Search PubMed⌕ Search

Biomedical subjects

W Risau

Publications and source records attributed to W Risau.

At least 91 records · Page 5Linked to original sources
PubMed1995

Differentiation of endothelium.

Vascular endothelial cells cover the entire inner surface of blood vessels in the body. They play an important role in tissue homeostasis, fibrinolysis and coagulation, blood-tissue exchange, vasotonus regulation, the vascularization of normal and neoplastic tissues, and blood cell activation and migration during physiological and pathological processes. It is therefore important to define the basic determinants of the endothelial phenotype and its modulation in response to different signals. Signal recognition, transduction, and processing are likely to be complex events dependent on the status of the target endothelial cell in a given organ or tissue. This status is a consequence of inductive and permissive interactions of a pluripotent cell with soluble and insoluble signaling molecules of the environment during embryonic and postnatal development. This review will focus on the biological mechanisms involved in the differentiation of endothelial cells from the mesoderm and their subsequent functional heterogeneity in different organs and tissues under physiological as well as pathological conditions.

Animals↗
PubMed1994

Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms.

We have previously suggested that tumor angiogenesis in human gliomas is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and flt-1 (VEGF-receptor 1). VEGF, an endothelial-cell-specific mitogen, is abundantly expressed in glioma cells which reside along necrotic areas, whereas flt-1, a tyrosine-kinase receptor for VEGF, is expressed in tumor endothelial cells, but not in endothelial cells in normal adult brain. Recently, a second tyrosine-kinase receptor which binds VEGF with high affinity, designated KDR or flk-1, has been described. We performed in situ hybridization for VEGF mRNA, flt-1 mRNA and KDR mRNA on serial sections of normal brain, low-grade and high-grade glioma specimens. We show that KDR mRNA is co-expressed with flt-1 in vascular cells in glioblastoma but not in low-grade glioma. Since flt-1 and KDR are not expressed in endothelial cells in the normal adult brain, the coordinate up-regulation of 2 receptors for VEGF appears to be a critical event which controls tumor angiogenesis. Immunocytochemistry with a monoclonal anti-VEGF antibody revealed significant amounts of VEGF protein in the same glioma cells that expressed VEGF mRNA. The largest amount of VEGF immunoreactivity, however, was detected on the vasculature of glioblastomas, the site where VEGF exerts its biological functions. These findings suggest that VEGF is produced and secreted by glioma cells and acts on tumor endothelial cells which express VEGF receptors. To further characterize VEGF-producer cells in vivo, we investigated cellular proliferation, immunoreactivity to the p53 tumor-suppressor gene product and epidermal-growth-factor-receptor (EGFR) expression on serial sections by immunocytochemistry. VEGF-producer cells did not show increased cellular proliferation, p53 immunoreactivity or EGFR immunoreactivity as compared with glioma cells which did not express VEGF. Our studies therefore do not demonstrate evidence for a growth advantage of VEGF-producer cells in vivo or VEGF induction by p53 mutation or EGFR over-expression.

Brain Neoplasms↗
PubMed1994

Normal and abnormal development of the blood-brain barrier.

The blood-brain barrier is responsible for the maintenance of the neuronal microenvironment. This is accomplished by isolation of the brain from the blood by the tight junctions that join endothelial cells in cerebral microvessels, and by selective transport and metabolism of substances from blood or brain by the endothelial cells. This review describes the growth and maturation of the brain vasculature, and the development of the special properties of the endothelia at the blood-brain interface. Evidence suggests that the development of the unique properties of the brain microvasculature is a consequence of tissue-specific interactions between endothelial cells of extraneural origin and developing brain cells. The cellular and molecular mechanisms that control these processes are as yet unknown but this review will include experimental studies which have used in vivo and in vitro systems to investigate what factors may be involved, and some pathological conditions in which abnormal barrier development is thought to be an important aspect of the disease process.

Animals↗
PubMed1994

Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant.

Angiogenesis, the sprouting of capillaries from pre-existing blood vessels, is a fundamental process in the formation of the vascular system during embryonic development. In adulthood, angiogenesis takes place during corpus luteum formation and in pathological conditions such as wound healing, diabetic retinopathy, and tumor-igenesis. Vascularization is essential for solid tumour growth and is thought to be regulated by tumour cell-produced factors, which have a chemotactic and mitogenic effect on endothelial cells. Vascular endothelial growth factor (VEGF), a homodimeric glycoprotein of relative molecular mass 45,000, is the only mitogen, however, that specifically acts on endothelial cells, and it may be a major regulator of tumour angiogenesis in vivo. Its expression has been shown to be upregulated by hypoxia, and its cell-surface receptor, Flk-1, is exclusively expressed in endothelial cells. Here we investigate the biological relevance of the VEGF/Flk-1 receptor/ligand system for angiogenesis using a retrovirus encoding a dominant-negative mutant of the Flk-1/VEGF receptor to infect endothelial target cells in vivo, and find that tumour growth is prevented in nude mice. Our results emphasize the central role of the Flk-1/VEGF system in angiogenesis in general and in the development of solid tumours in particular.

Animals↗
PubMed1994

Cek5, a tyrosine kinase of the Eph subclass, is activated during neural retina differentiation.

The expression of Cek5, a receptor-type tyrosine kinase of the Eph subclass, and its variant form Cek5+ were examined in the chick neural retina during development. Cek5 is present at high levels at all stages of retinal development examined, while Cek5+ is most abundant during differentiation. Cek5 mRNA expression and immunoreactivity are evenly distributed in the undifferentiated retina. With differentiation, Cek5 becomes concentrated in the inner and outer plexiform layers. While only moderate changes in Cek5 protein expression are observed throughout retinal development, Cek5 phosphorylation on tyrosine in vivo is dramatically increased during differentiation. This suggests that the Cek5 ligand is expressed at high levels and causes Cek5 activation. Thus, Cek5 is likely to play an active role in retinal morphogenesis, particularly during the establishment of interneuronal contacts.

Amino Acid Sequence↗
PubMed1994

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.

Animals↗
PubMed1994

Molecular biology of blood-brain barrier ontogenesis and function.

The vascular system of the central nervous system is derived from capillary endothelial cells, which have invaded the early embryonic neuroectoderm. This process is called angiogenesis and is probably regulated by brain-derived factors. Vascular endothelial cell growth factor (VEGF) is an angiogenic growth factor whose expression correlates with embryonic brain angiogenesis, i.e. expression is high in the embryonic brain when angiogenesis occurs and low in the adult brain when angiogenesis is shut off under normal physiological conditions. VEGF is also a vascular permeability factor (VPF) and, therefore, its expression is also consistent with the formation of the blood-brain barrier by brain endothelial cells, i.e. capillaries are leaky in the embryonic brain but are tight in the postnatal and adult brain. Thus, VEGF/VPF may be a key factor regulating endothelial cell growth and permeability. This notion is further supported by the observation that VEGF expression is induced and strongly upregulated in human malignant glioblastoma. This tumor is characterized by vascular proliferations, vascular leakage and edema. The differentiation of blood-brain barrier endothelial cells is probably regulated by astrocytes which form foot processes apposed to the abluminal vascular basement membrane. Blood-brain barrier endothelial cells express a set of cell surface proteins that are absent from permeable capillaries. We have characterized one such novel transmembrane glycoprotein which is a new member of the immunoglobulin superfamily. This protein and the analysis of the in vitro characteristics of brain endothelial cells may help to define the molecular mechanisms that are involved in blood-brain barrier induction and permeability.

Animals↗
PubMed1994

Quantification of tight junction complexity by means of fractal analysis.

The concept of fractal geometry provides an elegant tool for the quantitative and objective structural description of various objects, the fractal analysis. Fractal analysis quantifies the structural complexity of objects by a characteristic singular value, the fractal dimension (FD). It can be estimated, e.g. by the box-counting method and provides a highly integrated measure in the range 1 < FD < 2 for curves extending within a plane. In this study, fractal analysis is used for the first time to evaluate the complexity of the tight junction network between adjoining cells. Bovine brain endothelial cells were cultured under various experimental conditions and the tight junctions were drawn to scale as visualized by the freeze fracture technique. These drawings were analyzed by fractal analysis, and by two other methods commonly used in this field, viz. the strand counting (SC) and complexity index (CI) methods. In contrast to the latter methods, the FD shows no directional preference and therefore no assumptions on the dynamic properties of the network's complexity are required. Thus, FD is demonstrated to provide the most sensitive, reliable and complete measure of tight junction complexity. In combination with SC and CI, additional information can be achieved concerning the directionality of the altered arrangement of tight junctional strands. Our analysis allows for the following conclusions. (1) Defined experimental influences can modify the complexity of tight junctions that are formed between endothelial cells in vitro, and (2) these structural modifications of the tight junctions are mainly due to an altered strand branching pattern.

Animals↗
PubMed1994

Lysis of rat brain microvascular endothelial cells mediated by resting but not activated MBP-specific CD4+ T cell lines.

Previous work from this laboratory showed that the encephalitogenic potential of myelin basic protein (MBP)-specific T cells is inseparably associated with their cytotoxic potential. MBP-specific T cells lyse all cells that present autoimmunogenic MBP peptide in context of appropriate MHC class II determinants. Beside class II-induced glia cells, blood-brain barrier-derived endothelial cells were identified as highly susceptible target cells for cytotoxic MBP-specific T cells. Here we show that the cytotoxic reaction against endothelial cells essentially differs from cytotoxicity against other target cells. In contrast to classical T cell-mediated lytic responses, which are most efficiently executed by activated T cells, rat brain endothelium (RBE) lysis could only be mediated by resting T cells. Activated MBP-specific T cell blasts were not able to mediate strong RBE lysis. Furthermore, T cell lines with specificities for protein antigens other than MBP did not cause RBE lysis. A role of the cytolytic capacity of resting MBP-specific T cells in the pathogenesis of experimental autoimmune encephalomyelitis is probable.

Animals↗
PubMed1994

Molecular mechanisms of developmental and tumor angiogenesis.

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

Animals↗
PubMed1994

Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes.

Tight junctions between endothelial cells of brain capillaries are the most important structural elements of the blood-brain barrier. Cultured brain endothelial cells are known to loose tight junction-dependent blood-brain barrier characteristics such as macromolecular impermeability and high electrical resistance. We have directly analyzed the structure and function of tight junctions in primary cultures of bovine brain endothelial cells using quantitative freeze-fracture electron microscopy, and ion and inulin permeability. The complexity of tight junctions, defined as the number of branch points per unit length of tight junctional strands, decreased 5 hours after culture but thereafter remained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bilayer (P-face) decreased continuously with a major drop between 16 hours and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face association of tight junction particles was enhanced by elevation of cAMP levels and by coculture of endothelial cells with astrocytes or exposure to astrocyte-conditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together with astrocyte-conditioned medium synergistically increased transendothelial electrical resistance and decreased inulin permeability of primary cultures, thus confirming the effects on tight junction structure and barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood-brain barrier function that can be specifically modulated by astrocytes and cAMP levels. Our results suggest an important functional role for the cytoplasmic anchorage of tight junction particles for brain endothelial barrier function in particular and probably paracellular permeability in general.

Animals↗
PubMed1994

Angiogenesis and endothelial cell function.

Endothelial cell growth is tightly regulated. During embryonic development endothelial cells rapidly proliferate thereby forming new blood vessels. Two different mechanisms contribute to the development of the vascular system: vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the formation of capillaries from preexisting vessels. In the adult, endothelial cell turnover is very low but under a variety of pathological conditions such as tumor growth these cells quickly enter the cell cycle and divide. Vascular endothelial growth factor (VEGF) has been identified as a key regulatory paracrine growth factor for endothelial cells. Transient VEGF expression correlates with embryonic and tumor angiogenesis. On the other hand, constitutive expression of this factor in choroid plexus and kidney glomerular epithelium and its cognate receptors in adjacent fenestrated endothelium suggests a role for this ligand-receptor system in organotypic endothelial cell differentiation and capillary permeability of fenestrated endothelium.

Animals↗
PubMed1994

Oncogenes in the study of endothelial cell growth and differentiation.

During embryogenesis endothelial cells differentiate from mesodermal blood islands, proliferate and form new blood vessels throughout embryonic and early postnatal life by the processes of vasculogenesis and angiogenesis. Proliferation then ceases and is very low in the adult, although it resumes under certain physiological and pathological conditions, such as wound healing, tumor growth and hemangiomatous diseases. Expression of the polyoma middle T (PymT) oncogene in mouse endothelial cells leads to their rapid transformation and to the development of hemangiomas. These endothelial tumors allow the establishment of endothelioma (End) cell lines, which resemble normal endothelial cells yet exhibit a drastically altered proteolytic activity. The specific effects of PymT on the growth of endothelial cells appear in part to be mediated through the activation of cellular tyrosine kinases.

Animals↗
PubMed1994

Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium.

Two monoclonal antibodies (mAb), MEC 7.46 (IgG1) and MEC 13.3 (IgG2a) that specifically recognize mouse endothelial cells (EC) of blood vessels, were produced immunizing a Lewis rat with a polyoma middle T transformed EC line. Antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence on different cultured cell lines and by immunoperoxidase staining on frozen sections of various mouse normal and inflammatory tissues. Both mAbs reacted with eight transformed endothelial lines tested in vitro, but were consistently negative on various cell lines of different histological origin. Reactivity was not altered by preexposure of the cell lines to IL-1. Microscopic immunofluorescence analysis showed that the MEC mAbs localized at the cell-cell contacts in EC. Immunohistochemical staining of various mouse tissue was always restricted to the EC of all blood vessels of the organ considered. Staining of the endothelial lining of blood vessels was greater at cell-to-cell contacts. Weak reactivity was detected in bone marrow and spleen megakaryocytes. This picture was not altered in inflamed and tumor tissues. In the developing mouse embryo, MEC 13.3 specifically stained proliferating and sprouting endothelium in all organs and tissues examined. Both MEC 7.46 and MEC 13.3 mAbs were able to precipitate a molecule with an apparent molecular mass of 130 kDa from endothelioma lysates. The protein was synthesized by the cells and exposed on the cell surface. Immunodepletion analysis indicated that MEC 13.3 recognized a molecule related to the murine from of PECAM or CD31. We believe that these mAbs are promising tools for the identification of murine EC and for studying their ontogenesis and functions.

Animals↗
PubMed1993

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

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

Animals↗
PubMed1993

High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis.

Examination of flk-1 receptor tyrosine kinase mRNA expression by in situ hybridization analysis revealed specific association with endothelial cells at all stages of mouse development, including the blood islands in the yolk sac of day 8.5-10.5 embryos, in which the early progenitors of this lineage originate. flk-1 transcripts were abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased. Identification of the angiogenic mitogen, vascular endothelial growth factor (VEGF), as the high affinity ligand of Flk-1 and correlation of the temporal and spatial expression pattern of Flk-1 and VEGF suggest a major role of this ligand-receptor signaling system in vasculogenesis and angiogenesis.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗