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.     

Angiogenic synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in an in vitro quantitative microcarrier-based three-dimensional fibrin angiogenesis system

AIM: To develop an in vitro three-dimensional (3-D) angiogenesis system to analyse the capillary sprouts induced in response to the concentration ranges of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) and to quantify their synergistic activity.METHODS: Microcarriers (MCs) coated with human microvascular endothelial cells (HMVECs) were embedded in fibrin gel and cultured in 24-well plates with assay media. The growth factors bFGF, or VEGF, or both were added to the system. The wells (n = 8/group) were digitally photographed and the average length of capillary-like sprouts (ALS) from each microcarrier was quantitated.RESULTS: In aprotinin-stabilized fibrin matrix, human microvascular endothelial cells on the MCs invaded fibrin,forming sprouts and capillary networks with lumina. The angiogenic effects of bFGF or VEGF were dose-clependent in bhe range from 10 to 40 ng/mL. At d 1, 10 ng/mL of bFGF and VEGF induced angiogenesis with an ALS of 32.13±16.6 μm and 43.75±27.92 μm, respectively, which were significantly higher than that of the control (5.88±4.45 μm, P<0.01),and the differences became more significant as the time increased. In addition, the combination of 10 ng/mL of bFGF and VEGF each induced a more significant effect than the summed effects of bFGF (10 ng/mL) alone and VEGF (10 ng/mL) alone when analyzed using SPSS system for general linear model (GLM) (P= 0.011), and bhat also exceeded the effects by 20 ng/mL of either bFGF or VEGF.CONCLUSION: A microcarrier-based in vitro threedimensional angiogenesis model can be developed in fibrin.It offers a unique system for quantitative analysis of angiogenesis. Both bFGF and VEGF exert their angiogenic effects on HMVECs synergistically and in a dose-dependent manner.     

Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A

During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro. Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization. To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGF beta inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production. These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.     

Angiogenic factors stimulate mast-cell migration

Mast cells accumulate at sites of angiogenesis. The factor(s) that control mast-cell recruitment at these sites have yet to be defined. We sought to determine if angiogenic factors result in mast-cell chemotaxis. In this study, we observed that platelet-derived growth factor-AB (PDGF-AB), vascular endothelial cell growth factor (VEGF), and basic fibroblast growth factor (bFGF) each cause directed migration of murine mast cells at picomolar concentrations, with a typical bell- shaped dose-response curve. Another potent angiogenic factor, platelet- derived endothelial cell growth factor (PD-ECGF), appears to promote chemokinesis of mast cells, whereas tumor necrosis factor-alpha, a weak angiogenic factor, is less robust but still functions as a mast cell chemotactic factor. Epidermal growth factor (EGF), a growth factor with minimal angiogenic properties, was ineffective as a mast cell chemotactic factor. A checkerboard analysis confirmed the directional chemotactic response of PDGF-AB, VEGF, and bFGF, while indicating the chemokinetic response induced by PD-ECGF. Cross-desensitization of growth-factor-induced directed migration was observed between PDGF-AB and bFGF, and also between PDGF-AB and PD-ECGF. Tyrosine kinase- inhibitor genistein effectively dampened the chemotactic responses, whereas pertussis toxin had no effect. In summary, our findings suggest that factors known to act on endothelial cells and stimulate neovascularization may simultaneously serve to recruit mast cells to these sites. The local accumulation of mast cells is believed to facilitate new vessel formation through complex cell:cell interactions.     

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.     

Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.     

VEGF-C differentially regulates VEGF-A expression in ocular and cancer cells; promotes angiogenesis via RhoA mediated pathway

Vascular angiogenesis is regulated by a number of cytokines of which vascular endothelial growth factor (VEGF)-A/and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) play an indisputable role. Similarly lymphangiogenesis is regulated by VEGF-C and its receptor VEGFR3. Currently for treating vasculogenesis diseases such as proliferative retinopathies and cancer, a number of anti-VEGF-A therapies are approved for clinical use. Although clinical efficacies achieved are remarkable, they are found to be transitory in nature, followed by restoration of anti-VEGF therapy resistant angiogenesis. Recently the regulatory role of VEGF-C in initiating and potentiating neo-angiogenesis has been uncovered. Although the interactive nature of VEGF-A and C is known, the dynamics of their expression under knockdown conditions is yet to be established. Here in this study we have utilized siRNA to knockdown both VEGF-A and C either independently or in combination. Analysis of VEGF-A and C expression (only in cancer cell lines MCF7, A549 and H460 but not in the ocular cell line RPE19) has shown enhanced expression levels of VEGF-C with increase in knockdown of VEGF-A. However, VEGF-C knockdown has resulted in decreased expression levels of VEGF-A both in RPE19 and MCF7 cells in a dose dependent manner. In addition, VEGF-C knockdown also resulted in decreased expression of RhoA. Further, knockdown studies of RhoA even with supplementation of VEGF-C or A has resulted in decreased endothelial cell proliferation and stress fiber formation, indicating that VEGF-C does promote angiogenesis via RhoA mediated pathway.     

The periphery of the human fetal adrenal gland is a site of angiogenesis: zonal differential expression and regulation of angiogenic factors

CONTEXT: Although the inner fetal zone (FZ) of the mid-gestation human fetal adrenal (HFA) produces dehydroepiandrosterone sulfate, the function of the outer definitive zone (DZ) remains less clear. We have proposed that the DZ phenotype is that of a pool of progenitor cells, many of which are mitotically active. Recently, we studied HFA expression of a family of vascular endothelial cell-specific angiogenic factors, the angiopoietins (Angs), and demonstrated that Ang2 was localized predominantly in the periphery of the gland. Ang1 stabilizes, whereas Ang2 destabilizes, vessels, increasing responsiveness to angiogenic stimuli such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factor (FGF)-2. OBJECTIVE: Our objective was to test the hypothesis that the periphery of the HFA is a site of angiogenesis. DESIGN: Studies were conducted involving RNA, frozen sections, and primary cell cultures from midgestation HFAs. MAIN OUTCOME MEASURES: Immunofluorescence, laser capture microdissection, and real-time quantitative RT-PCR were used. RESULTS: Double immunostaining demonstrated that proliferating endothelial cells were limited to the DZ and DZ/FZ border. Ang2 mRNA was primarily expressed in the DZ, whereas Ang1 mRNA was primarily in the FZ. VEGF-A and FGF-2 mRNA levels were higher in the DZ. FGF-2 (10 ng/ml) induced Ang2 mRNA by 4-fold in both zones of cells (P < 0.01, at 24 h), but not Ang1 or VEGF-A mRNA. CONCLUSION: Data suggest that angiogenesis occurs at the periphery of the HFA. The DZ-predominant expression of Ang2 may be explained, in part, by the parallel pattern of FGF-2 expression.     

CCL16 activates an angiogenic program in vascular endothelial cells

Besides regulating leukocyte trafficking in normal and injured tissues, several chemokines may positively or negatively regulate angiogenesis. Here we report that CCL16 activates an angiogenic program in vascular endothelial cells by activating CCR1. CCL16 induces dose-dependent random and directional migration of endothelial cells isolated from large vessels and liver capillaries without inducing their proliferation. It also promotes endothelial differentiation into capillary-like structures in an in vitro assay and is angiogenic in the chick chorionallantoic membrane. These angiogenic activities are neutralized by a specific antibody against CCL16. The direct angiogenic activity of CCL16 is further amplified by its ability to prime endothelium to a mitogen signal induced by vascular endothelial growth factor A and to raise their basal production of CXCL8 and CCL2, 2 other angiogenic chemokines. BX471 (R-N-[5-chloro-2-[2-[4(4-fluorophenyl) methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl] urea hydrochloric acid salt), a CCR1 antagonist, inhibits angiogenic properties of CCL16, whereas blocking of CCR8 or desensitizing CCR2, which are both well known receptors for CCL16, did not abolish endothelial activation. CCL16 may be specifically cross-linked to CCR1 expressed on endothelial cells. The largely restricted CCL16 expression in the liver suggests that this chemokine may play a role in hepatic vascular formation during development and in angiogenesis associated to hepatic diseases.     

VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses

Optimal angiogenic and lymphangiogenic gene therapy requires knowledge of the best growth factors for each purpose. We studied the therapeutic potential of human vascular endothelial growth factor (VEGF) family members VEGF-A, VEGF-B, VEGF-C, and VEGF-D as well as a VEGFR-3-specific mutant (VEGF-C156S) using adenoviral gene transfer in rabbit hindlimb skeletal muscle. The significance of proteolytic processing of VEGF-D was explored using adenoviruses encoding either full-length or mature (DeltaNDeltaC) VEGF-D. Adenoviruses expressing potent VEGFR-2 ligands, VEGF-A and VEGF-DDeltaNDeltaC, induced the strongest angiogenesis and vascular permeability effects as assessed by capillary vessel and perfusion measurements, modified Miles assay, and MRI. The most significant feature of angiogenesis induced by both VEGF-A and VEGF-DDeltaNDeltaC was a remarkable enlargement of microvessels with efficient recruitment of pericytes suggesting formation of arterioles or venules. VEGF-A also moderately increased capillary density and created glomeruloid bodies, clusters of tortuous vessels, whereas VEGF-DDeltaNDeltaC-induced angiogenesis was more diffuse. Vascular smooth muscle cell proliferation occurred in regions with increased plasma protein extravasation, indicating that arteriogenesis may be promoted by VEGF-A and VEGF-DDeltaNDeltaC. Full-length VEGF-C and VEGF-D induced predominantly and the selective VEGFR-3 ligand VEGF-C156S exclusively lymphangiogenesis. Unlike angiogenesis, lymphangiogenesis was not dependent on nitric oxide. The VEGFR-1 ligand VEGF-B did not promote either angiogenesis or lymphangiogenesis. Finally, we found a positive correlation between capillary size and vascular permeability. This study compares, for the first time, angiogenesis and lymphangiogenesis induced by gene transfer of different human VEGFs, and shows that VEGF-D is the most potent member when delivered via an adenoviral vector into skeletal muscle.     

Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1

Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1alpha-null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirus-mediated expression of a constitutively active form of HIF-1alpha is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.     

Placental growth factor neutralising antibodies give limited anti-angiogenic effects in an in vitro organotypic angiogenesis model

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.     

Monocyte chemoattractant protein-1-induced angiogenesis is mediated by vascular endothelial growth factor-A

Monocyte chemoattractant protein-1 (MCP-1) has been recognized as an angiogenic chemokine. In the present study, we investigated the detailed mechanism by which MCP-1 induces angiogenesis. We found that MCP-1 up-regulated hypoxia-inducible factor 1 alpha (HIF-1 alpha) gene expression in human aortic endothelial cells (HAECs), which induced vascular endothelial growth factor-A(165) (VEGF-A(165)) expression in the aortic wall and HAECs through activation of p42/44 mitogen-activated protein kinase (MAPK). In vivo angiogenesis assay using chick chorioallantoic membrane (CAM) showed that MCP-1-induced angiogenesis was as potent as that induced by VEGF-A(165) and completely inhibited by a VEGF inhibitor, Flt(2-11). The inhibition of RhoA small G protein did not affect MCP-1-induced VEGF-A(165) production and secretion but completely blocked both MCP-1- and VEGF-A-induced new vessel formation, as determined by CAM assay. These results suggest that MCP-1-induced angiogenesis is composed largely of 2 sequential steps: the induction of VEGF-A gene expression by MCP-1 and the subsequent VEGF-A-induced angiogenesis.     

Oncostatin M induces angiogenesis in vitro and in vivo.

Neovascularization of the atherosclerotic plaque is responsible for its weakening and consequently for the complications of vascular disease. Macrophages are a source of growth factors that can modulate angiogenesis. In this study, we analyzed the effect of oncostatin M (OSM) on angiogenesis, as it could be involved in the development of atherosclerosis. The effect of OSM was compared with those of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). On human dermal microvasculature endothelial cells (HMEC-1s), OSM (22.5 to 112.5 pmol/L) induced a dose-dependent increase in cell proliferation greater than that induced by the classic angiogenic factors vascular endothelial growth factor (VEGF; 543 pmol/L) and basic fibroblast growth factor (bFGF; 1.1 nmol/L). LIF (19 to 475 pmol/L) induced only a 30% increase in cell proliferation, and IL-6 had no effect. Furthermore, in a modified Boyden-chamber model, OSM, LIF, and IL-6 were chemoattractant for HMEC-1s. In a tridimensional gel of fibrin, OSM increased tube formation and tube length, which were already noticeable by day 3. LIF and IL-6 induced a weaker effect that was only obvious by day 10. The angiogenic effect of OSM was also demonstrated in vivo in a rabbit corneal model: OSM was more potent than LIF, the length of the neovessels being longer with OSM than with LIF, whereas IL-6 was without effect. We tested factors that could be involved in the proliferative effect of OSM on HMEC-1s. OSM induced only a slight increase in the urokinase receptor and a 60% increase in VEGF secretion, whereas it does not modify IL-8 secretion or bFGF levels. The effect of OSM seems to depend on endothelial cell origin and cell species: OSM (up to 112.5 pmol/L) did not induce human umbilical vein endothelial cell proliferation and even had a small inhibitory effect (17%) on calf pulmonary artery endothelial cells. In conclusion, OSM induces an angiogenic effect on capillary endothelial cells, which could be, at least in part, implicated in pathological processes such as atherosclerosis or tumor growth.