document: angiogenesis , the process by which the existing vascular network expands to form new blood vessels , is required for the growth of solid tumors . for this reason , 
   vascular endothelial growth factor ( vegf ) , a primary stimulant of angiogenesis , binds and activates vegf receptor 1 ( vegfr1 ) and vegfr2 . vegf is an important and powerful factor increasing vascular permeability and promoting metastasis . without blood vessels , 
   the tumors can not be larger than a few millimeters , so the inhibition of angiogenesis with the use of several drugs could represent an important tool in cancer treatment for several reasons . 
   ( 1 ) angiogenesis occurs at high levels during fetal development , the menstrual cycle , and in wound healing . 
   therefore , the treatments should have low toxicity ; in fact , they might be expected to interfere with this process and should not harm most normal dividing cells . 
   ( 2 ) the antiangiogenic treatments should not be designed to attack directly the cancer cells . 
   the targets of several of these treatments are normal processes controlled by normal cells and not by the tumor cells themselves . 
   the high mutation rates of cancer cells that often render chemotherapy ineffective will not interfere with these drugs .   in this paper 
   , we underline the importance of inhibition of vegf as attractive therapeutic target in the treatment of cancer . 
   vegf is a primary stimulant for tumor angiogenesis , making it a critical target for cancer therapy [ 3 , 4 ] . in breast cancer , elevated levels of vegf 
   actually , bevacizumab , a humanized monoclonal antibody that binds human vegf and prevents vegf from binding vegfr1 and vegfr2 , is approved for the treatment of metastatic her2/neu - negative breast cancer . 
   anti - vegf therapy with bevacizumab , the phenethylamine of the 2c family 2c3 or the fully human antibody that inhibits vegf binding to vegfr2 r84 inhibits the growth of established orthotopic mda - mb-231 breast cancer cell line in severe combined immunodeficiency ( scid ) mice , reduces tumor microvessel density , and limits the infiltration of tumor - associated macrophages , but it is associated with elevated numbers of tumor - associated neutrophils [ 9 , 10 ] . 
   selective inhibition of vegfr2 with an anti - vegf antibody is sufficient for effective blockade of the protumorigenic activity of vegf in breast cancer xenografts . 
   these findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer . 
   inhibition of vegf binding to vegfr2 by 2c3 has been shown to reduce tumor size both in pancreatic [ 1113 ] and breast tumors . 
   also the effect on tumor growth after the treatment with r84 in an orthotopic breast cancer model , similar to 2c3 , has been evaluated . 
   in fact , mda - mb-231 cells ( 5  10 ) were injected into the mammary fat pad of nonobese diabetic nod / scid mice , and the therapy was initiated on day 26 after tumoral cell injection , when tumor volume reached 150 mm . in this orthotopic human breast cancer xenograft model , the chronic treatment with r84 , 2c3 , or bevacizumab significantly reduced ( p < 
   .001 ; days 44 and 48 versus control ) the tumoral growth , such that there was a 55% , 62% , and 58% decrease , respectively , in tumor volume compared with control - treated animals . 
   thus , these data show that inhibition of the vegf factor is sufficient to reduce the mass volume of mda - mb-231-derived tumors . to determine if the effect of r84 , 2c3 , and bevacizumab on mda - mb-231 tumor growth in vivo could be due directly to the block of vegf activation of tumor cells , the tumor cell proliferation and migration were also evaluated in vitro , demonstrating that mda - mb-231 cells migrated strongly toward vegf and , consequently , this migration was blocked by the addition of 2c3 , r84 , or bevacizumab . 
   vegf is an abundant cytokine in the tumor microenvironment and is known to stimulate immune cell chemotaxis ; however , few studies have looked directly at the effect of inhibitors of vegf on immune cell infiltration into tumors . 
   it has been shown previously that 2c3 inhibits macrophage infiltration in an orthotopic pancreatic cancer model [ 16 , 17 ] . in a mouse model of thyroid cancer , 
   in nonobese diabetic ( nod)/scid mice , inoculated with mda - mb-231 cells , anti - vegf therapy with 2c3 , r84 , or bevacizumab reduced tumor microvessel density by 50% , 45% , and 58% , respectively . cancer and inflammation are connected by two pathways : the intrinsic and the extrinsic pathways . 
   these two pathways converge , resulting in the activation of transcription factors , such as nuclear factor-b ( nf-b ) , signal transducer and activator of transcription 3 ( stat3 ) , and hypoxia - inducible factor 1 ( hif1 ) , in tumour cells . 
   these transcription factors modulate the production of inflammatory mediators , including cytokines and chemokines . these factors recruit and activate various leukocytes . 
   the cytokines activate the same key transcription factors in inflammatory cells , stromal cells , and tumor cells , resulting in more inflammatory mediators being produced and a cancer - related inflammatory microenvironment being generated . 
   the mediators and cellular effectors of inflammation are important constituents of the local environment of tumours ; in some cancers , inflammatory conditions are present before a malignant change occurs . to examine the importance of inflammatory microenvironment on tumor site , 
   where a massive macrophage infiltration is associated , breast tumors from bevacizumab , 2c3 , or r84 treatment groups were analyzed using three different macrophage markers : the mouse f4/80 antigen , a 160  kd glycoprotein expressed by murine macrophages , and cd16 + cells , a cluster of differentiation found on the surface of natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes , and macrophages . 
   immunofluorescence staining of tumor sections revealed that anti - vegf therapy with 2c3 , r84 , and bevacizumab reduced cluster of differentiation the infiltration of f4/80 +   and of cd16 + natural killer cells , neutrophil polymorphonuclear leukocytes , monocytes and macrophages . 
   the reduction of macrophage infiltration in the phase of treatment with 2c3 and r84 is presumably due to expression of vegfr2 on tumor - associated macrophages . 
   the major findings of this study were that 2c3 , r84 , and bevacizumab effectively decrease tumor size , microvessel density , and macrophage infiltration in an orthotopic model of breast cancer . 
   these data are consistent with previous findings [ 1618 ] and support the concept that reducing macrophage infiltration is an important aspect of anti - vegf therapy . in addition , these studies showed changes in other immune cell infiltrates : myeloid - derived suppressor cells ( mdscs ) , dendritic cells ( dcs ) , and neutrophils , following anti - vegf therapy . 
   in fact , infiltrating inflammatory cells constitute a large component of the overall tumor mass . 
   vegf is a major chemoattractant for inflammatory cells , including macrophages , neutrophils , dendritic cells , myeloid - derived suppressor cells , and t - cells [ 20 , 21 ] . to evaluate 
   if inhibition of vegf reduces angiogenesis and modulates immune cell infiltration of orthotopic breast cancer xenografts , roland et al . 
   use preclinical models of breast cancer to compare the effect of different anti - vegf therapies on breast cancer growth , vascular parameters , immune cell infiltration , and intratumoral cytokine levels . 
   they confirmed that the inhibition of vegf receptor activation resulted in changes in intratumoral levels of il-1 and cxcl1 that correlated with changes in immune cell infiltration . 
   in fact ,   ma et al . constructed a plasmid encoding vegf shrna to knock down vegf both in vitro and in vivo . in vitro , specificity and potency of the targeting sequence 
   were first validated in a549 lung adenocarcinoma cells by rtpcr and elisa assays . in vivo 
   the vegf shrna expressing plasmids were administered systemically in combination with low dose of cis - diclorodiamminoplatino ( ddp ) that is an antineoplastic chemotherapy agent that interferes with all phases of the cell cycle by binding to dna through the formation of crosslinks between complementary strands . 
   the combinated treatment of the two agents had a significantly enhanced antitumoral effect compared with the treatment with pshvegf or ddp alone , resulting in reduction of the tumor weight by 83.13% . to demonstrate that the therapeutic effects were related to downregulation of vegf expression instead of other nonspecific reactions , 
   it was analyzed the distribution of immunoreactive vegf in the tumors and observed a general decrease of vegf staining in the tumors belonging to the mice treated with pshvegf , whereas the tumors belonging to the mice treated with pshhk exhibited significantly more vegf staining . 
   consistently , the elisa assay showed that pshvegf caused significant reduction in intratumoral vegf expression compared with pshhk . 
   these data reinforce the hypothesis that the effect of anti - vegf agents extends beyond the inhibition of angiogenesis because many immune cells express vegfrs , including macrophages , neutrophils , mdscs , dcs , and t - cells [ 23 , 24 ] . 
   in fact , it was shown a reduction in macrophage number in tumors from animals treated with anti - vegf therapy with respect to the control mice . 
   vegf is a potent stimulator of endothelial cell survival , mitogenesis , migration , and differentiation [ 1 , 2 ] . 
   several vegf inhibitors have been approved by the us food and drug administration for the treatment of tumors or age - related macular degeneration [ 3 , 4 ] ( figure 1 ) . 
   recently , in many clinical trails , angioinhibitors were also being used in combination with conventional chemotherapy . 
   clinical trials generally combine very low dose of chemotherapy followed by angioinhibitor therapy . combination of angioinhibitors will need to be tested vigorously in the future , as single angioinhibitors are approved for use of cancer 
   .   preventive angioinhibitory therapy may also be possible in the future , because angioinhibitory therapy is generally less toxic and less susceptible to induction of acquired drug resistance .
summary: cancer is the second leading cause of death in the world after cardiovascular diseases . 
   some types of cancer cells often travel to other parts of the body through blood circulation or lymph vessels , where they begin to grow . 
   this process is recognized as metastasis . 
   angiogenesis is the formation of new blood vessels from existing vessel . 
   normally angiogenesis is a healthy process , that helps the body to heal wounds and repair damaged body tissues , whereas in cancerous condition this process supports new blood vessels formation that provide a tumor with its own blood supply , nutrients and allow it to grow . 
   the most important proximal factor for angiogenesis is the vascular endothelial growth factor vegf . 
   angioinhibition is a form of targeted therapy that uses drugs to stop tumors from making new blood vessels . 
   therefore , in this paper we analyse the importance of vegf as target of cancer therapy , analysing murine models .