Lymphangiogenesis and biological activity ov vascular endothelial growth factor-C]

Vascular endothelial growth factor (VEGF)-C is a new member of the VEGF family, a group of polypeptide growth factors which play key roles in the physiology and pathology of many aspects of the cardiovascular system, including vasculogenesis, hematopoiesis, angiogenesis and vascular permeability. VEGF signalling in endothelial cells occurs through three tyrosine kinase receptors (VEGFRs), expressed by endothelial cells and hematopoietic precursors. With respect to the first VEGF described, VEGF-A, which is an endothelial cell specific mitogen and key angiogenic factor, VEGF-C seems to play a major role in the development of the lymphatic system. This may reflect the different binding properties of VEGFs to VEGFRs, in that VEGF-A binds to VEGFR-1 and -2, whereas VEGF-C acts through VEGFR-3, whose expression becomes restricted to lymphatics and certain veins during development. However, the finding that VEGF-C also binds to and activates VEGFR-2 may explain why it induces angiogenesis under certain conditions, which makes it relevant to experimental or clinical settings in which one would wish to block or to stimulate angiogenesis. In this paper we briefly discuss current knowledge on the biological activity of VEGF-C, emphasizing that, as has already been shown for a number of other angiogenic factors, the biological effects of VEGF-C are strictly dependent on the activity of other angiogenic regulators present in the microenvironment of the responding endothelial cells.     

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.     

Exogenous recombinant dimeric neuropilin-1 is sufficient to drive angiogenesis

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.     

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

The comparison of VEGFR-1-binding domain of VEGF-A with modelled VEGF-C sheds light on receptor specificity

Receptor specificity determines the role of vascular endothelial growth factors (VEGFs), which either induce angiogenesis via VEGFR-1 and VEGFR-2 receptors or lymphangiogenesis via the VEGFR-3 receptor. Among the VEGFs, VEGF-A and VEGF-B predominantly induce angiogenesis while VEGF-C and VEGF-D induce lymphangiogenesis. The answer for the question of why VEGF-C and VEGF-D are not able to bind VEGFR-1 and behave as angiogenic growth factors may hide behind the details of the tertiary structures of these proteins. In the present study, the tertiary structure of human VEGF-C protein was modelled and the model was compared with the known human VEGF-A tertiary structure. In overall, the modelled structure highly resembled the structure of VEGF-A. The respective key residues that are involved in cysteine-knot motif formation in VEGF-A are similarly located and identically oriented in VEGF-C, indicating the presence of a VEGF-A-like homodimer. However, a VEGF-C homodimer created via monomer docking did not superimpose well with the VEGF-A homodimer. Rigid docking models of VEGF-C with the VEGFR-1 receptor revealed that in the VEGF-C–VEGFR-1 complex, the receptor–protein-interacting residues were not correctly oriented to induce angiogenesis via VEGFR-1. Mapping the electrostatic surface potentials to the protein surfaces revealed noteworthy number of dissimilarities between VEGF-A and VEGF-C, indicating that overall both proteins differ in their folding properties and stability.     

Vascular endothelial growth factor (VEGF)/VEGF-C mosaic molecules reveal specificity determinants and feature novel receptor binding patterns

Vascular endothelial growth factors (VEGFs) and their receptors play key roles in angiogenesis and lymphangiogenesis. VEGF activates VEGF receptor-1 (VEGFR-1) and VEGFR-2, whereas VEGF-C activates VEGFR-2 and VEGFR-3. We have created a library of VEGF/VEGF-C mosaic molecules that contains factors with novel receptor binding profiles, notably proteins binding to all three VEGF receptors ("super-VEGFs"). The analyzed super-VEGFs show both angiogenic and lymphangiogenic effects in vivo, although weaker than the parental molecules. The composition of the VEGFR-3 binding molecules and scanning mutagenesis revealed determinants of receptor binding and specificity. VEGFR-2 and VEGFR-3 showed striking differences in their requirements for VEGF-C binding; extracellular domain 2 of VEGFR-2 was sufficient, whereas in VEGFR-3, both domains 1 and 2 were necessary.     

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.     

Vascular endothelial growth factor (VEGF) receptor-2 signaling mediates VEGF-C(deltaNdeltaC)- and VEGF-A-induced angiogenesis in vitro

Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.     

Relative effects of VEGF-A and VEGF-C on endothelial cell proliferation, migration and PAF synthesis: Role of neuropilin-1

Vascular endothelial growth factor (VEGF-A) is an inducer of endothelial cell (EC) proliferation, migration, and synthesis of inflammatory agents such as platelet-activating factor (PAF). Recently, neuropilin-1 (NRP-1) has been described as a coreceptor of KDR which potentiates VEGF-A activity. However, the role of NRP-1 in numerous VEGF-A activities remains unclear. To assess the contribution of NRP-1 to VEGF-A mediated EC proliferation, migration, and PAF synthesis, we used porcine aortic EC (PAEC) recombinantly expressing Flt-1, NRP-1, KDR or KDR and NRP-1. Cells were stimulated with VEGF-A, which binds to Flt-1, KDR and NRP-1, and VEGF-C, which binds to KDR only. VEGF-A was 12.4-fold more potent than VEGF-C in inducing KDR phosphorylation in PAEC-KDR. VEGF-A and VEGF-C showed similar potency to mediate PAEC-KDR proliferation, migration, and PAF synthesis. On PAEC-KDR/NRP-1, VEGF-A was 28.6-fold more potent than VEGF-C in inducing KDR phosphorylation and PAEC-KDR/NRP-1 proliferation (1.3-fold), migration (1.7-fold), and PAF synthesis (4.6-fold). These results suggest that cooperative binding of VEGF-A to KDR and NRP-1 enhances KDR phosphorylation and its biological activities. Similar results were obtained with bovine aortic EC that endogenously express both KDR and NRP-1 receptors. In contrast, stimulation of PAEC-Flt-1 and PAEC-NRP-1 with VEGF-A or VEGF-C did not induce proliferation, migration, or PAF synthesis. In conclusion, the presence of NRP-1 on EC preferentially increases KDR activation by VEGF-A as well as KDR-mediated biological activities, and may elicit novel intracellular events. On the other hand, VEGF-A and VEGF-C have equipotent biological activities on EC in absence of NRP-1.     

Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on keratinocytes in human epidermis

Vascular endothelial growth factor (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRP-2) are high-affinity receptors for VEGF and are typically considered to be specific for endothelial cells. Here we showed expression of VEGFRs and NRPs on cultured epidermal keratinocytes at both mRNA and protein levels. We further localized these receptors by immunofluorescence (IF) staining in the epidermis of surgical skin specimens. We found positive staining for VEGFRs and NRPs in all layers of the epidermis except for the stratum corneum. VEGFR-1 and VEGFR-2 are primarily expressed on the cytoplasmic membrane of basal cells and the adjacent spinosum keratinocytes. All layers of the epidermis except for the horny cell layer demonstrated a uniform pattern of VEGFR-3, NRP-1, and NRP-2. Sections staining for NRP-1 and NRP-2 also showed diffuse intense fluorescence and were localized to the cell membrane and cytoplasm of keratinocytes. In another panel of experiments, keratinocytes were treated with different concentrations of VEGF, with or without VEGFR-2 neutralizing antibody in culture. VEGF enhanced the proliferation and migration of keratinocytes, and these effects were partially inhibited by pretreatment with VEGFR-2 neutralizing antibody. Adhesion of keratinocytes to type IV collagen-coated culture plates was decreased by VEGF treatment, but this reduction could be completely reversed by pretreatment with VEGFR-2 neutralizing antibody. Taken together, our results suggest that the expression of VEGFRs and NRPs on keratinocytes may constitute important regulators for its activity and may possibly be responsible for the autocrine signaling in the epidermis.     

比较NRP-1反义寡核苷酸与VEGFR-2反义寡核苷酸对人胃癌SGC7901细胞增殖和凋亡的影响

目的:研究比较神经纤毛蛋白1(NRP-1)反义寡核苷酸(ASODN)与血管内皮生长因子受体2(VEGFR-2)反义寡核苷酸(ASODN)对人胃癌SGC7901细胞增殖活性及凋亡水平的影响。 方法:分别及同时将不同浓度经硫代磷酸化修饰的NRP-1 ASODN 和 VEGFR-2 ASODN 转染入人胃癌SGC7901细胞,逆转录-聚合酶链反应(RT-PCR)检测NRP-1基因和VEGFR-2 基因mRNA的转录水平;MTT比色法测量细胞的增殖活性;流式细胞仪测量细胞的凋亡水平。 结果:转染NRP-1 ASODN和VEGFR-2 ASODN后,人胃癌SGC7901细胞NRP-1基因和VEGFR-2 基因mRNA的转录水平均出现降低;NRP-1 ASODN和VEGFR-2 ASODN对SGC7901细胞有明显抑制增殖和促进凋亡的作用,且随着ASODN浓度升高而增强;分别转染时其作用无显著差别,联合转染时其作用明显增强。结论:NRP-1 ASODN和VEGFR-2 ASODN可抑制人胃癌SGC7901细胞 NRP-1基因和VEGFR-2 基因mRNA的转录水平及细胞增殖活性,促进细胞凋亡;与分别转染相比,两者联合转染作用明显增强。     

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.     

VEGF、CD34、VEGF—C和VEGFR-3在胃癌中的表达及临床意义

目的：探讨胃癌组织中VEGF、CD34、VEGF-C和VEGFR-3的表达情况及临床意义。方法：采用免疫组化方法测定81例胃癌组织VEGF、CD34、VEGF—C和VEGFR-3表达情况,并结合患者的临床病理资料进行分析。结果：81例胃癌组织中MVD平均值为（42.95±14．79）个／视野,范围为13．00-68．33个／视野,VEGF、VEGF—C、VEGFR-3阳性表达率分别为74．1％、64．2％、67．9％。VEGF的表达与肿瘤的TNM分期、浸润深度、淋巴结转移有关,CD34的表达与肿瘤的分化程度、TNM分期、浸润深度、淋巴结转移有关,VEGF—C的表达与肿瘤的分化程度、浸润深度、淋巴结转移有关,VEGFR-3的表达与肿瘤的浸润深度、淋巴结转移有关。结论：VEGF、CD34、VEGF—C和VEGFR-3的表达与胃癌的浸润转移密切相关。     

A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.     

Neuropilin-1 forms complexes with vascular endothelial growth factor receptor-2 during megakaryocytic differentiation of UT-7/TPO cells

We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF₁₆₅-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF₁₆₅ promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF₁₆₅. These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF₁₆₅ in megakaryocytic cells.     

Involvement of lysosomal degradation in VEGF-C-induced down-regulation of VEGFR-3

The vascular endothelial growth factor (VEGF)-C-induced down-regulation of VEGF receptor (VEGFR)-3 is important in lymphangiogenesis. Here, we demonstrate that VEGF-C, -D, and -C156S, but not VEGF-A, down-regulate VEGFR-3. VEGF-C stimulates VEGFR-3 tyrosyl phosphorylation and transient phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinases in lymphatic endothelial cells. VEGF-C-induced down-regulation of VEGFR-3 was blocked by a VEGF-C trap, tyrosine kinase inhibitor, and leupeptin, pepstatin, and E64 (LPE), but was unaffected by Notch 1 activator and γ-secretase inhibitors. Our findings indicate that VEGF-C down-regulates VEGFR-3 in lymphatic endothelial cells through VEGFR-3 kinase activation and, in part, via lysosomal degradation.     

VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis

Blood vessels are crucial for normal development and growth by providing oxygen and nutrients. As shown by genetic targeting studies in mice, zebrafish and Xenopus blood vessel formation (or angiogenesis) is a multistep process, which is highly dependent on angiogenic growth factors such as VEGF, the founding member of the VEGF family. VEGF binds to the tyrosine kinase receptors VEGFR-1 and VEGFR-2, and loss of VEGF or its receptors results in abnormal angiogenesis and lethality during development. In contrast, PlGF, another member of this family, binds only to VEGFR-1, and appears to be crucial exclusively for pathological angiogenesis in the adult. However, the expression of VEGFR-1 and VEGFR-2 on non-vascular cells suggests additional biological properties for these growth factors. Indeed, the VEGF family and its receptors determine development and homeostasis of many organs, including the respiratory, skeletal, hematopoietic, nervous, renal and reproductive system, independent of their vascular role. These new insights broaden the activity spectrum of these "angiogenic" growth factors, and may have therapeutic implications when using these growth factors for vascular and/or non-vascular purposes.     

Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi's sarcoma cells.

Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype. In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration. Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion.     

Elevation of circulating interleukin-8 is related to lymph node and distant metastases in esophageal squamous cell carcinomas--implication for clinical evaluation of cancer patient

The presence of lymph node metastasis (LNM) is an important factor in clinical evaluation of esophageal cancer patients. Biological markers able to support detection of metastatic lymph nodes are sought after. Interleukin-8 (IL-8) is overexpressed by many cancers and involved in cancer dissemination. We investigated the relationship between circulating IL-8 and clinicopathological features of esophageal squamous cell carcinoma (ESCC), and evaluated the diagnostic potential of IL-8, with reference to the key angiogenic and lymphangiogenic factors: vascular endothelial growth factors A and C (VEGF-A and VEGF-C). We found elevated IL-8 levels in ESCC patients, correlated with tumor size and cancer dissemination, especially LNM. Circulating IL-8 correlated with lymphangiogenic VEGF-C rather then angiogenic VEGF-A. The association weakened in metastatic cancers, suggesting divergent mechanism of IL-8 involvement in the dissemination process. The cytokine levels correlated with platelets and neutrophils, pointing at these cells as possible sources of circulating IL-8. We demonstrated IL-8 that positively correlated with inflammation status of ESCC patients. Circulating IL-8 was a better indicator of ESCC dissemination than VEGF-A or VEGF-C. Yet, the detection rates were not satisfactory enough to allow for the recommendation of IL-8 determination as an adjunct to the clinical evaluation of lymph node involvement in ESCC patients.