Search PubMed⌕ Search

Biomedical subjects

W Risau

Publications and source records attributed to W Risau.

At least 73 records · Page 4Linked to original sources
PubMed1996

Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.

The endothelial cell-specific vascular endothelial growth factor (VEGF) and its cellular receptors Flt-1 and Flk-1 have been implicated in the formation of the embryonic vasculature. This is suggested by their colocalized expression during embryogenesis and the impaired vessel formation in Flk-1 and Flt-1 deficient embryos. However, because Flt-1 also binds placental growth factor, a VEGF homologue, the precise role of VEGF was unknown. Here we report that formation of blood vessels was abnormal, but not abolished, in heterozygous VEGF-deficient (VEGF+/-) embryos, generated by aggregation of embryonic stem (ES) cells with tetraploid embryos (T-ES) and even more impaired in homozygous VEGF-deficient (VEGF-/-) T-ES embryos, resulting in death at mid-gestation. Similar phenotypes were observed in F1-VEGF+/- embryos, generated by germline transmission. We believe that this heterozygous lethal phenotype, which differs from the homozygous lethality in VEGF-receptor-deficient embryos, is unprecedented for a targeted autosomal gene inactivation, and is indicative of a tight dose-dependent regulation of embryonic vessel development by VEGF.

Alleles↗
PubMed1996

Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo.

Angiogenesis, the sprouting of new blood vessels from existing vessels, occurs in many physiological and pathological processes, including embryonic development, wound healing, and tumor growth. It is required for tumor growth because new blood vessel formation is necessary for tumors to expand beyond a minimum volume. Several growth factor receptor tyrosine kinases have been implicated in angiogenesis, including receptors for epidermal, fibroblast, and platelet-derived growth factors, as well as the receptors Flk-1/KDR, Flt-1 Tek/Tie-2, and Tie-1. Endothelial cells in the vessels of tumors express Flk-1/KDR, a receptor for vascular endothelial growth factor. Flk-1 was previously shown to play a role in angiogenesis and tumor formation of s.c. xenografts of C6 glioma cells using dominant-negative methodology. We now demonstrate that Flk-1 seems to be generally involved in the growth of a wide range of solid tumors, including mammary, ovarian, and lung carcinoma, as well as glioblastoma. Furthermore, survival times in rats bearing intracerebral tumors were prolonged using the same dominant-negative methodology. The involvement of Flk-1 in a variety of tumor types suggests an important role for Flk-1 in tumor angiogenesis.

Animals↗
PubMed1996

Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system.

An early step in the formation of the extraembryonic and intraembryonic vasculature is endothelial cell differentiation and organization in blood islands and vascular structures. This involves the expression and function of specific adhesive molecules at cell-to-cell junctions. Previous work showed that endothelial cells express a cell-specific cadherin (vascular endothelial [VE]-cadherin, or 7B4/cadherin-5) that is organized at cell-to-cell contacts in cultured cells and is able to promote intercellular adhesion. In this study, we investigated whether VE-cadherin could be involved in early cardiovascular development in the mouse embryo. We first cloned and sequenced the mouse VE-cadherin cDNA. At the protein level, murine VE-cadherin presented 75% identity (90%, considering conservative amino acid substitutions) with the human homologue. Transfection of murine VE-cadherin cDNA in L cells induced Ca(++)-dependent cell-to-cell aggregation and reduced cell detachment from monolayers. In situ hybridization of adult tissues showed that the murine molecule is specifically expressed by endothelial cells. In mouse embryos, VE-cadherin transcripts were detected at the very earliest stages of vascular development (E7.5) in mesodermal cells of the yolk sac mesenchyme. At E9.5, expression of VE-cadherin was restricted to the peripheral cell layer of blood islands that gives rise to endothelial cells. Hematopoietic cells in the center of blood islands were not labeled. At later embryonic stages, VE-cadherin transcripts were detected in vascular structures of all organs examined, eg, in the ventricle of the heart, the inner cell lining of the atrium and the dorsal aorta, in intersomitic vessels, and in the capillaries of the developing brain. A comparison with flk-1 expression during brain angiogenesis revealed that brain capillaries expressed relatively low amounts of VE-cadherin. In the adult brain, the level of VE-cadherin transcript was further reduced. By immunohistochemistry, murine VE-cadherin protein was detected at cell-to-cell junctions of endothelial cells. Overall, these data demonstrate that VE-cadherin is an early, constitutive, and specific marker of endothelial cells. This distinguishes this molecule from other cadherins and suggests that its expression is associated with the early assembly of vascular structures.

Amino Acid Sequence↗
PubMed1996

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.

Journal Article↗
PubMed1996

Characterization of the endothelium-specific murine vascular endothelial growth factor receptor-2 (Flk-1) promoter.

Flk-1, a high-affinity signaling receptor for vascular endothelial growth factor (VEGF), is strongly and specifically expressed on endothelial cells during embryonic development of the vascular system and during tumor angiogenesis. Disruption of Flk-1 gene function has recently been shown to prevent completely endothelial cell differentiation during murine embryonic development. To gain insights into the mechanisms that regulate the endothelium-specific Flk-1 expression, we have isolated the 5'-flanking region of the murine Flk-1 gene. RNase protection and primer extension analyses revealed a single transcriptional start site located 299 bp upstream from the translational start site in an initiator-like pyrimidine-rich sequence. The 5'-flanking region is rich in GC residues and lacks a typical TATA or CAAT box. A luciferase reporter construct containing a fragment from nucleotides -1900 to +299 showed strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specific Flk-1 expression is stimulated by the 5'-untranslated region of the first exon, which contains an activating element between nucleotides +137 and +299. In addition, two endothelium-specific negative regulatory elements were identified between nucleotides -4100 and -623. Two strong general activating elements were present in the region between nucleotides -96 and -37, which contains one potential NF kappa B and three potential AP-2 binding sites. This study shows that Flk-1 expression in endothelial cells is mainly regulated by an endothelium-specific activating element in the long 5'-untranslated region of the first exon and by negative regulatory elements located further upstream.

Animals↗
PubMed1995

Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells.

Vascular endothelial growth factor (VEGF) is an endothelial specific angiogenic mitogen secreted from various cell types including tumor cells. Increasing evidence suggests that VEGF is a major regulator of physiological and pathological angiogenesis, and the VEGF/VEGF receptor system has been shown to be necessary for glioma angiogenesis. Hypoxia seems to play a critical role in the induction of VEGF expression during glioma progression. C6 glioma cells provide an in vivo glioma model for the study of tumor angiogenesis, and the expression of VEGF in C6 cells has been shown to be up-regulated by hypoxia in vitro. However, little is known about the molecular mechanism of hypoxic induction of VEGF. Here, we demonstrate that hypoxic induction of VEGF in C6 cells is due to both transcriptional activation and increased stability of mRNA. Nuclear run-on assays revealed a fast and lasting transcriptional activation, whereas the determination of mRNA half-life showed a slower increase of mRNA stability during hypoxia. Reporter gene studies revealed that hypoxia responsive transcription-activating elements were present in the 5'-flanking region of the VEGF gene. These results suggested that several distinct molecular mechanisms were involved in hypoxia-induced gene expression and were activated in a biphasic manner.

Animals↗
PubMed1995

Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation.

Tie-1 and Tie-2 define a new class of receptor tyrosine kinases that are specifically expressed in developing vascular endothelial cells. To study the functions of Tie-1 and Tie-2 during vascular endothelial cell growth and differentiation in vivo, targeted mutations of the genes in mice were introduced by homologous recombination. Embryos deficient in Tie-1 failed to establish structural integrity of vascular endothelial cells, resulting in oedema and subsequently localized haemorrhage. However, analyses of embryos deficient in Tie-2 showed that it is important in angiogenesis, particularly for vascular network formation in endothelial cells. This result contrasts with previous reports on Tie-2 function in vasculogenesis and/or endothelial cell survival. Our in vivo analyses indicate that the structurally related receptor tyrosine kinases Tie-1 and Tie-2 have important but distinct roles in the formation of blood vessels.

Animals↗
PubMed1995

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.

Adult↗
PubMed1995

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.

Base Sequence↗
PubMed1995

Coordinate expression of vascular endothelial growth factor receptor-1 (flt-1) and its ligand suggests a paracrine regulation of murine vascular development.

Vascular endothelial growth factor (VEGF) is a candidate regulator of blood vessel growth during embryonic development and in tumors. To evaluate the role of VEGF receptor-1/flt-1 (VEGFR1/flt-1) in the development of the vascular system, we have characterized the murine homolog of the human flt-1 gene and have analyzed its expression pattern during mouse embryogenesis. Receptor binding studies using transfected COS cells revealed that the murine flt-1 gene encodes a high affinity receptor for VEGF. The apparent Kd for VEGF binding, as determined by Scatchard analysis, was 114 pM, demonstrating that VEGFR1/flt-1 has a higher affinity to VEGF than VEGF receptor-2/flk-1 (VEGFR2/flk-1). By in situ hybridization, VEGFR1/flt-1 was detected in the yolk sac mesoderm already at the early stages of vascular development, while the receptor ligand was expressed in the entire endoderm of 7.5-day mouse embryos. A comparison with VEGFR2/flk-1 showed that the two receptors shared a common expression domain in the yolk sac mesoderm, but were expressed at different sites in the ectoplacental cone. The differential expression of the two VEGF receptors persisted in the developing placenta, where VEGFR1/flt-1 mRNA was detected in the spongiotrophoblast layer, whereas VEGFR2/flk-1 transcripts were present in the labyrinthine layer which is the site of VEGF expression. In the embryo proper, VEGFR1/flt-1 mRNA was specifically localized in blood vessels and capillaries of the developing organs, closely resembling the pattern of VEGFR2/flk-1 transcript distribution. In the developing brain, the expression of VEGF receptors in the perineural capillary plexus and in capillary sprouts which have invaded the neuro-ectoderm correlated with endothelial cell proliferation and brain angiogenesis. The data are consistent with the hypothesis that VEGF and its receptors have an important function both in the differentiation of the endothelial lineage and in the neovascularization of developing organs, and act in a paracrine fashion.

Animals↗
PubMed1995

Angiogenesis in malignant gliomas.

One event that accompanies glioma progression is the upregulation of angiogenesis. Low-grade gliomas are moderately vascularized tumors whereas high-grade gliomas show prominent microvascular proliferations and areas of high vascular density. To analyze the molecular mechanisms underlying glioma angiogenesis, we studied the expression of vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 during normal brain development and glioma-induced angiogenesis. Our results suggest a paracrine control of angiogenesis and endothelial cell proliferation that is tightly regulated and transient in the embryonic brain, switched off in the normal adult brain, and turned on in tumor cells (VEGF) and the host vasculature (VEGFR-1 and -2) during tumor progression. It is unknown how VEGF and VEGF receptors are upregulated during glioma angiogenesis, but there is recent evidence that VEGF as well as endogenous inhibitors of angiogenesis could be under control of the tumor suppressor genes p53 and VHL.

Animals↗
PubMed1995

Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (flk-1) are expressed during vasculogenesis and vascular differentiation in the quail embryo.

Vasculogenesis, the de novo formation of embryonic blood vessels from their angioblastic precursors in situ, is supposed to be under the control of polypeptide growth factors and their receptors. The receptor tyrosine kinase flk-1 and its high-affinity ligand vascular endothelial growth factor (VEGF) represent an endothelial specific signal transduction system expressed during embryonic vascular growth in the mouse. We have cloned the quail homologs of VEGF and flk-1 using PCR and have investigated their expression pattern in vivo. As shown by Northern analysis and reverse transcription PCR, VEGF and flk-1 mRNA (3.9 and 5.8 kb, respectively) were already present in the unincubated blastodisc at low levels and were largely upregulated during gastrulation at Embryonic Day 1. As detected by in situ hybridization, flk-1 mRNA was initially present in the entire mesoderm of Day 1 embryos but from Day 2 on was restricted to endothelial cells. At Day 2 VEGF was ubiquitously expressed in the embryo proper and was mainly restricted to the vascularized part (area vasculosa) in the yolk sac. Later on VEGF expression was detected in all organs. In the kidney VEGF mRNA was mainly localized to the glomeruli. This pattern of expression is consistent with the pattern found during mouse embryogenesis. We have recently established an in vitro model of vasculogenesis in which hemangioblastic precursors are induced in cell cultures from the unincubated quail blastodisc by basic fibroblast growth factor (bFGF) and give rise to blood vessels in vitro. Taking advantage of this in vitro model we examined whether FGF and VEGF act in concert during vasculogenesis. We found that the flk-1 receptor mRNA is dramatically upregulated within 24 hr upon the addition of FGF to quail blastodisc cell cultures. This inducibility in response to FGF is confined to the first 24 hr of culture. The early expression of the flk-1 mRNA may characterize the differentiation of hemangioblastic precursors from pluripotent epiblast cells which in vivo is initiated during gastrulation. Thus, the time course and the pattern of expression during embryogenesis in different species suggest a major role for the VEGF/flk-1 signal transduction system in vasculogenesis and angiogenesis.

Amino Acid Sequence↗
PubMed1995

Overexpression of vascular endothelial growth factor in the avian embryo induces hypervascularization and increased vascular permeability without alterations of embryonic pattern formation.

Vascular endothelial growth factor (VEGF)--also known as vascular permeability factor--has been implicated in the regulation of blood vessel formation, i.e., vasculogenesis and angiogenesis. High amounts of VEGF mRNA and protein have been detected during embryonic and tumor angiogenesis, but it remained unclear whether the level of VEGF correlated with the extent of vascularization in a given organ or tissue. We examined the role of VEGF and the high affinity, signal-transducing VEGF receptor-2 (flk-1) in the avian embryo. In a gain of function transgene-like approach the retroviral expression vector RCAS was used to increase the level of quail VEGF during critical periods of avian limb bud growth and morphogenesis. In contrast to basic fibroblast growth factor, which recently was demonstrated to induce morphogenetic alterations when overexpressed in this system, overexpression of VEGF in the limb bud exclusively resulted in hypervascularization as reflected by an increase in vascular density. However, cartilage expressing the construct was not vascularized prematurely. Thus hypervascularization was probably due to the augmentation of the VEGF signaling mechanism in a permissive environment. In addition to hypervascularization, vascular permeability was dramatically increased, leading to local and in some cases to general edema. This is the first indication of a link between the functions of VEGF as a vascular growth factor and as a permeability factor. VEGF receptor-2 (flk-1) was found to be upregulated only in those areas where VEGF was overexpressed. This implies a positive feedback system of the VEGF receptor on its own synthesis and would provide a basis for a paracrine system in which ligand concentration is critical for the extent of tissue vascularization. Our results show that the VEGF/VEGF-receptor system is specific and sufficient for the formation of new blood vessels. They also have implications for somatic gene therapy of diseases which are characterized by a lack of blood vessels such as chronic ischemic diseases of heart and brain.

Amino Acid Sequence↗
PubMed1995

The non-receptor tyrosine kinase Lyn is localised in the developing murine blood-brain barrier.

The blood-brain barrier, formed by brain endothelium, is critical for brain function. The development of the blood-brain barrier involves brain angiogenesis and endothelial cell differentiation, processes which require active signal transduction pathways. The differentiation of brain endothelial cells to the "blood-brain-barrier phenotype" involves cytoskeletal changes which modulate the tightness of the barrier. In order to identify signal transduction proteins involved in blood-brain barrier development, cDNA from bovine and murine brain endothelial cells was used in a polymerase chain reaction for cloning of DNA encoding Src homology 3 domains. Src homology 3 domains are structural domains found in many signal transduction proteins. These domains often mediate interaction of signaling proteins with the cytoskeleton and therefore may play a role in the regulation of the cytoskeletal changes which occur during blood-brain-barrier development. Unexpectedly, all bovine and murine clones analyzed from polymerase chain reactions encoded the Src homology 3 domain of one protein, namely the non-receptor tyrosine kinase, Lyn, which is involved in signal transduction in cells of the hemopoietic system. In situ hybridization analyses confirmed the presence of lyn mRNA in developing blood vessels in embryonic and early post-natal mouse brain, but not in endothelium outside the brain. In bovine brain endothelial cells in primary culture, p53lyn is highly abundant and present in two forms which have different patterns of tyrosine phosphorylation. These data suggest that Lyn may be involved in transduction of growth and differentiation signals required for blood-brain-barrier development.

Animals↗
PubMed1995

Vasculogenesis.

Induction by fibroblast growth factors of mesoderm during gastrulation leads to blood-forming tissue, including angioblasts and hemopoietic cells, that together constitute the blood islands of the yolk sac. The differentiation of angioblasts from mesoderm and the formation of primitive blood vessels from angioblasts at or near the site of their origin are the two distinct steps during the onset of vascularization that are defined as vasculogenesis. Vascular endothelial growth factor and its high-affinity receptor tyrosine kinase flk-1 represent a paracrine signaling system crucial for the differentiation of endothelial cells and the development of the vascular system. Specified cell adhesion molecules such as VE-cadherin and PECAM-1 (CD-31), and transcription factors such as ets-1, as well as mechanical forces and vascular regression and remodeling are involved in the subsequent events of endothelial cell differentiation, apoptosis, and angiogenesis.

Amphibians↗
PubMed1995

Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.

Vascular endothelial growth factor (VEGF) may modulate vascular permeability, chemotaxis for monocytes, and protease activity. In addition, VEGF may play a role in embryonic and tumor angiogenesis. In fetal mouse kidney, VEGF mRNA and protein expression have been demonstrated. This finding led to the hypothesis that VEGF might be involved in renal growth and development. To further elucidate the role of VEGF in human kidney, expression of VEGF and its receptors, the specific tyrosine kinase receptors, fit-1 and KDR, were studied. In fetal (6-24 gestational wk; mesonephros and metanephros) and adult kidney, VEGF mRNA and protein could be colocalized in glomerular epithelia and collecting duct cells by in situ hybridization and immunohistology. By reverse transcription-polymerase chain reaction, mRNA of three VEGF isoforms, VEGF121, VEGF165, and VEGF189, were found in fetal kidney and cortex, isolated glomeruli, and medulla of adult human kidney. KDR and flt-1 mRNA were coexpressed in endothelia of glomeruli and in peritubular capillaries in fetal and adult kidney. These data support the assumption that VEGF and its receptors may influence renal ontogenesis. We speculate that the constitutive expression of VEGF in adult kidney may be required for the function of VEGF receptor positive-fenestrated endothelia in glomeruli and postglomerular vessels. The expression of VEGF in collecting duct and of its receptors in medullary capillaries may in addition be relevant for maintaining medullary osmolality.

Aged↗