The role of vascular endothelial growth factor in blood vessel formation.

Angiogenic growth factors and their endothelial receptors function as signalling molecules during vascular growth and development. Vascular endothelial growth factor (VEGF) and its receptors represent a key regulatory system of embryonic vascular development and of both physiological and pathological neovascularization.

Vascular endothelial growth factor induces endothelial fenestrations in vitro.

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.

Polyoma middle T-induced vascular tumor formation: the role of the plasminogen activator/plasmin system.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

Tumor endothelium-specific transgene expression directed by vascular endothelial growth factor receptor-2 (Flk-1) promoter/enhancer sequences.

The receptor tyrosine kinase Flk-1 plays a pivotal role in the development of the vascular system and in the vascularization of a wide variety of tumors. We have investigated the activity of cis-acting sequences of the murine Flk-1 gene in the tumor endothelium of experimental tumor models in vivo. B16 melanoma, BFS-1 fibrosarcoma, and polyoma middle T-induced mammary adenocarcinoma were grown in transgenic mice that express the LacZ reporter gene under the control of a 939-bp Flk-1 promoter fragment and an enhancer element located in a 2.3-kb fragment of the first intron. In all experimental tumor models examined, strong endothelium-specific reporter gene expression was observed while being absent from most blood vessels in normal adult tissue. The expression patterns of the LacZ reporter gene correlate well between established tumors grown in Flk1-LacZ transgenic mice and tumors grown in Flk-1+/LacZ knock-in mice that express the LacZ reporter gene from the endogenous Flk-1 locus. The endothelium-specific activity of the Flk-1 promoter/enhancer sequences in three different experimental tumor models demonstrates that the regulatory sequences that mediate the up-regulation of Flk-1 in the tumor endothelium are contained in the Flk-1 promoter/enhancer sequences used, and that these elements function relatively independently of the tumor type. The Flk-1 promoter/enhancer sequences should allow the analysis of the signaling pathways that lead to the up-regulation of Flk-1 in the tumor endothelium and to specifically target therapeutic genes to the endothelium of tumors for antiangiogenic tumor therapy.

Up-regulation of flk-1/vascular endothelial growth factor receptor 2 by its ligand in a cerebral slice culture system.

Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but are down-regulated in the adult, when angiogenesis ceases. Up-regulation of VEGFR-2 and of VEGF are observed in many pathological conditions under which angiogenesis is reinduced. A major regulator of VEGF expression is hypoxia. Although the temporal expression pattern of VEGFR-2 parallels VEGF expression to a high extent, little is known about its regulation. Here, we show that VEGFR-2 is highly expressed in early postnatal mouse brain but is down-regulated commencing at postnatal day 15 (P15) of mouse brain development and is hardly detectable in P30 mouse brain. Using P30 mouse brain slices, we observed that hypoxia up-regulates VEGFR-2 in the slices but not in human umbilical vein endothelial cells, suggesting the presence of a hypoxia-inducible factor in the murine neuroectoderm that up-regulates VEGFR-2. To identify the factors involved, normoxic P30 cerebral slices were cultured with growth factors that are either hypoxia-inducible (e.g., PDGF-BB, erythropoietin, and VEGF) and/or are known to act on endothelial cells (e.g., PDGF-BB, VEGF, and PIGF). Exogenously added recombinant VEGF led to an up-regulation of VEGFR-2 expression, which could be inhibited by preincubation with a neutralizing anti-VEGF antibody. Addition of PDGF-BB, PIGF, and erythropoietin had no effect on VEGFR-2 expression. Our results suggest a differential but synergistic regulation by hypoxia of VEGF and VEGFR-2: a direct induction of VEGF that subsequently up-regulates VEGFR-2 in endothelial cells. This autoenhancing system may represent an important mechanism of tumor angiogenesis.

Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis.

We have recently shown that vascular endothelial growth factor (VEGF) is produced by human malignant glioma cells and acts on tumor endothelial cells, which express VEGF receptors, suggesting that VEGF is a regulator of tumor angiogenesis. To investigate the feasibility of antiangiogenic brain tumor therapy, we developed an intracerebral (i.c.) rat glioma model. We used two transplantable rat glioma cells lines, C6 and GS-9L, to analyze VEGF regulation in vitro and expression of VEGF and its high affinity tyrosine kinase receptors, flt-1 and flk-1, in vivo. Glioma cells were transplanted i.c. or s.c. into syngeneic rats. C6 gliomas exhibit morphological characteristics of human glioblastoma multiforme such as necroses with palisading cells. Immunocytochemistry with von Willebrand factor showed that C6 gliomas are highly vascularized and therefore show another prominent feature of human glioblastoma. GS-9L gliosarcomas were less vascularized. In situ hybridization showed that VEGF is expressed in vivo in rat glioma cells which reside along necrotic areas and therefore closely mimicks the expression pattern of VEGF observed in human glioblastoma. flt-1 and flk-1 are specifically expressed in endothelial cells in the tumor and at the border between tumor and normal brain but are absent from endothelial cells in the normal brain proper. The action of VEGF may therefore be restricted to tumor endothelium. Upregulation of VEGF, but not acid fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor B messenger RNA was observed in hypoxic C6 and GS-9L cells in vitro. These observations are consistent with a role for VEGF in tumor- and hypoxia-induced angiogenesis. Since the expression pattern of VEGF and its receptors in rat glioma appears to be indistinguishable from human glioblastoma multiforme, this model provides an excellent tool to study anti-angiogenic therapy.

Up-regulation of vascular endothelial growth factor and its receptors in von Hippel-Lindau disease-associated and sporadic hemangioblastomas.

Capillary hemangioblastoma is the most frequent manifestation of the autosomal dominantly inherited von Hippel-Lindau (VHL) disease but also presents as a nonfamilial, sporadic vascular tumor. Hemangioblastomas are characterized by a dense network of capillaries in association with cysts. To investigate the mechanisms underlying neovascularization and cyst formation, we analyzed eight VHL disease-associated and five sporadic hemangioblastomas. Histologically, both tumor types showed a similar phenotype. The capillaries expressed the endothelial cell markers von Willebrand factor and CD31 antigen. We investigated the expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen which is also known to induce vascular permeability in vivo, and its high affinity tyrosine kinase receptors flt-1 and KDR. Northern blot and in situ hybridization analysis revealed significant up-regulation of VEGF and VEGF receptor expression in VHL disease-associated and sporadic hemangioblastomas compared to normal brain and tumor stromal cells as sites of abundant VEGF transcription. Endothelial cells did not express detectable amounts of VEGF mRNA but coexpressed flt-1 and KDR. By immunohistochemistry, VEGF protein was detectable in the tumor interstitium and was found to be concentrated around capillaries. Performing reverse transcription-PCR, we demonstrated that VEGF121 and VEGF165 were the splice variants predominantly expressed, whereas mRNA encoding VEGF189 was present at smaller amounts. Our findings suggest that, in VHL disease-associated and sporadic hemangioblastomas, VEGF121 and VEGF165 are secreted by stromal cells and interact with the corresponding VEGF receptors expressed on tumor endothelial cells. This paracrine mechanism may mediate neovascularization and cyst formation in capillary hemangioblastomas.

Up-regulation of vascular endothelial growth factor expression in a rat glioma is conferred by two distinct hypoxia-driven mechanisms.

Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak beta-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene expression in the PNP cells, indicating that transcriptional activation of VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased beta-galactosidase staining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial pressure covered the same PNP cells that were stained for beta-galactosidase. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the perinecrotic palisading cells of gliomas, by two distinct hypoxia-driven regulatory mechanisms.

Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat.

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.

VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries.

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.

Molecular mechanisms of developmental and tumor angiogenesis.

Angiogenesis, the sprouting of capillaries from preexisting vessels, is of fundamental importance during embryonic development and is the principal process by which the brain and certain other organs become vascularized. Angiogenesis occurs during embryonic development but is almost absent in adult tissues. Transient and tightly controlled (physiological) angiogenesis in adult tissues occurs during the female reproductive cycle and during wound healing. In contrast, pathological angiogenesis is characterized by the persistent proliferation of endothelial cells, and is a prominent feature of diseases such as proliferative retinopathy, rheumathoid arthritis, and psoriasis. In addition, many tumors are able to attract blood vessels from neighbouring tissues. Tumor-induced angiogenesis requires a constitutive activation of endothelial cells. These endothelial cells dissolve their surrounding extracellular matrix, migrate toward the tumor, proliferate, and form a new vascular network, thus supplying the tumor with nutrients and oxygen and removing waste products. The onset of angiogenesis in human gliomas is characterized by the expression of genes encoding angiogenic growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) in tumor cells, and coordinate induction of genes in endothelial cells which encode the respective growth factor receptors. Developmental and tumor angiogenesis appear to be regulated by a paracrine mechanism involving VEGF and VEGF receptor-1 and -2.

Thrombopoietin stimulates VEGF release from c-Mpl-expressing cell lines and haematopoietic progenitors.

Vascular endothelial growth factor (VEGF) production was analysed in megakaryocytic cell lines and CD34+ haematopoietic progenitors following treatment with thrombopoietin (TPO). In CMK cells TPO caused a time- and dose-dependent increase in the levels of VEGF released into the medium. A similar effect was observed in UT-7/mpl cells transfected with the TPO receptor c-Mpl, but not in parental UT-7 cells. In CD34+ haematopoietic progenitor cell cultures TPO stimulated VEGF mRNA expression and VEGF protein release. Production of VEGF in CD34+ cultures increased with TPO-induced megakaryocytic differentiation, but not with erythroid or myelomonocytic differentiation induced respectively by erythropoietin and granulocyte-macrophage colony-stimulating factor. These results demonstrate that TPO stimulates VEGF release in c-Mpl-expressing cells and suggest that this process is an integral feature of the megakaryocytic differentiation programme.

Hypoxia and platelet-derived growth factor-BB synergistically upregulate the expression of vascular endothelial growth factor in vascular smooth muscle cells.

Vascular endothelial growth factor (VEGF) mRNA expression was analysed in rabbit vascular smooth muscle cells following exposure to hypoxia and platelet-derived growth factor-BB (PDGF-BB). Hypoxia potently upregulated VEGF mRNA steady-state levels in a time- and concentration-dependent manner reaching a maximum level (approximately 30-fold increase) after 12-24 h at 0% 0(2). In contrast, PDGF-BB caused a modest increase in VEGF expression. However, the combination of PDGF-BB and a threshold hypoxic stimulus (2.5% O2 for 4 h) had a marked synergistic effect. Synergy between hypoxia and PDGF-BB was selective for VEGF expression as hypoxia had no effect on the PDGF-induced upregulation of the proto-oncogene c-myc. These results raise the possibility that hypoxia and PDGF-BB may act in concert to induce VEGF expression in the arterial wall during the development of atherosclerosis.

Angiogenesis in embryos and ischemic diseases.

Angiogenic growth factors and their endothelial receptors are thought to function as major regulators of blood vessel formation. Vascular endothelial growth factor (VEGF) and its receptors, Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2), as well as Angiopoietin-1 and its receptor, Tie-2, represent key signal transduction systems involved in the regulation of embryonic vascular development. The expression of these molecules correlates with phases of blood vessel formation during embryogenesis. Inactivation of any of the genes encoding these molecules in mouse embryos results in defective vascular development and embryonic lethality around mid-gestation. In addition, the VEGF signal transduction system has been implicated in the regulation of pathological blood vessel growth during certain angiogenesis-dependent diseases that are often associated with tissue ischemia, such as proliferative retinopathy or solid tumor growth. This hypothesis is substantiated by experiments, in which the inhibition of VEGF signal transduction resulted in the the inhibition of neovascularization in these diseases. Thus, the VEGF signal transduction system represents a useful target for an anti-angiogenic therapy.

Angiogenesis in embryonic development--a review.

Recent studies, particularly of genetically altered mice, have greatly improved our understanding of the molecular basis underlying the development of the vascular system. Endothelial signalling systems, such as the vascular endothelial growth factor (VEGF)/VEGF receptor system and the angiopoietin/Tie2 system, were identified as central regulators of embryonic angiogenesis. The complex interactions between these and other endothelial signalling systems are beginning to emerge. Placenta (2000), 21, Supplement A, Trophoblast Research, 14, S11-S15.

Insights in vessel development and vascular disorders using targeted inactivation and transfer of vascular endothelial growth factor, the tissue factor receptor, and the plasminogen system.

VEGF has been proposed to participate in normal and pathological vessel formation. Surprisingly, lack of only a single VEGF allele resulted in embryonic lethality due to abnormal formation of intra- and extra-embryonic vessels. Homozygous VEGF-deficient embryos, generated by tetraploid aggregation, revealed an even more severe defect in vessel formation. These results (1) suggest a tight regulation of early vessel development by VEGF and, indirectly, the presence of other VEGF-like molecules; (2) reveal an unprecedented lethal phenotype associated with heterozygous deficiency of an autosomal gene, and (3) demonstrate that tetraploid aggregation was a valid and the only method to study the phenotype of the homozyogous VEGF-deficient embryos. The dominant and strict dose-dependent role of VEGF in vivo renders this molecule a desirable therapeutic target for promoting or preventing angiogenesis. Tissue factor (TF) is the principal cellular initiator of coagulation and its deregulated expression has been related to thrombogenesis in sepsis, cancer, and inflammation. However, TF appears to be also involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis. Surprisingly, TF deficiency resulted in embryonic lethality due to abnormal extra-embryonic vessel development and defective vitelloembryonic circulation. The abnormal yolk sac vasculature is reminiscent of that observed in embryos lacking VEGF, possibly suggesting that both gene functions are interconnected. These targeting studies extend the recently documented role of TF in tumor-associated angiogenesis and warrant further study of its role in angiogenesis during other pathological disorders. The plasminogen system, via its triggers, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), has been implicated in thrombosis, arterial neointima formation, and atherosclerosis. Studies in mice with targeted gene inactivation of t-PA, u-PA, PAI-1, the urokinase receptor (u-PAR), and plasminogen (Plg) revealed (1) that deficiency of t-PA or u-PA increase the susceptibility to thrombosis associated with inflammation and that combined deficiency of t-PA:u-PA or deficiency of Plg induces severe spontaneous thrombosis; (2) that vascular injury-induced neointima formation is reduced in mice lacking u-PA-mediated plasmin proteolysis, unaltered in t-PA- or u-PAR-deficient mice and accelerated in PAI-1-deficient mice, but that it can be reverted by adenoviral PAI-1 gene transfer; and (3) that atherosclerosis in mice doubly deficient in apolipoprotein E (apoE) and PAI-1 is reduced after 10 weeks of cholesterol-rich diet. Thus, the plasminogen system significantly affects thrombosis, restenosis, and atherosclerosis.

Upregulation of vascular endothelial growth factor in severe chronic brain hypoxia of the rat.

The vascular endothelial growth factor (VEGF) has been shown to be upregulated in acute hypoxia. Although an increase in blood vessel number has been described in severe chronic brain hypoxia, it is unclear whether VEGF is upregulated in this condition. We therefore investigated male inbred Wistar rats, which were exposed for 9 to 13 weeks to decreasing amounts of oxygen, down to 7% O2 (15%: 15 days; 12%, 10%, respectively; 8%: 1 day, 3 weeks, respectively; 7%: 4 weeks). The expression of VEGF was studied by Northern analysis and in situ hybridization in frozen sections of cerebral cortex, hippocampus and cerebellum in six chronic hypoxic and two control rats. We found a marked upregulation of VEGF mRNA in all brain regions investigated, being strongest in cerebral cortex and cerebellum. Our results suggest a potential role of VEGF for vascular growth and vascular permeability observed in chronic cerebral hypoxia.

Stress-stimulated volitional coping competencies and depression in multiple sclerosis.

OBJECTIVE: The present study examined the relationship between volitional modes of coping (self-regulation, volitional inhibition, and self-control) and depression in individuals with multiple sclerosis. METHODS: A cross-sectional study of 121 participants aged 22-60 years with clinically defined MS who were consecutively admitted to a neurological rehabilitation center during a 23-month period. Correlation analyses and hierarchical regressions were conducted to evaluate the predictive value of volitional competencies (Volitional Components Questionnaire, short form, VCQ-S) on depression (Centre for Epidemiologic Studies Depression Scale, CES-D), while controlling for demographic (age, gender, and education) and certain clinical variables (Expanded Disability Status Scale, EDSS; disease duration; and Modified Fatigue Impact Scale, MFIS). RESULTS: Hierarchical regression analyses of depression revealed a model in which 68% of the variance in the CES-D was explained by daily stress situations (VCQ-S), self-regulation (VCQ-S), fatigue (MFIS), and education. However, when the analysis included only participants who had scored above the cut-off of the CES-D (n=42), the VCQ-S factor volitional inhibition seemed to play a more relevant part in depression. In particular, the VCQ-S scales stimulation of self-access, stimulation of volitional inhibition, self-motivation, and emotional perseverance/state orientation after failure appear to be valuable predictors on CES-D. CONCLUSIONS: The results suggest that personality-accentuated volitional coping competencies elicited by daily stressful situations could be a relevant factor for depressive mood states in individuals with MS. However, to clarify the exact relationships of this rather circular framework, longitudinal study designs with objective measurements and a stronger focus on MS-specific stressors are needed.