Molecular Pattern of Angiogenesis Phenomenon and Inflammatory Cytokines in Rheumatoid Arthritis

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1 Molecular Pattern of Angiogenesis Phenomenon and Inflammatory Cytokines in Rheumatoid Arthritis Abstract Rheumatoid arthritis (RA) is an inflammatory disorder of the joints characterized by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. There is no firm cure for RA, however conventional medications can alleviate inflammation, mitigate the pain and slow joint damage.the chronic nature of this disease results in inflammation of the joints, with subsequent demolition of joint cartilage and bone erosion. Angiogenesis, the formation of new vessels, is important in the pathogenesis of RA. Recently, many studies have shown the importance of angiogenesis in a complex process involving different cell types and mediators. In this review, we discuss the role of angiogenesis and the most important angiogenic factors, as well as relevant cytokines and chemokines involved in RA. Keywords RA, angiogenesis, VEGF, FGF, PIGF, hypoxia, chemokines. A I. INTRODUCTION NGIOGENESIS is classically defined as the process of development and formation of new blood vessels from preexisting vasculature. Furthermore this phenomenon is a biological event and occurs during the growth, development and repair of tissues. In addition it can contribute to the pathogenesis of many diseases such as RA. [1, 2]. The requirement for blood vessels arises from the need to maintain oxygen homeostasis as well as to supply nutrients and to remove waste products. Angiogenesis play a key role in several physiological events, including embryonic development, reproduction and normal wound healing [3-5]. Angiogenesis is a series of successive events including activation, migration, proliferation and differentiation of ECs, and following formation and maturation of new blood vessels - VEGFR1(flt-1, fms-like tyrosine kinase-1) 1. Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran-14155, Box: 6446, Iran 2. Imam Hassan Mojtaba Hospital, Alborz University of Medical Sciences, Karaj, Iran * Correspondence to: Prof. Abbas Mirshafiey, Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran-14155, Box: 6446, Iran; mirshafiey@tums.ac.ir Roobina Boghozian 1, Gholamreza Azizi 2 and Abbas Mirshafiey 1 * Fax: , 57 [6, 7]. Several cells and mediators are involved in this process [8]. These elements can affect ECs proliferation and differentiation [9]. Cytokines, chemokines and cells, which interact to establish a specific microenvironment suitable for new vessel formation are vascular endothelial growth factor (VEGF), 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-β), tumor necrosis factor alpha(tnf-α), interleukin 1(IL-1), IL-6, IL-8, IL-13, IL-15, IL-18, IL-19, PAF, CXC chemokines, neutrophils, monocytes, macrophages (M) and lymphocytes [1, 10, 11]. Vascular endothelial growth factor (VEGF): One of the most important factors regulating angiogenesis is vascular endothelial growth factor; it is originally identified as an endothelial cell specific growth factor. VEGF can prevent the apoptosis of endothelial cells which is induced by serum starvation [8, 12]. VEGF stimulates the proliferation, differentiation, adhesion and migration of endothelial cells. Moreover, it induces the secretion and activation of proteolytic enzymes (matrixmetalloproteases (MMPs), plasminogen, etc). Therefore VEGF can promote vascularization by multiple mechanisms and it also plays a unique role in the regulation of the physiological and pathological growth of the blood vessels. VEGF is homodimeric kda protein produced by many cells such as macrophages, smooth muscle cells, tumor cells, epithelial cells of the retina and endothelial cells [6, 13]. VEGF is found in various isoforms which are VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E. These forms are derived from the alternative splicing of the same gene. Placental growth factor (PIGF) is the other member of VEGF family [6, 14]. VEGF-C and VEGF-D are capable of inducing lymph angiogenesis [15], while VEGF-A is the main regulator of angiogenesis [16, 17]. VEGF carries out its biological function through receptors which have tyrosine-kinase activity (RTK). These receptors which are expressed by endothelial cells are as follows: VEGFR2 (flk-1/kdr, fetal liver kinase-1/ kinase insert domain-containing receptor) VEGFR3 (flt-4) VEGFR1 is involved in vessels differentiation and can bind to VEGF-B, VEGF-A. VEGFR2 can bind to VEGF-D, VEGF-A

2 and VEGF-C. This receptor s affinity for VEGF is lower than VEGF1 but its tyrosine kinase activity is 10 fold higher than VEGF1. VEGFR2 regulates ECs proliferation, it is also necessary for ECs survival and it is thought to be the main mediator of angiogenesis [18]. VEGF binding stimulates dimerization and autophosphorilation of the receptors. Tyrosine phosphorilation can increase catalytic activity of the receptors and can induce generation of specific binding sites leading to activation of signaling pathway which results in the several responses [2]. VEGF3 (flt-4) is a 170 kda glycosylated protein, which is bind to mature form of VEGF-C [14, 18]. The function of ECs is mainly regulated by VEGFR1 and VEGFR2 [19]. VEGF can be both active on its own and in a synergetic way with other growth factors such as basic fibroblast growth factor (bfgf or FGF-2) [6]. Placenta growth factor (PIGF): The other angiogenic protein is placenta growth factor belonging to the VEGF family. PIGF can bind to VEGFR1 and contribute to angiogenesis. It can exert its angiogenic effect by synergizing with VEGF. However, it doesn t have an effect on lymphatic vessel functionality [20, 21]. PIGF can separate VEGF from VEGFR1, so make more available VEGF to create a bond with activated VEGFR2. By binding to VEGFR1, PIGF can directly affect ECs survival. PIGF can also promote the production of VEGF from monocytes and macrophages [22, 23]. Fibroblast growth factor (FGF): A heparin binding angiogenic growth factor consists of two members FGF1 and FGF2. Other members of the FGF family and their receptors have been identified. It can induce its biological activity through both high affinity tyrosine kinase family receptors FGFR 1-7) and low affinity receptors (heparin sulfate and neurophilin-1). FGF-2 has mitogenic and pro-migratory activity and can stimulate EC proliferation and differentiation [24-26]. Through binding to its receptors FGF can induce dimerization and activation of different signaling pathways [27]. Cytokines: TH1 and TH2 lymphocytes fully participate in the angiogenesis process via their different cytokines, which play an essential role in angiogenesis and can control this complex process. The balance between these two cells regulates neovascularization. Some cytokines such as interferon γ (IFN-γ) released from TH1 and IL-4, IL-5, IL-6, IL-10 and IL-13 which are produced by TH2 can affect ECs function during angiogenesis process [28, 29]. IFN-γ is a 50 kd protein and is produced by different cell types such as T lymphocytes, natural killer cells (NK cells), etc. IFN-γ inhibits ECs growth as well as the expression of MMP2 and MMP9. IFN-γ is a strong anti-proliferative cytokine [29]. It can also induce anti- angiogenic chemokines such as CXCL10 and CXCL11 and down-regulate pro-angiogenic CXCL12 chemokine expression [1]. IL-12 It is a heterodimeric cytokine produced mainly by macrophages, monocytes and dendritic cells (DC). This cytokine acts through IFN-γ and can also promote the production of this cytokine from T cells and NK cells through its receptors. Via IFN-γ, IL-12 not only can induce the production of anti-angiogenic chemokines, but also reduces the production of angiogenic chemokines [30]. IL-4 is a 20 kd cytokine and its angiogenesis related activities are controversial [29]. IL-4 can modulate neovascularization through pro-angiogenic and angiostatic mediators. It can also down-regulate IL-12R expression [31]. IL-6 is produced by many cells such as macrophages, ECs and identified as a B cell differentiation inducing factor [29]. It acting synergistically with TNF-α and IL-1 to induce the production of VEGF, a key regulator of angiogenesis. IL-6 also promotes increased production of metalloproteinases [32]. Moreover, oncostatin M as another member of IL-6 family can also induce VEGF production [1]. IL-10 potentially inhibits angiogenesis through preventing the production of IL-12 and other angiogenic mediators such as IL-8. It can also inhibit the proliferation of ECs, which is mediated by VEGF and FGF2 [29]. The release of IL-1, IL-6 and TNF-α can be inhibited by IL-10. IL-18 is a pro-inflammatory cytokine and have an effect on angiogenesis by inducing the secretion of angiogenic factors such as stromal cell derived factor (SDF-α), CXCL12, VEGF, etc [33]. IL-19 is a mitogenic and chemotactic agent for ECs and it can induce tube formation in angiogenesis process [34]. IL-17 is a 30 to 35 kda homodimeric polypeptide with pro-inflammatory activity which affects IL-1, TNF- α, IL-6 and IL-8 [35]. Transforming growth factor beta (TGF-β) consists of three cytokines that are TGF-β1, TGF-β2 and TGF-β3 produced by several cells. Macrophages are the main cell type which releases this cytokine. TGF-β stimulates neovascularization through attracting macrophages and increasing the production of many growth factors that act on ECs [16]. Macrophages: are mature myeloid cells which are derived from the migration of monocytes into the different tissues under the influence of specific chemokines. Sometimes, macrophages are divided into two classes: type 1 or TH1 derived macrophages which can produce TNF-α, IL-1, IL-6 and TGF-β and type 2 or TH2 derived macrophages (M2a, M2b and M2c). Activated macrophages produce many molecules such as IL-1, IL-6, TNF-α, TGF-β and MMPs, thus they can promote the reepithelisation [16, 28, 36]. Hypoxic conditions: hypoxic regions are present in various inflamed diseased tissues. Hypoxia can induce IL-6 in 58

3 endothelial and peripheral blood mononuclear cells. It can also induce the production of IL-1 from monocytes [29]. Hypoxia-reoxygenation increases TGF-β1 and decreases it s type1 receptors (TGF- βr1) in angiogenesis process[37]. Macrophages in hypoxic condition secrete angiogenic cytokines and enzymes such as MMP7 that stimulates ECs migration during angiogenesis [38]. Hypoxia can selectively stimulate the production of anti-angiogenic molecules from mononuclear phagocytes in granulocyte macrophage colony-stimulating factor (GM-CSF) rich environment. The svegfr1 (soluble VEGF receptor 1) is one of these molecules that acts as a negative regulator for VEGF activity through VEGFR2. Thus, it affects proliferation, migration and survival of ECs [39]. Chemokines: Chemotactic cytokines terms as chemokines are involved in angiogenesis. Chemokines such as IL-8/CXCL8, CXCL1 and CXCL2 which have ELR motif (glutamic acidleucine- argentine) are belong to CXC family of chemokines and have angiogenic property. Other members of this family such as CXCL4 with the exception of SDF-1 which have not this sequence are angiostatic. The C-C chemokines have also a role in angiogenesis such as CCL2 which can induce ECs migration [1, 40]. Out of chemokines receptors, CXCR2 is the most important one for ELR-containing CXC chemokines such as IL-8 which is expressed on ECs [40]. Matrix mettaloproteinases (MMPs): MMPs are a family of Zn-dependent endopeptides. They are involved in angiogenesis though inducing degradation of extracellular basement proteins, ECs differentiation and tube formation. The expression of MMPs increases in inflammatory cells [41-43]. Galectins (Gals): are a family of evolutionarily conserved animal lectins that bind to betagalactosides and participate in variety of biological functions [44]. Fifteen members of this group have been defined and divided into three classes according to their carbohydrate recognition domain (CRD): [7] -Prototype galectins with one CRD, they exist as monomers or dimmers. -Tandem repeat-type galectins, containing two different CRDs -Chimera type galectin 3 with one CRD which can form oligomers [45]. Galectins can be expressed by many cell types and have a role in angiogenesis. For example Gal-3 can bind to its ligands laminine and fibronectin in extracellular matrix (ECM) and can affect angiogenesis through inducing tube formation and ECs attachment and migration [9]. Gal-1 is another member of this family which can be involved in angiogenesis process. Gal-8 is associated with fibronectin in ECM and can regulate angiogenesis through binding to its receptors on ECs or ECM. It is expressed by primary human dermal lymphatic ECs [7]. Angiogenic Mediator in RA A prime example of immune-driven angiogenesis is seen in RA [46]. RA is an autoimmune disease which is characterized by painful joints, inflammatory cell infiltration in the synovium, angiogenesis and permanent damage of the joints. Angiogenesis leads to the inflammatory pannus formation and supplies the oxygen and nutrients in inflammatory region. Thus, its role could be important in the perpetuation of the RA and progressive destruction of the joints. RA is a chronic systemic autoimmune disease and synovial angiogenesis is considered to be a notable stage in its pathogenesis. Deregulation in immune responses through different immune cells (T cells, B cells, macrophages) and mediators (growth factors, cytokines, chemokines) results in a multistep complex process in angiogenesis reactions [12, 47, 48]. Angiogenesis is essential for maintaining RA progression because the formation of new blood vessels provides a supply of nutrients and oxygen to the augmented inflammatory cells and brings inflammatory cells and mediators inside the joints for progression of RA. [14, 49, 50]. Like many other diseases, various components of immune responses are involved in angiogenesis through T cell subsets, B cell, macrophage, fibroblasts and many cytokines and chemokines. Overall imbalance occurs in the main cytokine systems including VEGF, IL-1, TNF, IL-6, IL-18, IL-15, IL-13, etc. which can lead to angiogenesis as one of inflammatory reactions based on below brief explanation [51]. VEGF and Hypoxia: VEGF is expressed in RA synovial fluid along with its receptors (Flt-1, KDR and NRP-1). Furthermore, fluid and serum VEGF levels are also elevated in RA patients with hypoxic conditions [5, 14]. In RA synovium, expression of VEGF is not only up-regulated by hypoxia but also by IL-1 and TNF-α [52]. The vascular network in joints is dysfunctional, thus the synovium remains a hypoxic environment which in turn leads to the generation of reactive oxygen species and joint damage. Hypoxia can also induce the production of some angiogenic cytokines and chemokines in the joints from macrophages [38]. IL-1 and TNF-α: The activated macrophages are the main source of the TNF-α and IL-1 [53]. The TNF-α level is increased in RA patients and its soluble TNF receptor II (stnfrii) is also significantly increased before onset of the disease [54]. Moreover, TNF-α and IL-1 are synergized with IL-6 and IL-23 to maintain cytokine production from effecter TH17 cells [55, 56]. TNF-α with T cell cytokines such as IL-17 can induce the expression of pro-inflammatory cytokines in the synovium (IL-6 and IL-8) and degenerative enzymes, which lead to the joint inflammation [3, 55]. Moreover, IL-1 and TNF-α can up-regulate the synovium VEGF production [52] and stimulate the production of IL-23 from fibroblast like synovocytes (FLs) in RA [57]. IL-15: is produced by macrophages, ECs and fibroblasts in RA synovium. IL-15 regulates pro-inflammatory cytokines production such as IL-6, TNF-α, IL-1β in synovial tissue [58]. Also IL-15 is over expressed in RA synovium and can induce 59

4 the secretion of the IL-17 which in turn together with IL-6 can enhance IL-15 production from synovial fibroblasts [59]. IL-32: is produced by epithelial cells, NK cells, T cells and monocytes. It has six or more isoforms. IL-32γ isoform is detected in RA synovium which can induce the production of IL-6, IL-8 and CXCL12; therefore it can enhance the inflammation [57]. IL-12: IL-12 level increases in serum and synovial fluids of RA patients and is related to disease activity. Through increasing IFN-γ level, IL-12 can induce TH1 autoimmune responses [60]. IL-27: is a heterodimeric cytokine; it is expressed in monocytes, macrophages, etc. IL-27 has anti-inflammatory property and can enhances IFN-γ and IL-12 production, while it can inhibit IL-2 production [60]. TH17 cytokines; the main intermediate in RA Rheumatoid T cells are able in running the inflammatory process. T cells promote TNF-α, CC and CXC chemokines production in the synovium [3]. TH1 cytokines (IL-12, IFN-γ, IL-27, and IL-2) and TH2 cytokines (IL-4, IL13) inhibit the development of the TH17 cells. Moreover, IL-2 can induce regulatory T cells (Treg) generation [4, 61]. Th17 cells produce some cytokines, with pro-inflammatory properties, which appears to have a potential role in RA immunopathogenesis. Regarding these wide range secretion of cytokines by Th17 cells, it is anticipated that Th17 cell could have a pathogenic role in RA immunopathophysiology. TH17 related cytokines such as IL-17, IL-21, IL-22, IL-6 and TNF-α are implicated in RA and other autoimmune diseases, especially IL-17 has a key role in the progression of disease. IL-17 can induce inflammatory process within joints by secretion of some cytokines such as IL-6 and IL-1 leading to joint destruction through the induction of the matalloproteinases [60, 62]. Among the various isoforms, IL-17A is more potent in inducing IL-6 production [63]. As stated before, IL-17 can synergies with TNF-α and IL-1 in destructive arthritis. IL-17 can also inhibit TH1 through the inhibition of IL-12R beta2 expression on TH1 cells [64]. IL-21 is 15kDa cytokine and belongs to γ-chain cytokine family. It is mainly produced by activated T lymphocytes and TH17 cells and plays an important role in regulating both innate and acquired immunity through binding to its receptors on T cells, B cells and NK cells [58, 65, 66]. Furthermore, it induces TNF-α and IFN-γ production from T cells [65]. IL-21 can promote autoimmunity and be up-regulated in synovial tissues in RA patients. Similar to IL-6, IL-21 can in combination with TGF-β induce the TH17 generation and differentiation. IL-21 can also be induced by IL-6 [55, 59, 61]. Both IL-6 and IL-21 can enhance IL-17 function through the up-regulation of the IL-23 receptors on these cells [59]. IL-22 is another cytokine which is produced by TH17 cells and belongs to IL-10 family. This cytokine can induce the production of inflammatory cytokines and chemokines. Thus, it can enhance hyperplasia [55, 59]. 60 IL-23 is a survival factor for committed TH17 cells. This cytokine increases in peripheral blood and synovial fluid of RA patients [4, 67]. IL-23 is produced by macrophages, T cells, dendritic cells and ECs. It is an important cytokine in joint inflammation. The effect of this cytokine is exerted through TH17 cells [60]. IL-23 increases IL-17A and IL-17F production and inhibits TH1 and regulatory T cells specific transcription factors [55]. IL-6 has a wide range of biological activities and has a key role in RA pathogenesis with a negative effect on bones [68]. Together IL-6 with TGF-β can induce the TH17 cells differentiation from naive T cells. But IL-6 inhibits Tregs differentiation which is induced by TGF-β. IL-6 performs its biological functions through its receptors which exist in both transmembrane and soluble forms [56]. Additionally, other cell types can also have a role in disease progression such as Treg cells and B cells. Treg cells play an essential role in preventing autoimmune disease. Recently the various types of Tregs have been characterized. Treg is one of the components of self-tolerance. IL-6 has an inhibitory effect on Treg differentiation, whose absence causes organ autoimmunity [14, 56]. Although observations show an increased number of Treg cells in synovium but they are ineffective in controlling the inflammation and cartilage damage [43]. Activated B cells can act as antigen presenting cells (APCs), so that they can induce T cell activation. Moreover, through producing pro-inflammatory cytokines and different auto antibodies, these cells play an important role in RA disease development [3, 38, 47]. CONCLUSIONS Angiogenesis plays a main role in several rheumatic diseases, including RA. An imbalance between angiogenic inducers and inhibitors seems to be a critical factor in pathogenesis of this disease. Some specific components of the immune system are suitable targets for antiangiogenic therapies that may stop the joint destruction and disease progression in RA. Novel therapeutic approach and newest strategy for treatment of RA through inhibition of angiogenesis is transcriptional factors such as HIF-1α, LITAF and STAT6B. The VEGF regulation by transcription factors LITAF and/or STAT6B is important in angiogenesis signalling pathways and could be a useful target in the treatment of VEGF associated diseases as RA [69]. In addition, macrophages can produce a variety of pro-angiogenic factors that have been associated with the angiogenic response occurring during RA disease. Macrophages could be a target in the treatment of RA and other rheumatic diseases. 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