-Barr virus in rheumatoid arthritis.

The multifactorial role of neutrophils in rheumatoid arthritis.

Postpartum cervical repair in mice: a morphological characterization and potential role for angiogenic factors.

The Gm and Inv factors in rheumatoid arthritis.

Current cigarette use in rheumatoid arthritis patients: associated factors and a limited mediating role of depression.

Specificity of rheumatoid factors in relation to the disease state in rheumatoid arthritis.