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

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.

Recent in situ hybridization studies had demonstrated a strong increase in vascular endothelial growth factor (VEGF) mRNA expression in the hyperproliferative epithelium during wound healing. To determine potential mediators of VEGF induction during this process, we analyzed the regulation of VEGF expression in cultured human keratinocytes. We found a large induction of VEGF expression upon treatment of quiescent cells with serum, epidermal growth factor, transforming growth factor-beta 1, keratinocyte growth factor, or the proinflammatory cytokine tumor necrosis factor alpha, respectively. Since all these factors are present at the wound site during the early phase of wound healing, they might also be responsible for VEGF induction after cutaneous injury. To determine the importance of increased VEGF production for wound repair, we compared the time course of VEGF mRNA expression during wound healing of healthy control mice with the kinetics of VEGF expression during skin repair of genetically diabetic db/db mice which are characterized by impaired wound healing. In normal mice we found elevated VEGF mRNA levels during the period when granulation tissue formation occurs. In contrast, VEGF mRNA levels even declined during this period in db/db mice, suggesting that a defect in VEGF regulation might be associated with wound healing disorders.

Tissue factor, a member of the cytokine-receptor superfamily and high-affinity receptor and cofactor for plasma factor VII/VIIa (ref. 1), is the primary cellular initiator of blood coagulation. It is involved in thrombosis and inflammation associated with sepsis, atherosclerosis and cancer, and can participate in other cellular processes including intracellular signalling, metastasis, tumor-associated angiogenesis, and embryogenesis. Here we report that inactivation of the tissue factor gene (TF) results in abnormal circulation from yolk sac to embryo beyond embryonic day 8.5, leading to embryo wasting and death. Vitelline vessels from null mice were deficient in smooth-muscle alpha-actin-expressing mesenchymal cells, which participate in organization of the vessel wall. This implies that tissue factor has a role in blood vessel development.

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.