The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor

Angiogenesis has an essential role in many important pathological and physiological settings. It has been shown that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent cytokine expressed by most malignant tumors, has critical roles in vasculogenesis and both physiological and pathological angiogenesis. We report here that at non-toxic levels, the neurotransmitter dopamine strongly and selectively inhibited the vascular permeabilizing and angiogenic activities of VPF/VEGF. Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VPF/VEGF binding, receptor phosphorylation and subsequent signaling steps. The action of dopamine was specific for VPF/VEGF and did not affect other mediators of microvascular permeability or endothelial-cell proliferation or migration. These results reveal a new link between the nervous system and angiogenesis and indicate that dopamine and other D2 receptors, already in clinical use for other purposes, might have value in anti-angiogenesis therapy.     

Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology.     

Structural requirements for dimerization,glycosylation, secretion,and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosyaated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.     

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.     

The vesiculo-vacuolar organelle (VVO). A new endothelial cell permeability organelle.

A newly defined endothelial cell permeability structure, termed the vesiculo-vacuolar organelle (VVO), has been identified in the microvasculature that accompanies tumors, in venules associated with allergic inflammation, and in the endothelia of normal venules. This organelle provides the major route of extravasation of macromolecules at sites of increased vascular permeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), serotonin, and histamine in animal models. Continuity of these large sessile structures between the vascular lumen and the extracellular space has been demonstrated in kinetic studies with ultrastructural electron-dense tracers, by direct observation of tilted electron micrographs, and by ultrathin serial sections with three-dimensional computer reconstructions. Ultrastructural enzyme-affinity cytochemical and immunocytochemical studies have identified histamine and VPF/VEGF bound to VVOs in vivo in animal models in which these mediators of permeability are released from mast cells and tumor cells, respectively. The high-affinity receptor for VPF/VEGF, VEGFR-2, was localized to VVOs and their substructural components by pre-embedding ultrastructural immunonanogold and immunoperoxidase techniques. Similar methods were used to localize caveolin and vesicle-associated membrane protein (VAMP) to VVOs and caveolae, indicating a possible commonality of formation and function of VVOs to caveolae.     

Vascular Permeability Factor/Vascular Endothelial Growth Factor Inhibits Anchorage-Disruption-Induced Apoptosis in Microvessel Endothelial Cells by Inducing Scaffold Formation

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, requiredde novoprotein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to αvβ5 and vitronectin but not by antibodies to αvβ3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFβ did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.     

Paracrine VEGF/VE-cadherin action on ovarian cancer permeability

Ascites formation associated with neoplasms is most likely due to increased vascular permeability, a process in which vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) plays a pivotal role. We hypothesized that tumor-derived VEGF/VPF modulates ascites formation through a paracrine effect on both tumor and peritoneal vessels. We investigated human vascular endothelial permeability using a newly developed dual-chamber permeability assay by co-culturing human umbilical vein cells with and without ovarian cancer cell lines (OVCAR-3, Hey-A8, and OCC-1) in the presence or absence of a human VEGF monoclonal antibody and VE-cadherin function-blocking antibody. This method permits determination of mechanisms by which substances released from neoplasms and other sources of vascular endothelial cell secretagogues modulate vascular permeability and likely other pathologic states.     

Vascular endothelial growth factor modulates neutrophil transendothelial migration via up-regulation of interleukin-8 in human brain microvascular endothelial cells.

Hypoxia, a strong inducer for vascular endothelial growth factor (VEGF)/vascular permeable factor (VPF) expression, regulates leukocyte infiltration through the up-regulation of adhesion molecules and chemokine release. To determine whether VEGF/VPF is directly involved in chemokine secretion, we analyzed its effects on chemokine expression in human brain microvascular endothelial cells (HBMECs) by using a human cytokine cDNA array kit. Cytokine array analysis revealed a significant increase in expression of monocyte chemoattractant protein-1 and the chemokine receptor CXCR4 in HBMECs, a result similar to that described previously in other endothelial cells. Interestingly, we also observed that VEGF/VPF induced interleukin-8 (IL-8) expression in HBMECs and that IL-8 mRNA was maximal after 1 h of VEGF/VPF treatment of the cells. Enzyme-linked immunosorbent assay data and immunoprecipitation analysis revealed that although VEGF/VPF induced IL-8 expression at the translational level in HBMECs, basic fibroblast growth factor failed to induce this protein expression within 12 h. VEGF/VPF increased IL-8 production in HBMECs through activation of nuclear factor-KB via calcium and phosphatidylinositol 3-kinase pathways, whereas the ERK pathway was not involved in this process. Supernatants of the VEGF/VPF-treated HBMECs significantly increased neutrophil migration across the HBMEC monolayer compared with those of the untreated control. Furthermore, addition of anti-IL-8 antibody blocked this increased migration, indicating that VEGF/VPF induced the functional expression of IL-8 protein in HBMECs. Taken together, these data demonstrate for the first time that VEGF/VPF induces IL-8 expression in HBMECs and contributes to leukocyte infiltration through the expression of chemokines, such as IL-8, in endothelial cells.     

Tyrosine residues 951 and 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for vascular permeability factor/vascular endothelial growth factor-induced endothelium migration and proliferation, respectively.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exerts its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with two receptors intact, we, recently developed chimeric receptors (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR and Flt-1, respectively. With these fusion receptors, we have shown that KDR is solely responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration, whereas Flt-1 showed an inhibitory effect on KDR-mediated proliferation but not migration. To further characterize the VPF/VEGF-stimulated HUVEC proliferation and migration here, we have created several EGDR mutants by site-directed mutagenesis. We show that tyrosine residues 1059 and 951 of KDR are essential for VPF/VEGF-induced HUVEC proliferation and migration, respectively. Furthermore, the mutation of tyrosine 1059 to phenylanaline results in the complete loss of KDR/EGDR-mediated intracellular Ca(2+) mobilization and MAPK phosphorylation, but the mutation of tyrosine 951 to phenylanaline did not affect these events. Our results suggest that KDR mediates different signaling pathways for HUVEC proliferation and migration and, moreover, intracellular Ca(2+) mobilization and MAPK phosphorylation are not essential for VPF/VEGF-induced HUVEC migration.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) peceptor-1 down-modulates VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration, through phosphatidylinositol 3-kinase-dependent pathways

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) achieves its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGF receptor-1) and KDR (VEGF receptor-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with these two receptors intact, we developed a chimeric receptor system in which the N terminus of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR (EGDR) and Flt-1 (EGLT). We observed that KDR, but not Flt-1, was responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration. Moreover, Flt-1 showed an inhibitory effect on KDR-mediated proliferation, but not migration. We also demonstrated that the inhibitory function of Flt-1 was mediated through the phosphatidylinositol 3-kinase (PI-3K)-dependent pathway because inhibitors of PI-3K as well as a dominant negative mutant of p85 (PI-3K subunit) reversed the inhibition, whereas a constitutively activated mutant of p110 introduced the inhibition to HUVEC-EGDR. We also observed that, in VPF/VEGF-stimulated HUVECs, the Flt-1/EGLT-mediated down-modulation of KDR/EGDR signaling was at or before intracellular Ca(2+) mobilization, but after KDR/EGDR phosphorylation. By mutational analysis, we further identified that the tyrosine 794 residue of Flt-1 was essential for its antiproliferative effect. Taken together, these studies contribute significantly to our understanding of the signaling pathways and biological functions triggered by KDR and Flt-1 and describe a unique mechanism in which PI-3K acts as a mediator of antiproliferation in primary vascular endothelium.     

Ovarian steroids in endometrial angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is fundamental for human endometrial development and differentiation, which are necessary for implantation. This vascular process is supposed to be mainly mediated by the vascular endothelial growth factor (VEGF), also named vascular permeability factor (VPF). We report here the expression and modulation of VEGF and its receptors, Flk-1/KDR and Flt-1, in the functionalis throughout the menstrual cycle. Using immunocytochemistry, VEGF is localized in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles. The localization of VEGF on the endothelium correlates with the presence of Flt-1 and Flk-1/KDR receptors on vascular structures, including capillary strands that have not yet formed a lumen and that have been previously described in tumors as angiogenic capillaries. The strongest immunoreactivity for both VEGF and Flk-1/KDR receptor on endothelial cells is detected in the proliferative and midsecretory phases. Enhanced expression of VEGF and its Flk-1 receptors on narrow capillary strands during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration from the residual basal layer following menstrual shedding of the functionalis. The vascular expression of Flt-1 is more important in the secretory than in the proliferative phase, associated with a high microvascular density and an increase in vascular permeability in the implantation period. Consistently with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E(2)), or E(2) + progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner. These results demonstrate that the expression of VEGF and its receptors is cyclically modulated by ovarian steroids, and that this endothelial growth factor acts on the endothelium in a paracrine fashion to control endometrial angiogenesis and permeability.     

Requirement of different signaling pathways mediated by insulin-like growth factor-I receptor for proliferation,invasion, and VPF/VEGF expression in a pancreatic carcinoma cell line

Several oncogenes and growth factors are found to be mutated and overexpressed in adenocarcinoma of the pancreas, and may correlate with its highly aggressive nature. Insulin-like growth factor (IGF-I) and its receptor (IGF-IR) are highly expressed in this tumor type. We examined the IGF-IR-mediated signaling pathways in relation to cell proliferation, invasiveness, and expression pattern of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) in the pancreatic cancer line ASPC-1. Our findings show that IGF-IR is an important growth factor receptor for cell proliferation and invasion, and VPF/VEGF expression in ASPC-1. Further experiments indicate that IGF-IR mediates different signaling pathways to execute its functions. Activation of Ras by IGF-IR was found to be required for the cell invasion. On the other hand Src activation through IGF-IR is required for the cell proliferation, invasion, and also VPF/VEGF expression. Taken together, our data indicate the importance of IGF-IR in growth and invasiveness of the pancreatic cancer cell lines and also point out the multiple signaling pathways channeled through this receptor.     

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.