the   pathology   of   rheumatoid arthritis ( ra ) is characterized by the proliferation of synovial cells and angiogenesis , pannus formation .
multiple cell types , including lymphocytes , dendritic cells , macrophages , and synovial fibroblasts , contribute to the chronic inflammatory responses of ra , and comprise a major portion of the invasive pannus .
in addition , angiogenesis , the process of new blood vessel formation , is highly active in ra , particularly during the earliest stages of the disease [ 2 , 3 ] .
newly formed vessels can maintain the chronic inflammatory state by transporting inflammatory cells to sites of synovitis , and supply nutrients and oxygen to the pannus [ 2 , 3 ] .
angiogenesis is strictly regulated by many inducers and inhibitors , and a number of proangiogenic factors have been suggested to be involved in neovascularization in ra joints .
these include acidic and basic fibroblast growth factors , transforming growth factor ( tgf)- , angiopoietin , and placenta growth factor ( pigf ) in addition to vascular endothelial growth factor ( vegf ) [ 24 ] .  
the final goal of ra treatment is complete disease remission , and not symptomatic relief . at one end of the spectrum of ra treatment outcomes
lie a large group of patients who do not respond to single disease - modifying antirheumatic drugs ( dmards ) .
recent clinical trials have suggested that several biologic agents , such as tnf- blockers , rituximab , abatacept , and anakinra , are effective at retarding joint destruction and at alleviating ra activity [ 5 , 6 ] .
however , these biologic agents may have serious side effects , such as predispositions to tuberculosis , lymphoma , progressive multifocal leukoencephalopathy , and high cost , which limit their use .
it is also a concern that abrupt stoppages or reductions in these treatments may result in a relapse of disease activity .
moreover , the pathology of ra suggests that it is unlikely that a single biologic agent that targets a specific subset of immune cells is capable of effecting cure . in this review ,
we integrate current knowledge concerning how angiogenesis , specifically vegf , contributes to disease exacerbations in ra .
in addition , we present a new therapy for ra based on a synthetic anti - vegf hexapeptide that specifically targets the interaction between vegf and its receptor .
prospects for the development of pharmacologic regulators of placental growth factor , which is another angiogenic factor implicated in the pathogenesis of ra , also are discussed .
vegf is a dimeric glycoprotein that induces the proliferation and migration of endothelial cells to form new blood vessels , and which increases vascular permeability .
vegf plays important roles during wound healing , embryonic development , the growths of certain solid tumors , and during ascites formation .
several recent reports have demonstrated that vegf is also implicated in the pathogenesis of ra .
vegf in synovial fluids is significantly more increased in ra than in osteoarthritis [ 2 , 13 , 14 ] , and serum levels of vegf correlate well with ra disease activity , particularly with swollen joint counts . vegf protein and mrna are expressed by synovial macrophages and synovial fibroblasts in the synovial tissues of ra patients , and cultured synovial cells are able to secrete vegf under hypoxic conditions or when stimulated with il-1 , il-6 , il-17 , il-18 , -prostaglandin , or tgf- , or by cd40 ligation [ 24 , 1517 ] .
furthermore , vegf knockout mice showed reduced pathology and synovial angiogenesis in antigen - induced models of arthritis .
these findings strongly suggest that the inhibition of the angiogenic action of vegf is likely to suppress rheumatoid inflammation .
angiogenesis and inflammation are interdependent processes , and inflammatory mediators have significant effects on angiogenesis [ 2 , 3 ] .
furthermore , recent studies have suggested that the reverse is also true [ 14 , 19 ] .
for example , chronic transgenic delivery of placental growth factor ( plgf ) to murine epidermis resulted in a significant increase in inflammatory response .
in addition , in a previous study , we demonstrated that 165-amino acid form of vegf , vegf165 , has a direct proinflammatory role in the pathogenesis of ra . in this previous study
, recombinant vegf165 was found to increase the productions of tnf- and il-6 by human peripheral blood mononuclear cells ( pbmc ) .
moreover , the synovial fluid mononuclear cells of ra patients showed a greater response to vegf165 stimulation than the pbmc of healthy controls ( the major cell types that responded to vegf were monocytes ) .
these findings suggest that vegf165 may act as a proinflammatory mediator and as an angiogenic stimulator in ra joints , and thus , they indicate that vegf is an important link between angiogenesis and the inflammatory process .
a number of inflammatory cell types participate in maintaining a mutually activating network in ra joints , which leads to the establishment of a self - perpetuating cycle of autoimmunity .
it has been documented that vegf165 activates endothelial cells to produce chemokines , such as mcp-1 and il-8 [ 20 , 21 ] , which may recruit monocytes around endothelial cells in synovial membranes , where newly employed macrophages , in addition to resident synoviocytes , can produce tnf- and il-6 when stimulated by vegf165 ( as was evidenced by our work ) or via cell contact with activated endothelial cells .
tnf- and il-6 , in turn , further enhance the capacities of macrophages and synoviocytes to secrete vegf165 , and stimulate endothelial cells to induce cell - contact - mediated macrophage activation , which generates a positive feedback - loop ( figure 1 ) . thus , vegf165 may serve as a functional bridge between endothelial cells and macrophages / synoviocytes . in ra synovium ,
synovial fibroblasts proliferate abnormally and invade local environments , and in some ways exhibit the characteristics of tumor cells .
recently , we demonstrated that vegf is crucially required for the survival of rheumatoid synoviocytes . in this previous study , the ligation of recombinant vegf165 to its receptor prevented synoviocyte apoptosis induced by serum starvation or sodium nitroprusside ( snp ) .
vegf165 rapidly triggered pakt and perk activity , and then induced bcl-2 expression in rheumatoid synoviocytes .
furthermore , vegf165 completely blocked snp - induced bcl-2 downregulation and snp - induced bax translocation from the cytosol to mitochondria .
collectively , these results suggest that vegf functions as an important synoviocyte survival factor in ra . as mentioned above , vegf165 is present at higher levels in sera , synovial fluid , and in the inflamed synovial tissues of ra patients than in those of osteoarthritis patients [ 13 , 14 ] .
therefore , ra synoviocytes are more likely to be stimulated by vegf165 than osteoarthritis synoviocytes , which causes synoviocyte hyperplasia .
moreover , hyperplastic synoviocytes in ra joints secrete more vegf165 , and thus , generate positive feedback that promotes their survival ( figure 1 ) .
vegf165 exerts its biological effects by binding with its receptor subtypes , that is , fms - like tyrosine kinase ( flt-1 ) , kinase insert domain - containing receptor ( kdr ) and neuropilin-1 ( np-1 ) .
flt-1 and kdr exhibit tyrosine kinase activity , and both are expressed in the majority of vascular endothelial cells [ 8 , 24 , 25 ] .
kdr is a primary mediator of endothelial cell proliferation in response to vegf165 , whereas unlike kdr , flt-1 is present in inflammatory cells , such as , macrophages and monocytes [ 8 , 24 , 25 ] . therefore , in addition to its proangiogenic action , flt-1 is critically involved in monocyte activation , and in addition , it also promotes the mobilization of myeloid progenitors from bone marrow to the blood [ 8 , 24 , 25 ] . on the other hand ,
np-1 has been demonstrated to function as a nontyrosine kinase receptor for vegf165 , and specifically , for the heparin - binding domain of vegf165 [ 26 , 27 ] .
np-1 was initially characterized as a receptor for semaphorin 3a , which mediates the guidance of neuronal cells . in endothelial cells , np-1 is also a coreceptor of vegf , and has been shown to regulate kdr - dependent angiogenesis [ 26 , 27 ] . furthermore ,
we previously demonstrated that np-1 , rather than flt-1 or kdr , is the major vegf165 receptor in ra synoviocytes .
np-1 was found to be highly expressed in the lining layer , and on infiltrating leukocytes and endothelial cells of the rheumatoid synovium .
furthermore , the downregulation of np-1 transcripts by short interfering rna caused spontaneous synoviocyte apoptosis , which was associated with both a decrease in bcl-2 expression and an increase in bax translocation to mitochondria .
in addition , more recently , we found that flt-1 is highly expressed in the sublining of leukocytes in ra synovium , and in monocytes from the pbmc of active ra patients .
furthermore , flt-1 expression levels were found to be well correlated with erythrocyte sedimentation rates , a marker of disease activity , indicating that they reflect inflammatory activity of ra .
these findings suggest that chronic inflammatory milieux , such as those generated by high concentrations of proinflammatory cytokines , may upregulate flt-1 expression on ra monocytes .
first , vegf165 binding to kdr may lead to an increase in angiogenesis , and thereby , the recruitment of peripheral leukocytes to inflamed synovium , which diminishes the growing burden of synoviocytes by supplying the oxygen and nutrients required for tissue metabolism .
second , via interaction with flt-1 , vegf165 may directly stimulate the productions of cytochemokines , such as tnf- , il-6 , mcp-1 , and il-8 , which are essential for the perpetuation of chronic inflammation in joints .
third , np-1 could hamper synoviocyte apoptosis upon ligation of vegf165 , and thus , function as a survival factor , in an autocrine or paracrine fashion . in this manner
, vegf165 would simultaneously regulate the developments of synovial inflammation , hyperplasia , and angiogenesis in ra joints ( figure 1 ) .
the success of anti - vegf antibody ( ab ) treatment in cancer patients raises the possibility of applying antiangiogenic therapies in other diseases , such as retinopathy , ra , and other inflammatory disorders .
given the pleiotropic roles played by vegf and its receptor in ra inflammation [ 2 , 3 , 8 , 24 ] , it can be postulated that anti - vegf treatment retards chronic synovitis in several ways , as follows : ( a ) it may decrease nutrient supply to the tumor - like synovium ; ( b ) inhibit leukocyte adhesion and migration by decreasing endothelial cell surface area ; ( c ) decrease chemokine and cytokine productions by activated endothelial cells ; ( d ) reduce the vegf - induced productions of tnf- and il-6 by monocytes / macrophages ; ( e ) abrogate vegf - induced increases in synoviocyte survival .
these different mechanisms may occur independently , but it remains to be determined which mechanism plays a dominant role in the quenching of ra inflammation .
bevacizumab , a humanized form of anti - vegf ab , was the first antiangiogenic agent approved by the fda in the us for the treatment of metastatic colon cancer , and it was also found to be beneficial for the treatment of lung and renal cell cancer [ 33 , 34 ] .
although bevacizumab is generally well tolerated , it has some serious toxic effects , for example , hypertension , bleeding , and arterial thromboembolism , which occur infrequently [ 3234 ] . currently , various different developmental approaches to inhibit vegf and its receptors are in progress .
the fda approved pegaptanib , an anti - vegf rna aptamer , for the treatment of neovascular age - related macular degeneration ( amd ) .
notably , pegaptanib was found to reduce vision loss in amd patients by about 50% during the first treatment year and to stabilize vision during the second year .
other agents which target vegf receptors , such as , chimerized anti - kdr antibody , vegf - trap , and a synthetic ribozyme of flt-1 , are also undergoing phase i or ii trials for the treatment of solid tumors and cancer .
the effects of vegf and of its receptor antagonists have also been tested in experimental models of ra . neutralizing ab to vegf
was found to prevent collagen - induced arthritis and to ameliorate established disease in mice , and treatment with a soluble form of flt-1 receptor significantly attenuated the severity of murine collagen - induced arthritis .
interestingly , the failure of anti - kdr ab , but not of anti - flt-1 ab , to block arthritis and atherosclerosis [ 24 , 25 , 39 ] , indicates that anti - inflammation , rather than antiangiogenesis , may be primarily responsible for the observed effects of anti - vegf ab . considering that flt-1 tyrosine kinase signaling promotes ra via monocyte / macrophage activation [ 31 , 40 ] ,
the selective inhibition of flt-1 may be effective at blocking vegf - induced inflammation and angiogenesis with minimal toxicity .
however , no clinical trial on anti - vegf inhibitors has been undertaken in ra .
indeed , some antirheumatic drugs with well - known clinical efficacy in ra , such as cyclosporin and anti - tnf- ab , have been reported to inhibit vegf production in ra patients [ 41 , 42 ] . through the screening of positional scanning synthetic peptide libraries
, we identified a soluble arginine - rich hexapeptide sequence , rrkrrr ( arg - arg - lys - arg - arg - arg ) , which binds to vegf165 , and thereby prevents it from interacting with its receptor . to increase the in vivo stability of this peptide , we changed its peptide structure from the l- to the d - form , and accordingly , were able to increase its half life to more than 24 hours , which makes the peptide more suitable for therapeutic applications . in mice ,
the hexapeptide rrkrrr significantly inhibited vegf - induced angiogenesis , and also retarded the growth and metastasis of colon carcinoma cells .
in addition , it strongly inhibited ongoing paw inflammation in arthritic mice without apparent side effects . when compared with several known vegf antagonists , such as anti - vegf antibody and aptamer , rrkrrr is advantageous from the clinical standpoint because it is a short peptide that is easily synthesized and because it has low immunogenicity . in a similar manner ,
bae et al . , also found that the novel anti - flt-1 hexapeptide , gnqwfi ( gly - asn - gln - trp - phe - iie ) , inhibited angiogenesis and tumor growth without side effects .
this peptide selectively binds to flt-1 , and thereby , blocks the interaction between flt-1 and vegf or plgf .
investigations on the effect of anti - flt-1 peptide gnqwfi on an experimental model of arthritis are under way .
plgf is a member of the vegf family , which was first identified in placenta , but is also known to be present in heart , lung , and joints . as a specific ligand for flt-1 , plgf has potent angiogenic properties , and it also induces the growth and migration of endothelial cells [ 24 , 25 ] .
in addition , plgf stimulates tissue factor production and chemotaxis in monocytes , and also increases tnf- , il-1 , il-6 , il-8 , and mcp-1 productions by normal and/or rheumatoid monocytes [ 31 , 46 ] , which suggests that it directly modulates the inflammatory process .
plgf concentrations were reported to be increased in ra synovial fluids , and to induce vegf production by mononuclear cells . moreover , genetic ablation of plgf prevented the development of antibody - induced arthritis in mice   suggesting the critical role of plgf in ra inflammation .
plgf exhibits functions that are distinct from those of vegf , in that it regulates the angiogenic switch during the diseased state .
it was recently reported that neutralizing ab to plgf inhibits the growth and metastasis of various tumors , including those resistant to vegf inhibitors , and that it enhances the efficacies of chemotherapy and that of anti - kdr ab .
unlike anti - kdr ab , anti - plgf ab prevented the infiltration of angiogenic macrophages and severe tumor hypoxia , and thus , did not switch on the angiogenic rescue program responsible for resistance to anti - kdr ab . furthermore , it did not cause or enhance anti - kdr ab - related side effects , such as , the inhibition of placental vascular development .
similar suppressive effects of plgf antagonist on tumor growth were observed in another study , in which an antagonistic plgf peptide ( shryrlaiqlhasdsssscv ) inhibited the growth and metastasis of human breast cancer xenografts . taken together , plgf antagonists may prevent angiogenesis and tumor growth without affecting normal physiology , and thus , are ideal candidates for ra treatment .
we are currently investigating whether synthetic anti - plgf peptides inhibit the severity of arthritis and angiogenesis .
we and others have demonstrated that proangiogenic factors , such as vegf and plgf , exert direct proinflammatory [ 14 , 1921 , 31 , 40 , 45 , 46 ] and antiapoptotic effects [ 23 , 29 ] . in this regards , the developments of synovial inflammation , hyperplasia , and angiogenesis in the joints of ra patients may all be regulated by vegf . given the importance of vegf in the pathology of ra , antiangiogenic therapies , particularly those involving an anti - flt-1 blocking agents , could when administered as a monotherapy or in combination with other biologic agents selectively ameliorate ra symptoms and reverse its fundamental pathology .
the antiangiogenic peptides , rrkrrr and gnqwfi , introduced here represent a promising development in the antiangiogenic field .
 recent experimental and clinical studies have placed new emphasis on the role of angiogenesis in chronic inflammatory disease . 
 vascular endothelial growth factor ( vegf ) and its receptors are the best characterized system in the regulation of rheumatoid arthritis ( ra ) by angiogenesis . 
 furthermore , in addition to its angiogenic role , vegf can act as a direct proinflammatory mediator during the pathogenesis of ra , and protect rheumatoid synoviocytes from apoptosis , which contributes to synovial hyperplasia . 
 therefore , the developments of synovial inflammation , hyperplasia , and angiogenesis in the joints of ra patients seem to be regulated by a common cue , namely , vegf . 
 agents that target vegf , such as anti - vegf antibody and aptamer , have yielded promising clinical data in patients with cancer or macular degeneration , and in ra patients , pharmacologic modulations targeting vegf or its receptor may offer new therapeutic approaches . in this review , 
 the authors integrate current knowledge of vegf signaling and information on vegf antagonists gleaned experimentally and place emphasis on the use of synthetic anti - vegf hexapeptide to prevent vegf interacting with its receptor . 