Georg Breier

The Vascular Endothelial Growth Factor Receptor Flt-1 Mediates Biological Activities IMPLICATIONS FOR A FUNCTIONAL ROLE OF PLACENTA GROWTH FACTOR IN MONOCYTE ACTIVATION AND CHEMOTAXIS

Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF-stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.

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.

Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2

Tumors have been thought to initiate as avascular aggregates of malignant cells that only later induce vascularization. Recently, this classic concept of tumor angiogenesis has been challenged by the suggestion that tumor cells grow by co-opting preexisting host vessels and thus initiate as well-vascularized tumors without triggering angiogenesis. To discriminate between these two mechanisms, we have used intravital epifluorescence microscopy and multi-photon laser scanning confocal microscopy to visualize C6 microglioma vascularization and tumor cell behavior. To address the mechanisms underlying tumor initiation, we assessed the expression of VEGF, VEGF receptor-2 (VEGFR-2), and angiopoietin-2 (Ang-2), as well as endothelial cell proliferation. We show that multicellular aggregates (<< 1 mm(3)) initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Host blood vessels are not co-opted by tumor cells but rather are used as trails for tumor cell invasion of the host tissue. Our data further suggest that the established microvasculature of growing tumors is characterized by a continuous vascular remodeling, putatively mediated by the expression of VEGF and Ang-2. The results of this study suggest a new concept of vascular tumor initiation that may have important implications for the clinical application of antiangiogenic strategies.

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

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

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

Vascular endothelial growth factor (VEGF) mRNA expression was analysed in rabbit vascular smooth muscle cells following exposure to hypoxia and platelet‐derived growth factor‐BB (PDGF‐BB). Hypoxia potently upregulated VEGF mRNA steady‐state levels in a time‐ and concentration‐dependent manner reaching a maximum level (∼30‐fold increase) after 12–24 h at 0% 02. In contrast, PDGF‐BB caused a modest increase in VEGF expression. However, the combination of PDGF‐BB and a threshold hypoxic stimulus (2.5% O2 for 4 h) had a marked synergistic effect. Synergy between hypoxia and PDGF‐BB was selective for VEGF expression as hypoxia had no effect on the PDGF‐induced upregulation of the proto‐oncogene c‐myc. These results raise the possibility that hypoxia and PDGF‐BB may act in concert to induce VEGF expression in the arterial wall during the development of atherosclerosis.

p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but also is a potent inducer of vascular permeability. Although a dissection of the molecular pathways between angiogenesis‐ and vascular permeability‐inducing properties would be desirable for the development of angiogenic and anti‐angiogenic therapies, such mechanisms have not been identified yet. Here we provide evidence for a role of the p38 MAPK as the signaling molecule that separates these two processes. Inhibition of p38 MAPK activity enhances VEGF‐induced angiogenesis in vitro and in vivo, a finding that was accompanied by prolonged Erk1/2 MAPK activation, increased endothelial survival, and plasminogen activation. Conversely, the same inhibitors abrogate VEGF‐induced vascular permeability in vitro and in vivo. These dualistic properties of p38 MAPK are relevant not only for therapeutic angiogenesis but also for reducing edema formation and enhancing tissue repair in ischemic diseases.

Spatial regulation of VEGF receptor endocytosis in angiogenesis

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

Impaired brain angiogenesis and neuronal apoptosis induced by conditional homozygous inactivation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is essential for the differentiation of the primitive embryonic vascular system and has been implicated in the vascularization of organs. Recently, VEGF has also been proposed to play a role in neural development, neuroprotection, and adult neurogenesis. Here we have investigated the function of VEGF in the developing brain by cre-lox technology. We show that VEGF produced by the embryonic neuroectoderm is required for the vascularization and the development of the brain. Both the invasion and the directed growth of capillaries were severely impaired in the fore-, mid- and hindbrain of VEGF(lox/lox)/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate that VEGF, via local secretion by neural progenitors, induces brain angiogenesis and guides the growth of capillaries toward the ventricular zone. VEGF deficiency led to developmental retardation and progressive destruction of neural tissue in all brain regions. The defect was most pronounced in telencephalic structures, such as the hippocampus, and caused microcephaly. Taken together, the findings establish the critical importance of neuroectoderm-derived VEGF in the morphogenesis of the brain. VEGF acts as a key regulator of brain angiogenesis and provides instructive cues for the correct spatial organization of the vasculature.

Insights in Vessel Development and Vascular Disorders Using Targeted Inactivation and Transfer of Vascular Endothelial Growth Factor, the Tissue Factor Receptor, and the Plasminogen System

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

Circulating Vascular Progenitor Cells Do Not Contribute to Compensatory Lung Growth

Abstract— The biological principles that underlie the induction and process of alveolization in the lung as well as the maintenance of the complex lung tissue structure are one of the major obstacles in pulmonary medicine today. Bone marrow–derived cells have been shown to participate in angiogenesis, vascular repair, and remodeling of various organs. We addressed this phenomenon in the lung vasculature of mice in a model of regenerative lung growth. C57BL/6 mice were transplanted with bone marrow from one of three different reporter gene–transgenic strains. flk-1+/lacZ mice, tie-2/lacZ transgenic mice (both exhibiting endothelial cell–specific reporter gene expression), and ubiquitously enhanced green fluorescent protein (eGFP)-expressing mice served as marrow donors. After hematopoietic recovery, compensatory lung growth was induced by unilateral pneumonectomy and led to complete restoration of initial lung volume and surface area. The lungs were consecutively investigated for bone marrow–derived vascular cells by lacZ staining and immunohistochemistry for phenotype identification of vascular cells. lacZ- or eGFP-expressing bone marrow–derived endothelial cells could not be found in microvascular regions of alveolar septa. Single eGFP-positive endothelial cells were detected in pulmonary arteries at very low frequencies, whereas no eGFP-positive vascular smooth muscle cells were observed. In conclusion, we demonstrate in a model of lung growth and alveolization in adult mice the absence of significant bone marrow–derived progenitor cell contribution to the concomitant vascular growth and remodeling processes.