VEGF-A,HGF and bFGF are involved in IL-17A-mediated migration and capillary-like vessel formation of vascular endothelial cells

Background: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. Objective: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. Methods: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. Results: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. Conclusion: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.     

Role of growth factors in coronary morphogenesis

This communication briefly reviews the role of angiogenic growth factors in myocardial vessel formation during development. The earliest signs of vascularization are the migration and differentiation of angioblasts from the epicardium and subepicardium into the myocardium. A regulator of this process is vascular endothelial growth factor (VEGF), which is probably triggered by hypoxia. The subsequent formation of vascular tubes is regulated by multiple growth factors: VEGF family members, fibroblast growth factors (FGFs), and angiopoietins and their receptors. Our studies on explanted quail hearts reveal that these growth factors are interdependent. We also have shown that a harmonic interplay of growth factors characterizes early postnatal development in rats. Neutralizing antibodies to either basic FGF (bFGF) or VEGF inhibit capillary formation, whereas arteriolar growth is markedly inhibited by bFGF, but not VEGF, neutralizing antibodies. Arteriolar diameter is also increased when anti-bFGF and anti-VEGF are administered in combination. Thus, the hierarchical development of the arteriolar vasculature depends on both of these growth factors; however, the establishment of arterioles, as reflected by length density, is dependent on bFGF but not on VEG. Finally, stretch of cardiac myocytes and endothelial cells serves as a stimulus for increases in growth factor and receptor proteins. We have shown that cyclic stretch of either cell type increases VEGF, and that endothelial cells respond to stretch by up-regulation of VEGF receptor-2 (VEGFR-2), and Tie-2 receptor. These results indicate that both mechanical and metabolic factors are primary stimuli for coronary angiogenesis.     

Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.     

Angiogenic synergy of bFGF and VEGF is antagonized by Angiopoietin-2 in a modified in vivo Matrigel assay

A murine modification of the Matrigel chamber assay originally developed for use on rats is presented. This modified assay permits improved quantification due to subcutaneous Matrigel implants of constant shape and volume. We have quantitatively assessed the angiogenic potential of the growth factors basic fibroblast growth factor (bFGF), VEGF, and Angiopoietin-2 (Ang-2) with special emphasis on their mutual interactions. A reproducible dose-response relationship for bFGF was established for doses between 150 and 1000 ng per chamber, whereas VEGF did not display angiogenic activity on its own in the tested dose of up to 200 ng per chamber. Conversely, we found a strong synergistic action of bFGF and VEGF when combined in a 3:1 ratio. Two other combinations (ratios) with greater VEGF doses were also tested, but the synergistic effect was only observed when 50 ng of VEGF was added to 150 ng per chamber of bFGF. This synergistic effect of bFGF and VEGF was significantly reduced by further addition of 100 ng Angiopoietin-2. Inhibition of the response to bFGF and VEGF was confirmed by in vitro EC migration experiments, which, together with our in vivo results, indicates that Ang-2 may target chemotactic responses to bFGF and VEGF in vivo.     

The c-kit receptor ligand functions as a mast cell chemoattractant.

Mast cells accumulate at sites of neovascularization, solid tumors, and many immune reactions. Such accumulation requires directed migration of mature mast cells or their precursors. The nature of the chemoattractants that regulate mast cell motility and the identity of the receptors that mediate the chemotactic response are poorly understood. We have tested the ability of stem cell factor (SCF), a mast cell growth factor, to stimulate mast cell migration. Our results show that SCF is a potent mast cell attractant that stimulates directional motility of both mucosal and connective tissue-type mast cells. The activity is potentiated by costimulation with interleukin-3 (IL-3), another mast cell chemoattractant. SCF, a known ligand for the c-kit tyrosine kinase receptor, was unable to stimulate motility in W42 mutant mast cells, which have a defective c-kit tyrosine kinase. However, W42 mast cells were still able to migrate in response to IL-3. These results show that SCF is a chemotactic factor as well as a growth factor and that the c-kit receptor can transduce signals leading to both cell proliferation and increased directional cell motility.     

A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices

Interactions between migratory endothelial cells (ECs) and surrounding extracellular matrix (ECM) are of central importance to vascular growth. Here, we present a new model of EC migration and morphogenesis within three-dimensional ECM termed "radial invasion of matrix by aggregated cells" (RIMAC). In the RIMAC model, single aggregates of defined numbers of bovine aortic ECs were embedded within small, lenticular gels of type I collagen supported by annuli of nylon mesh. Culture of the gels in nutrient media resulted in quantifiable, reproducible, radial migration of ECs into the collagen. The angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) each stimulated migration of ECs in a concentration-dependent manner. In combination, bFGF and VEGF stimulated migration synergistically. In contrast, transforming growth factor-beta1 inhibited migration of ECs. Low concentrations (0.1-0.5 ng/ml) of VEGF induced ECs to form multicellular sprouts, some of which possessed lumen-like spaces. Mitomycin C, an inhibitor of cell proliferation, did not affect the migration of ECs into collagen induced by 0.5 ng/ml VEGF but moderately inhibited migration induced by 5 ng/ml VEGF. Increasing the density (concentration) of the collagen gel inhibited the migration of single ECs and increased the branching and anastomosis of multicellular sprouts. We conclude that the RIMAC model is a highly efficacious assay for the screening of potentially angiogenic and angiostatic compounds and, moreover, is advantageous for mechanistic studies of vascular morphogenesis.     

Platelet-derived endothelial cell growth factor: structure and function.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa single chain polypeptide, which stimulates the growth and chemotaxis of endothelial cells in vitro and angiogenesis in vivo. Purification from human platelets and cDNA cloning of PD-ECGF disclosed that it is a novel type of angiogenic factor without sequence similarity to hitherto known proteins. PD-ECGF is present in human platelets as well as in placenta. Amino acid sequencing of PD-ECGF from human placenta revealed that the placental form has an additional 5 amino acids at the N-terminus. In cultured cells, it is produced by normal fibroblasts as well as some transformed cell lines. PD-ECGF lacks a hydrophobic signal sequence and remains inside the producer cells. PD-ECGF may act at sites of injury as a wound hormone and thus play an important role under several physiological and pathological conditions.     

Fibrin fragment E stimulates the proliferation,migration and differentiation of human microvascular endothelial cells in vitro

Various factors involved in haemostasis also regulate the development of new blood vessels by a process called angiogenesis. Enzymatic cleavage of fibrin yields a variety of fibrin degradation products, particularly in areas of intense angiogenesis such as in healing wounds and active atherosclerotic plaques. One of these, fibrin fragment E (FnE), is a potent angiogenic factor in the chick chorioallantoic membrane assay of angiogenesis. Here, we extend these studies to show that FnE stimulates the proliferation, migration and differentiation of human dermal microvascular endothelial cells (HuDMECs) in vitro, both in the absence and presence of such additional endothelial growth factors as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We also show that these stimulatory effects occur at concentrations of the protein known to be present in angiogenic tissues in vivo. FnE enhanced the angiogenic effects of VEGF or bFGF, indicating a possible synergy between the signalling pathways used by these three angiogenic factors. This revised version was published online in June 2006 with corrections to the Cover Date.     

Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase Enhanced Human IgE Production

BackgroundAngiogenesis is one pathogenesis of allergic airway disease.Methodspotent angiogenic factor is platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) in the field of cancer-associated research. Vascular endothelial growth factor (VEGF) is another representative angiogenic factor. Both factors were added to the culture system of human peripheral blood mononuclear cells (PBMC) with IL-4 and anti-CD40 monoclonal antibody (mAb). Total IgE levels in the supernatants and signal transduction of stimulated PBMC were evaluated.ResultsAddition of PD-ECGF enhances in vitro IgE production by PBMC in the presence of IL-4 and anti- CD40 mAb, but VEGF does not enhance IgE production. Although PD-ECGF catalyzes the reversible phosphorolysis of thymidine to 2-deoxy-D-ribose-1-phosphate (2DDR), treatment of 2DDR has no effect on IgE production by human PBMC. Both IL-4 and anti-CD40 mAb induce PD-ECGF by human PBMC. Thymidine phosphorylase inhibitor (TPI), 5-chloro-6-[1- (2-iminopyrrolidinyl) methyl] uracil hydrochloride reduce IgE production via blocking of STAT6- phosphorylation.ConclusionsTaken together, these results suggest TP involvement in the enhancement of IgE production and suggest that TPI is a novel strategy against IgE-related allergic disease.     

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.     

Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture.

Platelet-derived endothelial cell growth factor (PD-ECGF) is a 45-kDa endothelial cell mitogen which has angiogenic properties in vivo. We report here that human foreskin fibroblasts, a human squamous cell carcinoma cell line, and 2 out of the 3 human thyroid carcinoma cell lines investigated produce PD-ECGF, whereas 21 other cell lines examined do not. The positive cell lines contained a 1.8-kilobase PD-ECGF mRNA, and a 45-kDa protein could be demonstrated in lysates of the cell lines by immunoblotting and immunoprecipitation using a specific antiserum against PD-ECGF. Furthermore, the cell lysates contained mitogenic activity for endothelial cells that was neutralized by the PD-ECGF antiserum. PD-ECGF was found to be secreted only slowly from the producer cells, consistent with the previous finding that the primary translation product lacks a signal sequence. The restricted expression and intracellular sequestration of PD-ECGF imply a strictly controlled function in endothelial cell proliferation and angiogenesis. Aberrant production of PD-ECGF may play a role in tumor angiogenesis.     

Distinct regulation of genes by bFGF and VEGF-A in endothelial cells

A finely tuned balance of angiogenic inhibitors and inducers controls the activity of angiogenesis characterized by proliferation, migration and differentiation of endothelial cells. Among many angiogenic factors, basic fibroblast growth factor (bFGF) was first identified to be angiogenic whereas vascular endothelial growth factor A (VEGF-A) is an endothelial cell specific mitogen. In addition to being a specific mitogen, VEGF-A is also known as a vascular permeability factor. The majority of growth factors transduce their mitogenic signals from cell surface to nucleus where gene expression occurs. Whether these ligands utilize a distinct or a common molecular pathway to exert their biological effects on human endothelial cells remains elusive. We thus studied the expression profile of 884 human genes under the influence of either bFGF or VEGF-A alone in the context of human endothelial cells. A total of ninety-four genes were differentially regulated by more than two folds. The expression patterns of 32 genes are similar between the treatment of either factor alone whereas those of the remaining 62 genes are only regulated by one but not the other factor. Their function in the control of angiogenesis will be discussed and apoptotic signaling in the regulation of angiogenesis is also implicated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Expression of platelet-derived endothelial cell growth factor in inflammatory bowel disease

Background: Platelet-derived endothelial cell growth factor (PD-ECGF) is reported to be highly expressed in tumors and inflammatory tissues, but its expression and role in inflammatory bowel disease (IBD) are still unclear. In this study we examined the location and tissue density of cells immunoreactive for PD-ECGF in the colonic mucosa of IBD. Methods: Paraffin-embedded sections of colonic tissue from patients with ulcerative colitis (UC) or Crohn's disease (CD) were immunostained for PD-ECGF. As controls, noninflamed mucosa of IBD, as well as normal colonic mucosa from patients with colorectal cancer, were used. Also, cancer tissues were evaluated. In addition, changes in the expression of PD-ECGF in human umbilical vein endothelial cells (HUVEC) after treatment with inflammatory cytokines and angiogenic factors, as well as after coculture with colon cancer cell lines, were evaluated by flow cytometry. Results: In normal colonic mucosa and noninflamed mucosa of IBD, PD-ECGF expression was negligible. In inflamed colonic mucosa, strong expression was observed, predominantly in macrophages and fibroblasts. Vascular endothelial cells of the inflamed colonic mucosa, but not of normal colonic mucosa or of neoplastic tissues, stained for PD-ECGF, and the microvessel density was significantly increased in the severely inflamed mucosa. Flow cytometry demonstrated that PD-ECGF was constitutively expressed in HUVEC. Inflammatory cytokines and vascular endothelial growth factor (VEGF) increased its expression, whereas basic fibroblast growth factor (bFGF) decreased it. Coculture with colon cancer cell lines in direct contact, but not in those without contact, also resulted in an important decrease in the expression of PD-ECGF in HUVEC. Conclusions: Autocrine production of PD-ECGF by endothelial cells may be a mechanism of inflammatory angiogenesis, but not tumor angiogenesis, and may be particularly important for the maintenance of damaged vasculature in IBD. Received: September 17, 2001 / Accepted: September 6, 2002 Acknowledgments. This study was supported partly by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and partly by a grant from the Ministry of Health, Labour, and Welfare of Japan. Reprint requests to: S. Saito, Present address: Department of Surgery, Chigasaki Municipal Hospital, 5-15-1 Honson, Chigasaki 253-0042, Japan     

Reduced pathological angiogenesis and tumor growth in mice lacking GPR4, a proton sensing receptor

The G protein-coupled receptor GPR4 is activated by acidic pH and recent evidence indicates that it is expressed in endothelial cells. In agreement with these reports, we observe a high correlation of GPR4 mRNA expression with endothelial marker genes, and we confirm expression and acidic pH dependent function of GPR4 in primary human vascular endothelial cells. GPR4-deficient mice were generated; these are viable and fertile and show no gross abnormalities. However, these animals show a significantly reduced angiogenic response to VEGF (vascular endothelial growth factor), but not to bFGF (basic fibroblast growth factor), in a growth factor implant model. Accordingly, in two different orthotopic models, tumor growth is strongly reduced in mice lacking GPR4. Histological analysis of tumors indicates reduced tumor cell proliferation as well as altered vessel morphology, length and density. Moreover, GPR4 deficiency results in reduced VEGFR2 (VEGF Receptor 2) levels in endothelial cells, accounting, at least in part, for the observed phenotype. Our data suggest that endothelial cells sense local tissue acidosis via GPR4 and that this signal is required to generate a full angiogenic response to VEGF.     

Endothelial cell growth factors and the vessel wall

The role of endothelial cell growth factors in the maintenance of the blood vessel wall is, as we have described here, much more complex than merely stimulating the mitogenesis of endothelial cells. The FGFs are capable of eliciting an array of responses in endothelial cells, some, or all, of which are important for neovascularization and the control of clot dissolution. These endothelial cell responses include protease elaboration, chemotaxis, and mitogenesis. That these growth factors seem neither to be constitutively released into the medium of cultured cells that synthesize bFGF, nor released into the bloodstream in vivo suggests that the temporal and local control of neovascularization may involve the regulation of growth factor release from cells such as endothelial cells, fibroblasts, and macrophages. Although there is no known example of this for bFGF, it is well known that both thrombin and Factor Xa stimulate the release of a mitogenic protein from endothelial cells and that low oxygen tension stimulates the release of macrophage-derived angiogenesis factor. In addition, both TGF beta and heparin alone appear to play a role in wound healing and vessel wall maintenance. The work of Roberts et al suggests that TGF beta is not only angiogenic, but also stimulates the growth of fibrotic tissue as well. Studies on mast cells demonstrated that released heparin is chemotactic for endothelial cells and can potentiate tumor angiogenesis. An attractive hypothesis is that these molecules not only act as FGF potentiators or inhibitors but that they also may exert their angiogenic effects by inducing FGF release from cells. Perhaps angiogenin, an angiogenic molecule with no mitogenic activity, works in this way. However, no evidence as yet exists concerning this point. A second level of control of neovascularization may involve the interaction of FGF with other molecules released into the same microenvironment. For example, thrombin and TGF beta released from platelets, as well as heparin released from mast cells, have all been demonstrated to affect bFGF activity in vitro and may act as modifiers of FGF activity in vivo. Since bFGF can modulate fibrinolytic activity, one could imagine that its release into a wound region of the vasculature could have detrimental effects on clot formation and subsequent wound healing. Thus, the transient inhibition of bFGF activity by TGF beta would allow clot formation before the induction of neovascularization by bFGF, TGF beta thereby playing a role in the regulation of the sequence in which events occur.(ABSTRACT TRUNCATED AT 400 WORDS)