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Discusses the ability of the vascular endothelial growth factor (VEGF) to increase cell permeability through direct action on endothelial cells. Physiological mechanisms of VEGF; Medical significance of the growth factor; Intracellular permeability effect of VEGF realized.

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

Formation of a correctly organised vasculature and subsequently embryonic survival is critically dependent on the dosage and site-specific expression of VEGF. Murine VEGF exists in three common isoforms (viz. 120, 164 and 188 amino acids) having different organ specific distribution levels. Gene knock-in studies show that expression of any of the individual isoforms of VEGF extends survival until birth, although each is associated with distinct organ-specific abnormalities. Comparison of the effects of VEGF isoform expression is complicated by the general lethality of mis-expression, in addition to cumulative effects of adjacent tissues from the inappropriately patterned vasculature. Here we investigate the effects of over-expression of individual VEGFA isoforms from the lens-specific alphaA-Crystallin promoter and characterise their effects on the vessel morphology of the hyaloid and developing retinal vasculature. Since the hyaloid vasculature is an anatomically distinct, transient vasculature of the eye, comprising 3 cell types (endothelium, pericytes and macrophages) it is possible to more readily interpret the role of individual VEGF-A isoforms in vascular pattern formation in this model. The severity of the vascular phenotype, characterised by a hyperplastic hyaloid at E13.5 and subsequently retinal vascular patterning and ocular defects, is most severe in transgenics over-expressing the more diffusible forms of VEGFA (120 and 164), whereas in VEGFA(188) transgenics the hyaloid vascular defects partially resolve post-natally. The results of this study indicate that individual isoforms of VEGFA induce distinct vascular phenotypes in the eye during embryonic development and that their relative doses provide instructive cues for vascular patterning.

In the central nervous system (CNS) complex endothelial tight junctions (TJs) form a restrictive paracellular diffusion barrier, the blood-brain barrier (BBB). Pathogenic changes within the CNS are frequently accompanied by the loss of BBB properties, resulting in brain edema. In order to investigate whether BBB leakiness can be monitored by a loss of TJ proteins from cellular borders, we used an in vitro BBB model where brain endothelial cells in co-culture with astrocytes form a tight permeability barrier for 3H-inulin and 14C-sucrose. Removal of astrocytes from the co-culture resulted in an increased permeability to small tracers across the brain endothelial cell monolayer and an opening of the TJs to horseradish peroxidase as detected by electron microscopy. Strikingly, opening of the endothelial TJs was not accompanied by any visible change in the molecular composition of endothelial TJs as junctional localization of the TJ-associated proteins claudin-3, claudin-5, occludin, ZO-1 or ZO-2 or the adherens junction-associated proteins beta-catenin or p120cas did not change. Thus, opening of BBB TJs is not readily accompanied by the complete loss of the junctional localization of TJ proteins.

Vascular endothelial growth factor A (VEGFA) plays a pivotal role in the first steps of endothelial and haematopoietic development in the yolk sac, as well as in the establishment of the cardiovascular system of the embryo. At the onset of gastrulation, VEGFA is primarily expressed in the yolk sac visceral endoderm and in the yolk sac mesothelium. We report the generation and analysis of a Vegf hypomorphic allele, Vegf(lo). Animals heterozygous for the targeted mutation are viable. Homozygous embryos, however, die at 9.0 dpc because of severe abnormalities in the yolk sac vasculature and deficiencies in the development of the dorsal aortae. We find that providing 'Vegf wild-type' visceral endoderm to the hypomorphic embryos restores normal blood and endothelial differentiation in the yolk sac, but does not rescue the phenotype in the embryo proper. In the opposite situation, however, when Vegf hypomorphic visceral endoderm is provided to a wild-type embryo, the 'Vegf wild-type' yolk sac mesoderm is not sufficient to support proper vessel formation and haematopoietic differentiation in this extra-embryonic membrane. These findings demonstrate that VEGFA expression in the visceral endoderm is absolutely required for the normal expansion and organisation of both the endothelial and haematopoietic lineages in the early sites of vessel and blood formation. However, normal VEGFA expression in the yolk sac mesoderm alone is not sufficient for supporting the proper development of the early vascular and haematopoietic system.

The formation of new microvasculature by capillary sprouting, or angiogenesis, is a prerequisite for solid tumor growth. The genetic alterations required to activate the angiogenic program in tumor angiogenesis are still only vaguely known, but dominantly acting oncoproteins may have a much greater impact than previously realized. Here we have studied the consequences of oncogenic transformation on tumor angiogenesis in a mouse mammary carcinoma model. We provide evidence that the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptor-2 (Flk-1), a signaling system centrally involved in tumor angiogenesis, occurs efficiently in tumors formed by Ras-transformed mammary epithelial cells and that both TGF-beta1 and hypoxia are potent inducers of VEGF expression in these cells. VEGF induction in the tumor periphery is mainly triggered by TGF-beta1, whereas VEGF expression in perinecrotic areas is regulated by both hypoxia and TGF-beta1. As the Ras-transformed tumor cells convert into migrating, fibroblastoid cells that start to produce TGF-beta during tumor progression, the TGF-beta effect on VEGF expression becomes propagated throughout the tumor tissue. Thus, in progressed tumors, areas of TGF-beta1 activation and hypoxia may overlap and hence cooperate to induce VEGF expression and angiogenesis. Nevertheless, the overexpression of VEGF in non-Ras-transformed mouse mammary epithelial cells was not sufficient to promote vascularization in vivo. Based on these findings, we conclude that amongst the multiple mutations that render a normal cell tumorigenic, oncogenic Ras is a major player that in conjunction with the tumor's micro-environment sets the stage for tumor cell invasion and angiogenesis., (Copyright 2002 Wiley-Liss, Inc.)

Sequencing of rat and human vascular endothelial growth factor (VEGF) cDNA clones has previously identified a 3' untranslated region of approximately 1.9 kb, although the apparent site of polyadenylation differed in the two species, despite a high degree of sequence conservation in the region. Neither site is preceded by a canonical AAUAAA polyadenylation signal, a situation frequently found in genes that are subject to alternative polyadenylation. We have sequenced 2.25 kb of the 3' region of the mouse VEGF gene and mapped the usage of potential polyadenylation sites in fibroblasts cultured under both normoxic and hypoxic conditions. We find that two sites for polyadenylation are present in the mouse VEGF gene but the majority of transcripts contain the longer form of the 3'UTR and that their usage is not effected by environmental oxygen tension.

Vascular endothelial growth factor (VEGF) induces both angiogenesis and an increase in vascular permeability, 2 processes that are considered to be important for both tumor growth and the delivery of drugs to the site of tumors. This study demonstrates that transmembrane expression of tumor necrosis factor (tmTNF) is up-regulated in the endothelium of a murine methylcholanthrene (meth A)-induced sarcoma in comparison to the adjacent normal dermal vasculature and is also present on cultivated human endothelial cells. It is further shown that tmTNF is required for VEGF-mediated endothelial hyperpermeability in vitro and in vivo. This permissive activity of TNF appears to be selective, because anti-TNF antibodies ablated the VEGF-induced permeability but not proliferation of cultivated human endothelial cells. Furthermore, tnf gene-deficient mice show no obvious defects in vascularization and develop normally but failed to respond to administration of VEGF with an increase in vascular permeability. Subsequent studies indicated that the tmTNF and VEGF signaling pathways converge at the level of a secondary messenger, the "stress-activated protein kinase-2" (SAPK-2)/p38: (1) up-regulated endothelial expression of tmTNF resulted in the continuous activation of SAPK-2/p38 in vitro, and (2) an inhibitor of SAPK-2/p38 activation abolished the vascular permeability activity of VEGF in vivo. In conclusion, the study's finding that continuous autocrine signaling by tmTNF sensitizes endothelial cells to respond to VEGF by increasing their vascular permeability provides new therapeutic concepts for manipulating vascular hyperpermeability.

Tumor growth is angiogenesis-dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia-mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 are up-regulated in the tumor endothelium. A second endothelial cell-specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin-1 and the inhibitory ligand angiopoietin-2, has been characterized. We have examined 6 human primary breast-cancer samples and 4 murine breast-cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin-1, angiopoietin-2 and tie2 was compared to VEGF and VEGFR-2 expression. Human tumors expressed VEGFR-2 and tie2 but varied considerably in VEGF and angiopoietin-1/-2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR-2 and angiopoietin-2 but not tie2 or angiopoietin-1, suggesting activation of VEGFR-2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR-2, tie2 and angiopoietin-1 but little angiopoietin-2, suggesting activation of both VEGFR-2 and tie2 signaling pathways. In vivo studies using truncated dominant-negative tie2 and VEGFR-2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR-2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR-2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR-2 but suggest that, in addition, tie2-dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in the Flk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed with Flk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream of Flk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.

Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs.

We investigated the hypothesis that hypoxia induces angiogenesis and thereby may counteract the detrimental neurological effects associated with stroke. Forty-eight to seventy-two hours after permanent middle cerebral artery occlusion we found a strong increase in the number of newly formed vessels at the border of the infarction. Using the hypoxia marker nitroimidazole EF5, we detected hypoxic cells in the ischemic border of the neocortex. Expression of vascular endothelial growth factor (VEGF), which is the main regulator of angiogenesis and is inducible by hypoxia, was strongly up-regulated in the ischemic border, at times between 6 and 24 hours after occlusion. In addition, both VEGF receptors (VEGFRs) were up-regulated at the border after 48 hours and later in the ischemic core. Finally, the two transcription factors, hypoxia-inducible factor-1 (HIF-1) and HIF-2, known to be involved in the regulation of VEGF and VEGFR gene expression, were increased in the ischemic border after 72 hours, suggesting a regulatory function for these factors. These results strongly suggest that the VEGF/VEGFR system, induced by hypoxia, leads to the growth of new vessels after cerebral ischemia. Exogenous support of this natural protective mechanism might lead to enhanced survival after stroke.

Proteolysis mediated by the ubiquitin-proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) -induced apoptosis at low concentrations (EC(50)=24 nM), whereas at least 340-fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact-inhibited cells. PSI-mediated cell death could be blocked by a caspase-3 inhibitor (Ac-DEVD-H), but not by a caspase-1 inhibitor (Ac-YVAD-H), suggesting that a caspase-3-like enzyme is activated during PSI-induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti-angiogenic and anti-neoplastic substances.

During development of the vertebrate vascular system essential signals are transduced via protein-tyrosine phosphorylation. Null-mutations of receptor-tyrosine kinase (RTK) genes expressed in endothelial cells (ECs) display early lethal vascular phenotypes. We aimed to identify endothelial protein-tyrosine phosphatases (PTPs), which should have similar importance in EC-biology. A murine receptor-type PTP was identified by a degenerated PCR cloning approach from endothelial cells (VE-PTP). By in situ hybridization this phosphatase was found to be specifically expressed in vascular ECs throughout mouse development. In experiments using GST-fusion proteins, as well as in transient transfections, trapping mutants of VE-PTP co-precipitated with the Angiopoietin receptor Tie-2, but not with the Vascular Endothelial Growth Factor receptor 2 (VEGFR-2/Flk-1). In addition, VE-PTP dephosphorylates Tie-2 but not VEGFR-2. We conclude that VE-PTP is a Tie-2 specific phosphatase expressed in ECs, and VE-PTP phosphatase activity serves to specifically modulate Angiopoietin/Tie-2 function. Based on its potential role as a regulator of blood vessel morphogenesis and maintainance, VE-PTP is a candidate gene for inherited vascular malformations similar to the Tie-2 gene.

Angiogenic growth factors and their endothelial receptors function as major regulators of blood vessel formation. The VEGF/VEGFR and the Angiopoietin/Tie2 receptor systems represent key signal transduction pathways involved in the regulation of embryonic vascular development. Inactivation of any of the genes encoding these molecules results in defective vascular development and lethality between embryonic day 8.5 and 12.5. In addition, VEGF and its receptors are also critically involved in the regulation of pathological blood vessel growth in the adult during various angiogenesis-dependent diseases that are associated with tissue hypoxia, such as solid tumor growth and ischemic diseases. It is now well established that therapeutic angiogenesis can be achieved in animal models of hind limb and myocardial ischemia by exogenously adding VEGF and/or other angiogenic growth factors. Available clinical data from human trials also suggests that patients with severe cardiovascular diseases could potentially benefit from such therapies. However, much more work needs to be done to compare the potency of different angiogenic factors or the combination thereof, as well as the best way of delivery, either as recombinant proteins, as naked DNA or via adenoviral vectors. Nevertheless, the therapeutic efficacy of simply injecting naked plasmid DNA or proteins into ischemic tissue to deliver secreted angiogenic factors is an encouraging finding. Time will show whether the adverse side effects of therapeutic angiogenesis, mainly vascular permeability and edema formation, can be minimized and angiogenic factors can be used as an effective therapy in patients for the treatment of ischemic diseases such as arterial occlusive disease, myocardial infarction, and, eventually, also stroke.

With increasing size tumors are continually dependent on a functional blood vessel system to guarantee the supply with oxygen and nutrients. Vascular endothelial growth factor (VEGF) is a key mediator not only of developmental but also of hypoxia-mediated and tumor-induced angiogenesis. Gene therapy using antisense VEGF with the aim to inhibit tumor angiogenesis may be a successful strategy for the treatment of highly vascular and invasive malignant gliomas. We investigated whether retrovirus producer cells encoding antisense VEGF can be used for in vivo gene transfer. The full length mouse VEGF164 cDNA was cloned in a sense and antisense direction into the retroviral expression vector pLEN. pLEN-VEGF (sense) and pLEN-FGEV (antisense) expression vectors were used to transfect the packaging cell line GP + E86 and to establish ecotropic virus producer cell lines. GP + E86:LEN-FGEV (#5) cells showed high expression of antisense VEGF mRNA, whereas GP+ E86:LEN-VEGF (#8) showed high expression of sense VEGF mRNA and active VEGF protein. Co-implantation of GS-9L cells with retrovirus producing cells containing the antisense VEGF construct into the brains of syngeneic rats showed a statistically significant inhibition of tumor growth and prolongation of survival time, while co-implantation of retrovirus producer cells containing the sense VEGF expression vector resulted in an increasing tumor growth and reduced survival time of the rats compared to control animals. Histological analysis of the tumors co-implanted with GP + E86:LEN-FGEV (#5) cells showed the suppression of angiogenesis, high degree of necrosis and no evidence of a significant immune response. Expression of antisense VEGF mRNA in these tumors was confirmed by in situ hybridization analysis. This is the first report demonstrating the potential utility of virus producer cells as in vivo gene transfer vehicles for antisense VEGF gene therapy of malignant gliomas.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells leading to vascular malformations reminiscent of endothelial tumors or hemangiomas. Flk-1, a receptor tyrosine kinase which is activated upon binding of its ligand VEGF, is predominantly expressed in endothelial cells and essential for the formation of blood vessels since absence of Flk-1 prevents the development of mature endothelial cells in mice and in ES-cell differentiation experiments. To investigate the role of Flk-1 in PymT-induced vascular tumor formation, we studied the expression of Flk-1 and VEGF in PymT-transformed endothelial cells (Endothelioma cells, END. cells). The receptor and its ligand were both expressed in END. cells suggesting that a VEGF/Flk-1 autocrine loop might be causally involved in the formation of vascular tumors. To test this hypothesis, ES cells lacking Flk-1 were generated and the transforming potential of PymT was analysed after in vitro differentiation. Flk-1(-/-) END. cell lines were established which are morphologically identical to flk-1(+/+) END. cells and which express several markers characteristic for endothelial cells. This result suggests that PymT functionally replaces the requirement of Flk-1 in expansion and/or survival of endothelial progenitor cells. Therefore, flk-1(-/-) END. cells provide a powerful tool to dissect the downstream signaling pathways of Flk-1.

The vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) is the first endothelial receptor tyrosine kinase to be expressed in angioblast precursors, and its function is essential for the differentiation of endothelial cells and hematopoietic precursors. We have identified cis-acting regulatory elements of the murine Flk-1 gene that mediate endothelium-specific expression of a LacZ reporter gene in transgenic mice. Sequences within the 5'-flanking region of the Flk-1 gene, in combination with sequences located in the first intron, specifically targeted transgene expression to angioblasts and endothelial cells of transgenic mice. The intronic regulatory sequences functioned as an autonomous endothelium-specific enhancer. Sequences of the 5'-flanking region contributed to a strong, uniform, and reproducible transgene expression and were stimulated by the transcription factor HIF-2alpha. The Flk-1 gene regulatory elements described in this study should allow the elucidation of the molecular mechanisms involved in endothelial cell differentiation and angiogenesis.

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

Malignant gliomas are a prominent target for cancer gene therapy approaches because of their poor prognosis despite all currently available therapies. Gene therapy strategies developed to interfere with the normal function of vascular endothelial growth factor receptors have been successfully used in different experimental models to block tumor angiogenesis and to inhibit tumor growth. In this study we examined whether retroviruses encoding a mutant VEGF receptor 2 (VEGFR-2) could suppress tumor angiogenesis and thereby prolong the survival of rats bearing syngeneic intracerebral glioma tumors. Survival time of rats with intracerebral tumors was significantly prolonged in a dose-dependent manner when retroviruses carrying a VEGFR-2 mutant were cotransplanted with tumor cells. No effect on survival was observed in rats that received virus-producing cells or virus supernatant intracerebrally after 5 days of tumor injection. In established subcutaneous tumors treatment with multiple injections of virus-producing cells also inhibited tumor growth in a dose-dependent manner. After implantation of tumor cells stably transfected with a truncated form of VEGFR-2, rats exhibited a rate of survival similar to that of animals treated with high numbers of virus-producing cells encoding the truncated form of VEGFR-2. Morphologically, tumors showed signs of impaired angiogenesis, such as extensive necrosis and reduced tumor vascular density. These results suggest a dual mode of function of truncated VEGFR-2, namely dominant-negative inhibition of VEGFR-2 function and VEGF depletion by receptor binding. We further explored the safety of retrovirus-mediated gene transfer. Although virus sequences were found in different tissues after intracerebral injection of virus-producing cells, no morphological changes were observed in any tissue after a follow-up time of 6 months. Our results indicate that VEGFR-2 inhibition is useful for the treatment of malignant gliomas.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a mitogen and chemotactic factor for endothelial cells in vitro and an angiogenesis and vascular permeability factor in vivo. Due to its properties, VEGF is a candidate for both angiogenesis and vascular permeability/oedema induction which typically occur in glioblastomas. In this study we test the hypothesis that the antioedema effect of dexamethasone is mediated by downregulation of VEGF or VEGF receptor expression. VEGF mRNA and protein levels of two rat glioma cells lines, C6 and GS-9L, were determined after incubation with dexamethasone under normoxic and hypoxic conditions. In normoxic C6 and GS9L cells, we observed 50-60% downregulation of VEGF mRNA by dexamethasone (P=0.015 and P=0. 01, respectively). This effect was dependent on glucocorticoid-receptor (GR) function. The inhibitory effect of dexamethasone on VEGF gene expression by tumour cells was markedly reduced by hypoxia which suggests that the upregulation of VEGF driven by hypoxia overcomes the effect of the dexamethasone. Dexamethasone did not alter VEGFR-2 mRNA levels in human umbilical endothelial cells. In a subcutaneous glioma tumour model, we observed only a 15% decrease in VEGF mRNA expression in dexamethasone treated animals (n = 12) compared with controls animals (P = 0.24). We conclude that dexamethasone may decrease brain tumour-associated oedema by reduction of VEGF expression in tumour cells. However, the highly reduced activity on hypoxic tumour cells suggests that dexamethasone efficacy may be limited by hypoxia in rapidly growing tumours.

Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3' untranslated region (3'UTR), but also contains destabilizing elements in the 5'UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5'UTR, coding region, and 3'UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention.

Eph receptor tyrosine kinases and their cell-surface-bound ligands, the ephrins, regulate axon guidance and bundling in the developing brain, control cell migration and adhesion, and help patterning the embryo. Here we report that two ephrinB ligands and three EphB receptors are expressed in and regulate the formation of the vascular network. Mice lacking ephrinB2 and a proportion of double mutants deficient in EphB2 and EphB3 receptor signaling die in utero before embryonic day 11.5 (E11.5) because of defects in the remodeling of the embryonic vascular system. Our phenotypic analysis suggests complex interactions and multiple functions of Eph receptors and ephrins in the embryonic vasculature. Interaction between ephrinB2 on arteries and its EphB receptors on veins suggests a role in defining boundaries between arterial and venous domains. Expression of ephrinB1 by arterial and venous endothelial cells and EphB3 by veins and some arteries indicates that endothelial cell-to-cell interactions between ephrins and Eph receptors are not restricted to the border between arteries and veins. Furthermore, expression of ephrinB2 and EphB2 in mesenchyme adjacent to vessels and vascular defects in ephB2/ephB3 double mutants indicate a requirement for ephrin-Eph signaling between endothelial cells and surrounding mesenchymal cells. Finally, ephrinB ligands induce capillary sprouting in vitro with a similar efficiency as angiopoietin-1 (Ang1) and vascular endothelial growth factor (VEGF), demonstrating a stimulatory role of ephrins in the remodeling of the developing vascular system.

SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.

The development of the embryonic lens is dependent on the formation and regression of the tunica vasculosa lentis (TVL), which is a transiently occurring capillary plexus that surrounds the posterior part of the lens. In this study, by using the terminal deoxy-nucleotidyl transferase mediated nick end-labelling technique (TUNEL), electron microscopy, radioactive end-labelling of DNA extracted from TVL, and the Comet assay, we show that widespread apoptosis of the endothelial cells that constitute the TVL is occurring already at embryonic day 17.5 (E17.5) of mouse development, much earlier than was reported previously (Jack [1972a] Am. J. Ophthalmol. 74:261-272; Lang [1997] Cell Death Diff. 4:12-20). In addition to apoptotic cell death, regression of this structure is associated with loss of capillary integrity, leakage of erythrocytes into the vitreal compartment, and phagocytosis of the apoptotic endothelium by tissue macrophages (hyalocytes). In situ hybridization experiments with probes for the flk-1 receptor and its high-affinity ligand, vascular endothelial growth factor (VEGF; Terman et al. [1992] Biochem. Biophys. Res. Commun. 187:1579-1586; Millauer et al. [1993] Cell 72:835-846), revealed strong endothelial cell expression for flk-1 in the eyes of E13.5-E17.5 embryos. VEGF mRNA was detected in lens epithelial cells located at the posterior pole of the developing lens in E13.5 embryos, in close proximity to the TVL capillaries. At later times (E14.5-E17.5), when the lens epithelial cells have differentiated into primary lens fiber cells, and a thick lenticular capsule is formed, the expression of VEGF mRNA becomes restricted to the anterior and equatorial portions of the lens. The physical separation of the VEGF-producing cells from the flk-1-expressing endothelium (due to the differentiation of the lens epithelial cells into lens fiber cells and the formation of the lenticular capsule) may deprive the endothelium of an essential survival factor and, thus, may constitute the primary mechanism that is responsible for the induction of endothelial cell apoptosis in this model.

Endothelial monocyte-activating polypeptide II (EMAP II) is a proinflammatory cytokine and a chemoattractant for monocytes. We show here that, in the mouse embryo, EMAP II mRNA was most abundant at sites of tissue remodeling where many apoptotic cells could be detected by terminal deoxynucleotidyltransferase-mediated dUTP end labeling. Removal of dead cells is known to require macrophages, and these were found to colocalize with areas of EMAP II mRNA expression and programmed cell death. In cultured cells, post-translational processing of pro-EMAP II protein to the mature released EMAP II form (23 kDa) occurred coincidentally with apoptosis. Cleavage of pro-EMAP II could be abrogated in cultured cells by using a peptide-based inhibitor, which competes with the ASTD cleavage site of pro-EMAP II. Our results suggest that the coordinate program of cell death includes activation of a caspase-like activity that initiates the processing of a cytokine responsible for macrophage attraction to the sites of apoptosis.

Vascular endothelial growth factor (VEGF), an essential regulator of angiogenesis during early development as well as during the growth of solid tumours, bears an unusually large 5' untranslated region (5'-UTR) in the mRNA of over 1000 nucleotides. We found that the VEGF 5'-UTR, despite being GC-rich and containing an upstream short open reading frame, promotes efficient translation of a luciferase reporter. The VEGF 5'-UTR also allowed translation of luciferase from a dicistronic mRNA when placed between the two cistrons, demonstrating that it contains an internal ribosome entry site. Deletion analysis indicated that the IRES resides towards the 3' end of the 5'-UTR.

Vascular endothelial cells play an important role in tissue homeostasis, fibrinolysis, and coagulation; blood-tissue exchange, vasotonus regulation, blood cell activation, and migration; and the vascularization of tissues. The formation of new blood vessels comprises two distinct steps: vasculogenesis, the in situ assembly of capillaries, and angiogenesis, the sprouting of capillaries from preexisting ones. Vascular endothelial growth factor (VEGF) is essential for vasculogenesis and angiogenesis. Its expression is high in the embryonic brain and kidney when angiogenesis occurs and low in the adult brain when angiogenesis is absent. In the kidney, VEGF expression remains high in glomerular podocytes even in the adult. VEGF receptors 1 and 2 (fit-1 and flk-1) are endothelial-specific receptor tyrosine kinases. Similar to the ligand, expression of these receptors is high during brain and kidney angiogenesis, low in adult brain endothelium, but high in adult glomerular endothelium. Because VEGF is also a vascular permeability factor, the expression in the adult correlates with the low permeability of blood-brain barrier endothelium and the high permeability of fenestrated glomerular endothelium. Although fenestrae formation can be induced in vitro by VEGF and a basal lamina-type extracellular matrix, blood-brain barrier characteristics seem to require the presence of still unknown brain-derived factors.

FAS associated factor 1 (FAF1) has been described as an unusual protein that binds to the intracellular portion of the apoptosis signal transducing receptor FAS/Apo-1 and potentiates apoptosis in L-cells. By means of mRNA differential display we have identified the avian homologue (qFAF) as a fibroblast growth factor-inducible gene in pluripotent cells from E0 quail embryos during mesoderm induction in vitro. Later during embryonic development, qFAF expression is ubiquitous. We confirm that qFAF is associated with FAS, and show that it is phosphorylated on serine residues and localized to the nucleus. By in vitro mutagenesis we have delimited a novel nuclear targeting domain to a short 35 amino acid &agr ;-helical region in the amino-terminal half of the protein. The nuclear function of qFAF remains unclear. However, a probably dominant negative deletion mutant of qFAF causes apoptosis of transfected cells. This function resides in the carboxy-terminal domain of qFAF which shares remarkable sequence homologies with a putative ubiquitin conjugating enzyme from Caenorhabditis elegans. Our data indicate a complex function for FAF, which may be executed during FAS signalling and/or in the ubiquitination pathway, and may be essential for cell differentiation and survival.

Interendothelial junctions play an important role in the regulation of endothelial functions, such as vasculogenesis, angiogenesis, and vascular permeability. In this paper we show that vascular endothelial growth factor (VEGF), a potent inducer of new blood vessels and vascular permeability in vivo, stimulated the migration of endothelial cells after artificial monolayer wounding and induced an increase in paracellular permeability of human umbilical vein endothelial cells (HUVECs). Furthermore, VEGF increased phosphotyrosine labeling at cell-cell contacts. Biochemical analyses revealed a strong induction of VEGF-receptor-2 (flk-1/KDR) tyrosine-autophosphorylation by VEGF which was maximal after 5 minutes and was followed by receptor downregulation. 15 minutes to 1 hour after VEGF stimulation the endothelial adherens junction components VE-cadherin, beta-catenin, plakoglobin, and p120 were maximally phosphorylated on tyrosine, while alpha-catenin was not modified. PECAM-1/CD31, another cell-cell junctional adhesive molecule, was tyrosine phosphorylated with similar kinetics in response to VEGF. In contrast, activation of VEGF-receptor-1 (Flt-1) by its specific ligand placenta growth factor (PlGF) had no effect on the tyrosine phosphorylation of cadherins and catenins. Despite the rapid and transient receptor activation and the subsequent tyrosine phosphorylation of adherens junction proteins the cadherin complex remained stable and associated with junctions. Our results demonstrate that the endothelial adherens junction is a downstream target of VEGFR-2 signaling and suggest that tyrosine phosphorylation of its components may be involved in the the loosening of cell-cell contacts in established vessels to modulate transendothelial permeability and to allow sprouting and cell migration during angiogenesis.

Sprouting of new capillaries from pre-existing blood vessels is a hallmark of angiogenesis during embryonic development and solid tumor growth [1]. In addition to the vascular endothelial growth factor (VEGF) and its receptors, the Tie receptors and their newly identified ligands, the angiopoietins, have been implicated in the control of blood vessel formation [2,3]. Although 'knockouts' of the gene encoding the Tie2 receptor, or its activating ligand angiopoietin-1 (Ang1), result in embryonic lethality in mice due to an absence of remodeling and sprouting of blood vessels [4,5], biological activity in vitro has not yet been described for this receptor-ligand system. In an assay in which a monolayer of endothelial cells were cultured on microcarrier beads and embedded in three-dimensional fibrin gels, recombinant Ang1 (0.5-10 nM) induced the formation of capillary sprouts in a dose-dependent manner that was completely inhibited by soluble Tie2 receptor extracellular domains. In contrast with VEGF, which also induced sprouting of capillaries, Ang1 was only very weakly mitogenic for endothelial cells. Suboptimal concentrations of VEGF and Ang1 acted synergistically to induce sprout formation. Thus, the biological activity of Ang1 in vitro is consistent with the specific phenotype of mice deficient in Tie2 or Ang1. The data suggest that, like in other developmental systems, blood vessel formation requires a hierarchy of master-control genes in which VEGF and angiopoietins, along with their receptors, are amongst the most important regulators.

Smad proteins are intracellular signalling molecules and putative transcription factors that transduce signals elicited by members of the transforming growth factor beta (TGF-beta) superfamily. By comparing the expression of Smad1 and Smad2 during embryonic development, we show that mRNAs of both Smad isoforms are present in a variety of tissues. The major sites of expression of both Smads can be correlated with the expression domains of several members of the TGF-beta superfamily. Our expression data suggest that Smad proteins are involved in organ development, particularly that of organs arising from mesenchymal-epithelial interactions. A second site of strong expression is the central nervous system. Transcriptional control mediated by Smad1 and Smad2, therefore, may exert an important function in differentiation processes of embryonic development that are controlled by ligands of the TGF-beta superfamily.

The vascular system of the central nervous system is derived from capillary endothelial cells, which have invaded the early embryonic neuroectoderm from the perineural vascular plexus. This process is called angiogenesis and is probably regulated by brain-derived factors. Vascular endothelial cell growth factor (VEGF) is an angiogenic growth factor whose expression correlated with embryonic brain angiogenesis, i.e. expression is high in the embryonic brain when angiogenesis occurs and low in the adult brain when angiogenesis is shut off under normal physiological conditions. VEGF receptors 1 and 2 (flt-1 and flk-1) as well as the recently identified angiopoietin receptors (tie-1 and tie-2) are receptor tyrosine kinases specifically expressed in endothelial cells. Expression of these receptors is high during brain angiogenesis but low in adult blood-brain barrier endothelium. They are required for the proper development of a vascular system, and particularly tie-2 is necessary for brain angiogenesis. Signal transduction by these receptors regulates endothelial cell growth, permeability and differentiation. Blood-brain barrier endothelial cell characteristics (complex tight junctions, low number of vesicles, specialized transport systems) are induced by the local brain environment, e.g. neurons and astrocytes. Tight junctions between brain endothelial cells are the structural basis for the paracellular impermeability and high electrical resistance of blood-brain barrier endothelium. Association of tight junction particles with the P-face rather than the number or branching frequency of tight junction stands correlated with blood-brain barrier development and function suggesting that the cytoplasmic anchoring of the tight junctions plays an important role. During inflammation, leukocytes migrate through blood-brain barrier endothelium. ICAM-1 and VCAM-1 on blood-brain barrier endothelial cells appear to be the major mediators of these processes while the selectins are absent from brain endothelium in vivo.

Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1-specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.

In gastrulating embryos, various types of cells are generated before differentiation into specific lineages. The mesoderm of the gastrulating mouse embryo represents a group of such intermediate cells. PDGF receptor alpha (PDGFRalpha), c-Kit and fetal liver kinase 1 (Flk1) are expressed in distinctive mesodermal derivatives of post-gastrulation embryos. Their expressions during gastrulation were examined by whole mount immunostaining with monoclonal antibodies against these three receptors. The antibodies stained different mesodermal subsets in gastrulating embryos. Flow cytometry of head fold stage embryos revealed that Flk1+ mesodermal cells could be further classified by the level of c-Kit expression. To examine the possibility that hematopoietic cell differentiation is initiated from the Flk1+ mesoderm, embryonic stem (ES) cells were cultured on the OP9 or PA6 stromal cell layer; the former but not the latter supported in vitro hematopoiesis from ES cells. Flk1+ cells were detected only on the OP9 cell layer from day 3 of differentiation before the appearance of hematopoietic cells. Thus, Flk1+ cells will be required for in vitro ES cell differentiation into hematopoietic cells. The results suggest that these three receptor tyrosine kinases will be useful for defining and sorting subsets of mesodermal cells from embryos or in vitro cultured ES cells.

The endothelial cell-specific mitogen vascular-endothelial growth factor (VEGF) plays a key role in both physiological and pathological angiogenesis. The up-regulation of VEGF expression in response to reduced oxygen tension occurs through transcriptional and post-transcriptional mechanisms. To investigate the molecular mechanisms of transcriptional activation by hypoxia (1% oxygen), fine mapping of a hypoxia-responsive region of the human VEGF promoter was carried out using luciferase reporter-gene constructs in C6 glioma cells. Here, we report that the binding site of hypoxia-inducible factor 1 (HIF1) is crucial for the hypoxic induction of VEGF gene expression. However, an enhancer subfragment containing the HIF1 binding site was not sufficient to confer full hypoxia responsiveness. Addition of upstream sequences restored the full sensitivity to hypoxia induction. This potentiating effect is due to activator protein 1 binding. The 'potentiating' sequences are unable to confer hypoxia responsiveness on their own. Our results strongly suggest that in C6 glioma cells a complex array of trans-acting factors facilitates full transcriptional induction of VEGF gene expression by hypoxia.

Thickening of the arterial intima and smooth muscle cell (SMC) proliferation remain major problems after vascular surgery and other types of vascular manipulations. We studied the effect of endothelial cell (EC)-specific vascular endothelial growth factor (VEGF) gene transfer on the thickening of the intima using a silicone collar inserted around carotid arteries that acted both as the agent that caused intimal SMC growth and as a reservoir for the transfected gene. The model preserved EC integrity and permitted direct extravascular gene transfer without any intravascular manipulation. Compared to beta-galactosidase (lacZ)-transfected control arteries, plasmid/liposome-mediated VEGF gene transfer significantly reduced intimal thickening 1 week after the gene transfer. Administration to the experimental animals of the nitric oxide (NO) synthase inhibitor L-NAME abolished the difference in intimal thickening between VEGF and lacZ-transfected arteries. Furthermore, VEGF caused NO release from cultured human umbilical vein EC. It is concluded that extravascular VEGF gene transfer attenuates intimal growth and could be useful for the prevention of intimal thickening during vascular surgery. Our results further suggest that VEGF may reduce SMC proliferation via a mechanism that involves VEGF-induced NO production from the endothelium.