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PDF file - 108K, S3. VEGF levels are not changed in PHD4-overexpressing LM8 tumors.

Solid tumor growth is intimately associated with angiogenesis, a process that is efficiently triggered by hypoxia. Therefore, oxygen-sensitive signaling pathways are thought to play a critical role in tumor angiogenesis and progression. Here, the function of prolyl hydroxylase-4 (PHD4), a relative of the prolyl hydroxylase domain proteins 1–3 that promote the degradation of hypoxia-inducible factors (HIF), was interrogated. To test the hypothesis that PHD4 might inhibit tumor angiogenesis, it was overexpressed in osteosarcoma cells, and unexpectedly, this manipulation led to increased tumor blood vessel density. However, the newly formed blood vessels were smaller than normal and appeared to be partially nonfunctional, as indicated by poor vessel perfusion. PHD4 overexpression in tumor cells stimulated the expression of TGF-α, which was necessary and sufficient to promote angiogenic sprouting of endothelial cells. On the other hand, PHD4 overexpression reduced HIF-2α protein levels, which in turn inhibited in vivo tumor growth. Combined, elevated PHD4 levels deregulate angiogenesis via increased TGF-α expression in vitro and in vivo. These data support the hypothesis that tumor growth can be uncoupled from vessel density and that the individual PHD family members exert distinct functions in tumors.Implications: PHD4 influences tumor growth and vascularization through discrete mechanisms and molecular pathways that likely have therapeutic potential. Mol Cancer Res; 11(11); 1337–48. ©2013 AACR.

PDF file - 133K, S5. Lentivirus-mediated silencing of HIF-2alpha in LM8 control cells.

PDF file - 72K, S1. PHD4 overexpression in eEPCs reduces HIF-2alpha levels under hypoxia.

PDF file - 101K, S4. Lentivirus-mediated silencing of TGF-alpha in LM8-PHD4 cells.

PDF file - 40K, S2. Overexpression of PHD4 in LM8 osteosarcomas has no effect on the relative endothelial area.

Supplementary Materials, Table 1, Figure Legends 1-7 from Prolyl Hydroxylases 2 and 3 Act in Gliomas as Protective Negative Feedback Regulators of Hypoxia-Inducible Factors

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases; but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists; for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.

Small-molecule inhibitors of hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) are currently under clinical development as novel treatment options for chronic kidney disease (CKD) associated anemia. Inhibition of HIF-PH mimics hypoxia and leads to increased erythropoietin (EPO) expression and subsequently increased erythropoiesis. Herein we describe the discovery, synthesis, structure-activity relationship (SAR), and proposed binding mode of novel 2,4-diheteroaryl-1,2-dihydro-3H-pyrazol-3-ones as orally bioavailable HIF-PH inhibitors for the treatment of anemia. High-throughput screening of our corporate compound library identified BAY-908 as a promising hit. The lead optimization program then resulted in the identification of molidustat (BAY 85-3934), a novel small-molecule oral HIF-PH inhibitor. Molidustat is currently being investigated in clinical phase III trials as molidustat sodium for the treatment of anemia in patients with CKD.

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

In 2008, the University of Cologne and Bayer Schering Pharma together decided to establish a "privileged partnership" in order to foster interactions in the field of preclinical and clinical drug discovery and development. As one of the activities of this partnership, we launched a graduate program run by the University and supported by Bayer funds. Founded in 2009, the program now provides administrative and personal support as well as a scientific curriculum for 16 students. They carry out their doctoral thesis (PhD or MD) work either at a research lab at Bayer, or at the Medical or the Natural Sciences Faculty of the University. Research topics span the field of shared scientific interests of the partners (e.g., cardiovascular diseases, acute care, oncology). Project selection, student recruitment, and curriculum design are carried out by a program committee, which equally represents Bayer and the two participating University Faculties. Within a 2- or 3-year curriculum, students meet regularly for journal clubs, soft-skills workshops, and special courses. They carry out a lab rotation over several weeks at the partner institution - students working at the university are then hosted in a lab at Bayer and vice versa. Students receive scientific and personal support from their thesis committee (supervisor plus two mentors, again representing both Faculties and Bayer). Feedback from students, principal investigators and external reviewers indicates that an environment combining partners from academia and industry is highly appreciated and carries the potential to attract particularly gifted students. Compared with other, publicly funded graduate programs in Germany, the research topics within our program are rather diverse, which require that a common thread of important aspects of pharmacology is implemented within the core curriculum. Ultimately, our program will be evaluated with regard to academic output and career opportunities for its graduates.

The hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha are activated in hypoxic tumor regions. However, their role in tumorigenesis remains controversial, as tumor growth promoter and suppressor activities have been ascribed to HIF-1alpha, while the role of HIF-2alpha remains largely unknown. Here, we show that overexpression of HIF-2alpha in rat glioma tumors enhances angiogenesis but reduces growth of these tumors, in part by increasing tumor cell apoptosis. Moreover, siRNA knockdown of HIF-2alpha reduced apoptosis in hypoxic human malignant glioblastoma cells. Furthermore, inhibition of HIF by overexpression of a dominant-negative HIF transgene in glioma cells or HIF-2alpha deficiency in teratomas reduced vascularization but accelerated growth of these tumor types. These findings urge careful consideration of using HIF inhibitors as cancer therapeutic strategies.

Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

Interactions between Ets family members and a variety of other transcription factors serve important functions during development and differentiation processes, e.g. in the hematopoietic system. Here we show that the endothelial basic helix-loop-helix PAS domain transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha) (but not its close relative HIF-1alpha), cooperates with Ets-1 in activating transcription of the vascular endothelial growth factor receptor-2 (VEGF-2) gene (Flk-1). The receptor tyrosine kinase Flk-1 is indispensable for angiogenesis, and its expression is closely regulated during development. Consistent with the hypothesis that HIF-2alpha controls the expression of Flk-1 in vivo, we show here that HIF-2alpha and Flk-1 are co-regulated in postnatal mouse brain capillaries. A tandem HIF-2alpha/Ets binding site was identified within the Flk-1 promoter that acted as a strong enhancer element. Based on the analysis of transgenic mouse embryos, these motifs are essential for endothelial cell-specific reporter gene expression. A single HIF-2alpha/Ets element conferred strong cooperative induction by HIF-2alpha and Ets-1 when fused to a heterologous promoter and was most active in endothelial cells. The physical interaction of HIF-2alpha with Ets-1 was demonstrated and localized to the HIF-2alpha carboxyl terminus and the autoinhibitory exon VII domain of Ets-1, respectively. The deletion of the DNA binding and carboxyl-terminal transactivation domains of HIF-2alpha, respectively, created dominant negative mutants that suppressed transactivation by the wild type protein and failed to synergize with Ets-1. These results suggest that the interaction between HIF-2alpha and endothelial Ets factors is required for the full transcriptional activation of Flk-1 in endothelial cells and may therefore represent a future target for the manipulation of angiogenesis.

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin–angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

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.

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.

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 α-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.

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.

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.

Solid tumor growth is intimately associated with angiogenesis, a process that is efficiently triggered by hypoxia. Therefore, oxygen-sensitive signaling pathways are thought to play a critical role in tumor angiogenesis and progression. Here, the function of prolyl hydroxylase-4 (PHD4), a relative of the prolyl hydroxylase domain proteins 1–3 that promote the degradation of hypoxia-inducible factors (HIF), was interrogated. To test the hypothesis that PHD4 might inhibit tumor angiogenesis, it was overexpressed in osteosarcoma cells, and unexpectedly, this manipulation led to increased tumor blood vessel density. However, the newly formed blood vessels were smaller than normal and appeared to be partially nonfunctional, as indicated by poor vessel perfusion. PHD4 overexpression in tumor cells stimulated the expression of TGF-α, which was necessary and sufficient to promote angiogenic sprouting of endothelial cells. On the other hand, PHD4 overexpression reduced HIF-2α protein levels, which in turn inhibited in vivo tumor growth. Combined, elevated PHD4 levels deregulate angiogenesis via increased TGF-α expression in vitro and in vivo. These data support the hypothesis that tumor growth can be uncoupled from vessel density and that the individual PHD family members exert distinct functions in tumors. Implications: PHD4 influences tumor growth and vascularization through discrete mechanisms and molecular pathways that likely have therapeutic potential. Mol Cancer Res; 11(11); 1337–48. ©2013 AACR.

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.

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.

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. © 1993 Wiley-Liss, Inc.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

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-β, TGF-α 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.

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.

Adaptive responses to hypoxia in tumors are transcriptionally regulated by the hypoxia inducible factors (HIF-1α/HIF-2α), which are tightly controlled by the HIF-prolyl hydroxylases (PHD). Hypoxia induces expression of the PHD2 and PHD3 proteins in tumors but the pathobiological significance of these events is uncertain. Here, we show that PHD2 and PHD3 induction acts within a negative feedback loop to limit the hypoxic HIF response. In glioblastomas, PHD2 and PHD3 are hypoxia-inducible in vitro and expressed in hypoxic areas of tumors in vivo. Comparison with other PHDs revealed distinct cytoplasmatic and nuclear localization patterns of PHD2 and PHD3. Gain and loss of function experiments defined PHD2 and PHD3 as HIF target genes that remained operative even at low oxygen concentrations. We found that increased PHD levels could compensate for reduced oxygen availability to regulate the HIF response. This negative feedback loop protected tumor cells against hypoxia-induced cell death, functionally implicating this pathway in the control of the tumor-suppressive components of the HIF system in glioblastoma. Moreover, PHD inhibition facilitated cell death induction by staurosporine or tumor necrosis factor–related apoptosis-inducing ligand, hinting at a more general protective role of PHD in the regulation of cell viability. In summary, our findings recognize the PHD/HIF regulatory axis as a novel therapeutic target to disable a tumor's ability to adjust to hypoxic conditions and control cell survival, helping to potentially overcome therapeutic cell death resistance in glioblastomas. Cancer Res; 70(1); 357–66

The present study describes a method of culturing chick embryos together with their surrounding area vasculosa on two different culture media in succession. Embryos in the 2nd day of incubation (stages 13, 14, 15 according to Hamburger and Hamilton, 1951) were explanted from the yolk with the aid of a ring of filter paper and transferred dorsal side up to a silicone culture dish containing the first culture medium (89.5% L-15, 10% fetal calf serum, 0.5% Antibiotics). The paper ring was clamped onto the wall of the culture dish by a steel ring so that the embryo was fixed for the culture period. After 4 +/- 1, 8 +/- 1, 12 +/- 1 hrs, the embryos were taken from the culture dishes and transferred to others containing a yolk-albumen mixture as culture medium; 81.2% of embryos survived the first phase of culture. On the second medium 50.3% of explanted embryos were still alive at stage 20 (HH), and 7.9% of them reached the 5th day of development (St 25 HH). The average length of survival in vitro was found to be influenced by both the length of the first culture phase and the stage at which embryos were explanted. This culture method may be useful for teratological tests, since in the first phase of culture, concentrations of test substances and the time of exposure can be exactly adjusted, and in the second phase, the embryo is allowed to develop quite normally, under conditions similar to those in ovo.

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin–sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17×103Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.

The abundance of the transcription factor hypoxia-inducible factor is regulated through hydroxylation of its alpha-subunits by a family of prolyl-hydroxylases (PHD1-3). Enzymatic activity of these PHDs is O2-dependent, which enables PHDs to act as cellular O2 sensor enzymes. Herein we studied endogenous PHD activity that was induced in cells grown under hypoxia or in the presence of nitric oxide. Under such conditions nuclear extracts contained much higher PHD activity than the respective cytoplasmic extracts. Although PHD1-3 were abundant in both compartments, knockdown experiments for each isoenzyme revealed that nuclear PHD activity was only due to PHD2. Maximal PHD2 activity was found between 120 and 210 microm O2. PHD2 activity was strongly decreased below 100 microm O2 with a half-maximum activity at 53 +/- 13 microm O2 for the cytosolic and 54 +/- 10 microm O2 for nuclear PHD2 matching the physiological O2 concentration within most cells. Our data suggest a role for PHD2 as a decisive oxygen sensor of the hypoxia-inducible factor degradation pathway within the cell nucleus.

N-nitrosomelatonin (NOMela) is well-known for its capabilities of transnitrosating nucleophiles such as thiols and ascorbate, thereby generating nitric oxide (NO)-releasing compounds. It is unknown, however, whether NOMela can be successfully applied as a precursor of NO in a complex biological environment like a cell culture system. NO donors may be useful to induce the transcription factor hypoxia inducible factor 1 (HIF-1), which coordinates the protection of cells and tissues from the lack of oxygen (hypoxia). In this study, the effects of NOMela in an in vitro cell-free assay [NO-release, inhibition of prolylhydroxylase1 (PHD1)] and in living cells (upregulation of HIF-1, reduction of HIF-1 hydroxylation, upregulation of the HIF-1-target gene PHD2) were compared with those of the frequently applied NO donor S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NOMela released NO in a predictable manner and this release in vitro was found to be independent of the composition of the buffer system. The NOMela-mediated effects in oxygenated cells were in all cases comparable to the hypoxic response, whereas unphysiological strong effects were observed with GSNO. Probably, because of the antioxidative power of the NOMela-dependent formation of melatonin, cells were completely protected against the attack of reactive nitrogen oxygen species, which are generated by autoxidation of NO. In conclusion, NOMela had to be an excellent NO precursor for cells in culture and potentially tissues.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen levels. By stimulating the expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), they trigger the neovascularization of tissues under physiologic and pathologic conditions. Here, we have investigated the endothelial cell–autonomous HIF function in blood vessel growth and development by expressing a dominant-negative HIF mutant (HIFdn) that inhibits the transcriptional responses mediated by both HIF-1 and HIF-2, specifically in endothelial cells of transgenic mice. HIFdn transgenic embryos were growth retarded and died around E11.5. Primitive vascular networks were established, but vascular remodeling in the yolk sac and in the embryo proper was defective, and vascular sprouts failed to invade the neuroepithelium. In addition, heart looping was incomplete, and the ventricles of the heart were thin-walled and lacked trabeculation. Similar cardiovascular defects have been observed in Tie2–deficient mouse embryos. Consistently, HIFdn transgenic embryos expressed reduced levels of the endothelial angiopoietin receptor, Tie-2, whereas other endothelial markers, such as PECAM-1, Tie-1, and VE-cadherin were not affected. These results show that HIFs in endothelial cells are essential for embryonic heart and blood vessel development and control angiogenesis and vascular remodeling.

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.

Hypoxia-inducible transcription factors (HIFs) are important for transcriptional adaptation to hypoxia. Availability of HIFs is regulated via posttranslational modification of their alpha subunits (HIF-1alpha and HIF-2alpha). Under normoxia, two highly conserved proline residues within the oxygen-dependent degradation domain (ODDD) are hydroxylated by oxoglutarate-dependent proline 4-hydroxylases EGLN1-3. Hydroxylated HIF-alpha interacts with the pVHL-E3 ubiquitin ligase complex and, subsequently, is degraded via the proteasomal pathway. We identified a novel putative proline 4-hydroxylase, PH-4, with an aminoterminal EF-hand motif and a carboxyterminal catalytic domain, which was highly expressed in most organs, and-unlike EGLNs which localize to the cytoplasm and nucleus-was associated with the endoplasmic reticulum. Like EGLNs, PH-4 overexpressed in cellular reporter assays suppressed the HIF transactivation activity, dependent on the consensus ODDD proline residues. Suppression of transactivation was correlated with decrease of cellular contents of HIF. Thus, PH-4 might be related to cellular oxygen sensing.

The cardiovascular system of vertebrates emanates from the mesodermal layer of the primitive embryo. Angioblasts giving rise to endothelial cells and hematoblasts giving rise to blood cells differentiate from their fibroblast-like precursors shortly after having migrated through the primitive streak during gastrulation (Gonzalez Crussi, 1971). Nascent angioblasts and their assembly into the primordial vascular plexus were made visible for the first time in the quail embryo by means of monoclonal antibodies, which recognize epitopes on endothelial and hematopoietic cells (MB-1 and QH-1) (Peault et al., 1983;Pardanaud et al., 1987). The first angioblasts originate at the periphery of the extraembryonic mesoderm, but a little later (when the head fold is formed at the one-somite stage) in the embryo proper (Pardanaud et al., 1987;Coffin and Poole, 1988;Coffin and Poole, 1989). Along the anterior intestinal portal they establish the primordia of the endocardium, and along the lateral edges of the somites they establish the primordia of large body vessels, which become interconnected to the extraembryonic vasculature at the two-somite stage. The morphogenesis of the early vasculature has been described in a series of comprehensive articles (His, 1900;Evans, 1909;Sabin, 1917,Sabin, 1920;Gonzalez Crussi, 1971;Haar and Ackerman, 1971;Lanot, 1980;Hirakow and Hiruma, 1981; Pardanaud et al., 1987; Coffin and Poole, 1989;De Ruiter et al., 1991,De Ruiter et al., 1993).

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.

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 murineFlk-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-2. 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.

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.

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.

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. Specific 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.

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.

The precursor of each definitive blood vessel is a capillary. During embryogenesis, the pattern of adult blood vessels is preformed by patterning at the capillary level. Under pathological conditions such as inflammation, collateralization in chronic ischemia, wound healing and tumor growth, capillary vessels precede the definitive newly formed blood vessels. Therefore, the mechanisms that are involved in the formation of capillary blood vessels are crucial for the development of a vascular system. Suitable models for studying capillary formation in vivo are rare, because in most newly vascularized tissues observation and interpretation of capillary growth are hampered by a complex spatial structure.

Expression of alpha-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No alpha-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of alpha-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little alpha-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on alpha-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of alpha-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the alpha-sm-actin and alpha-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.