Up-regulation of hypoxia-inducible factors HIF-1alpha and HIF-2alpha under normoxic conditions in renal carcinoma cells by von Hippel-Lindau tumor suppressor gene loss of function.

Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix-loop-helix PAS family, composed of alpha and beta subunits. HIF-1alpha shares 48 per cent identity with the recently identified HIF-2alpha protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von Hippel-Lindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2alpha mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in a range of VHL wildtype and VHL deficient RCC cell lines. In the presence of functional VHL protein, HIF-1alpha mRNA levels are elevated, whereas HIF-2alpha mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL deficient cell lines, both HIF-alpha subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1alpha and HIF-2alpha proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1alpha and HIF-2alpha in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1alpha and HIF-2alpha proteins.

Role of SCL/Tal-1, GATA, and ets transcription factor binding sites for the regulation of flk-1 expression during murine vascular development.

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.

Expression and proteolysis of vascular endothelial growth factor is increased in chronic wounds.

Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Flt-1 and KDR, in chronic venous leg ulcerations. Immunohisto- chemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.

mRNA cloning and expression studies of the quail homologue of HIF-2alpha.

Hypoxia inducible factors (HIF) are candidate transcriptional regulators of vascular development. Unlike HIF-1alpha - the founding member of the HIF family - which is expressed more or less ubiquitously, HIF-2alpha (also called HRF, HLF and EPAS1) is highly expressed by vascular endothelial cells and was shown to activate the transcription of endothelial cell-specific receptor tyrosine kinases (tie-2 and flk-1/VEGF receptor 2) and of vascular endothelial growth factor (VEGF). Therefore HIF-2alpha is a candidate dual regulator of vascular development. Here we describe the quail homologue of HIF-2alpha. Sequence analysis reveals that HIF-2alpha is highly conserved between birds and mammals. Like the murine HIF-2alpha, the quail molecule is highly expressed by endothelial cells but also detectable in certain epithelial cells such as in the endoderm.

Identification of vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) promoter/enhancer sequences sufficient for angioblast and endothelial cell-specific transcription in transgenic mice.

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.

Characterization of novel nuclear targeting and apoptosis-inducing domains in FAS associated factor 1.

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.

Up-regulation of vascular endothelial growth factor in stromal cells of hemangioblastomas is correlated with up-regulation of the transcription factor HRF/HIF-2alpha.

Hemangioblastomas, the most frequent manifestation of the hereditary von Hippel-Lindau disease (VHL), are highly vascularized tumors of the central nervous system. In previous studies, the endothelial-specific mitogen vascular endothelial growth factor (VEGF) was shown to be up-regulated in the stromal cells, the putative neoplastic cells in hemangioblastomas. Therefore, it was suggested that secretion of VEGF by stromal cells is the pathogenetic cause of the vascular lesions in hemangioblastomas. The novel basic helix loop helix transcription factor HRF/HIF-2alpha is a candidate regulator of VEGF expression during development. We therefore investigated expression of HRF/HIF-2alpha in hemangioblastomas and found the overexpression of VEGF mRNA in stromal cells to be highly correlated with elevated expression levels of HRF/HIF-2alpha mRNA. This finding is suggestive for a role of HRF in VEGF-dependent vascular growth in hemangioblastomas and could provide a link between transcriptional activation of the VEGF gene and loss of function of the VHL gene product.

The in vivo activity of vascular endothelial growth factor isoforms in the avian embryo.

The activity of the different isoforms of quail vascular endothelial growth factor (VEGF; 122, 146, 166 and 190 amino acids) were quantitatively examined in vivo by overexpression in the chicken embryo using the retroviral expression vector RCAS. All isoforms were potent inducers of vascularization and permeability. A linear relationship between expression of their mRNA and induction of vascularization and permeability was observed for all isoforms. Pattern formation and morphogenesis was otherwise not altered. Overexpression of qVEGF122 and 146 in the eye of chicken embryos did not induce vascularization of either the cornea or retina-which are avascular tissues in birds. We conclude that all isoforms of VEGF are potent inducers of angiogenesis dependent on a permissive environment.

Thrombomucin, a novel cell surface protein that defines thrombocytes and multipotent hematopoietic progenitors.

MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets-transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571-amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein-1, a rabbit cell surface glycoprotein of kidney podocytes. Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell-specific proteins with possibly overlapping functions in early hematopoietic progenitors.

HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels.

Transcription factors of the bHLH-PAS protein family are important regulators of developmental processes such as neurogenesis and tracheal development in invertebrates. Recently a bHLH-PAS protein, named trachealess (trl) was identified as a master regulator of tracheogenesis. Hypoxia-inducible factor, HIF-1 alpha, is a vertebrate relative of trl which is likely to be involved in growth of blood vessels by the induction of vascular endothelial growth factor (VEGF) in response to hypoxia. In the present study we describe mRNA cloning and mRNA expression pattern of mouse HIF-related factor (HRF), a novel close relative of HIF-1 alpha which is expressed most prominently in brain capillary endothelial cells and other blood vessels as well as in bronchial epithelium in the embryo and the adult. In addition, smooth muscle cells of the uterus, neurons, brown adipose tissue and various epithelial tissues express HRF mRNA as well. High expression levels of HRF mRNA in embryonic choroid plexus and kidney glomeruli, places where VEGF is highly expressed, suggest a role of this factor in VEGF gene activation similar to that of HIF-1 alpha. Given the similarity between morphogenesis of the tracheal system and the vertebrate vascular system, the expression pattern of HRF in the vasculature and the bronchial tree raises the possibility that this family of transcription factors may be involved in tubulogenesis.

Flk-1 as a target for tumor growth inhibition.

A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.

Developing brain cells produce factors capable of inducing the HT7 antigen, a blood-brain barrier-specific molecule, in chick endothelial cells.

Homeostasis of the neural microenvironment is maintained by the blood-brain barrier (BBB). To analyze the molecular mechanisms by which the BBB is induced during embryonic development, we have taken advantage of an in vivo model of BBB induction based on the expression of the HT7 cell surface protein. This protein is a transmembrane glycoprotein of the immunoglobulin superfamily. It is expressed in the chick BBB-forming endothelial cells, but not in peripheral endothelial cells. Here we show that the HT7 protein is induced in vessels which had vascularized a quail embryonic brain graft transplanted in the coelomic cavity of chick embryo. The quail brain graft was vascularized by both chick and quail-derived vessels. All chick host-derived vessels in the brain transplant were found to express HT7 while the neighboring chick vessels were negative. We conclude that the invading host endothelial cells differentiated into BBB-forming vessels under the influence of developing quail brain cells. This model reproduces the BBB induction during development. It may be useful for further approaches to study the molecular mechanisms involved in BBB induction.

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.

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.

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.

Induction of vasculogenesis in quail blastodisc-derived embryoid bodies.

We have previously developed an in vitro culture system in which dissociated cells from unincubated quail blastodiscs formed in vivo-like blood islands consisting of blood and endothelial cells in response to fibroblast growth factors (FGFs). Here we demonstrate that the same quail blastodisc cells grown in suspension culture in the presence of basic FGF (bFGF) reaggregated and formed three-dimensional spherules (embryoid bodies, EBs) which underwent vasculogenesis and hematopoiesis within 3 days. In contrast to murine embryoid bodies, which undergo vasculogenesis spontaneously, the formation of vascular structures in quail blastodisc cultures was absolutely dependent on bFGF. While about 75% contained blood islands and about 50% formed capillaries in the presence of bFGF, only 0.2% of the embryoid bodies formed blood islands in control cultures without bFGF. Vascular channels were gradually encoated by primitive smooth muscle cells within 5 days. Ultrastructural examinations revealed capillary blood vessels and blood islands indistinguishable from their yolk sac counterparts. Mesodermal tissue was present in cultures both with and without bFGF, but consisted of an avascular undifferentiated mesenchyme in control cultures. Since the entire sequence of vasculogenesis from the formation of endothelial cells to their assembly into a vascular plexus is observed in response to the inducer bFGF, this culture system is a suitable model for studying the molecular events that initiate the emergence of endothelial cells and the formation of a vascular plexus during vasculogenesis.

A new model of vasculogenesis and angiogenesis in vitro as compared with vascular growth in the avian area vasculosa.

In cultures of dissociated quail epiblast the basic constituents of the vascular system, blood cells and endothelial cells can be induced by basic fibroblast growth factor (Flamme and Risau, Development, 116: 435-439, 1992). As we show here, in those cultures three types of vascular plexus differentiate spontaneously under different culture conditions: At the 3rd day a vascular plexus appears in situ closely resembling the vascular plexus of the quail area opaca vasculosa (vasculogenesis). Vascular sprouts are formed, extending long filopodia at their tips. Such filopodia are shown to build the first intervascular bridges in the growing vascular plexus of the area vasculosa at embryonic day 3. Connections of filopodia turn out to be precursors of new capillaries interconnecting pre-existing blood vessels (angiogenesis). Two further types of in vitro capillary plexus differentiate in long term endothelial cell cultures derived from induced angioblasts. Whereas one closely resembles so-called angiogenesis in vitro, the third type comprises mainly multinucleated giant endothelial cells lining loop like capillaries and represents a differentiation of aging endothelial cell culture. Thus, the present in vitro model is an approach to the sequence of angioblast induction, vasculogenesis, and angiogenesis.

Induction of vasculogenesis and hematopoiesis in vitro.

Despite a large number of investigations of embryonic vascular development, in particular in avian embryos, the conditions under which the endothelial and hematopoietic cell lineages emerge remain unknown. As we demonstrate here, both endothelial and hematopoietic cells can be induced by treatment of dissociated quail epiblast with fibroblast growth factors in vitro. These cells aggregate in characteristic blood islands. In long-term culture, the induced endothelial cells gave rise to vascular structures in vitro, i.e. vasculogenesis. No induction was observed in the absence of fibroblast growth factors, and other growth factors like TGF-beta, TGF-alpha and EGF were not capable of inducing blood island formation. Thus, the dissociated quail epiblast provides a remarkably simple test system to investigate cell lineage diversification in higher vertebrates.

A comparative study on the effects of tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo.

The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax carriers on the CAM: Tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominantly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation.