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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.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells, leading to the formation of vascular tumors in newborn mice. Transformed endothelial (End.) cell lines established from such tumors exhibit altered proteolytic activity as a result of increased expression of urokinase-type plasminogen activator (uPA) and are capable of inducing vascular tumors efficiently when injected into adult mice. In this study we have used mice lacking components of the PA/plasmin system to analyze the role of this system in the transformation process and in tumor growth. We found that the proteolytic status of the host is not a critical determinant for PymT-induced vascular tumor formation. In addition, the lack of either uPA or tissue-type PA (tPA) activity is not limiting for the establishment and proliferation of End. cells in vitro, although the combined loss of both PA activities leads to a marked reduction in proliferation rates. Furthermore, the in vitro morphogenetic properties of mutant End. cells in fibrin gels could only be correlated with an altered proteolytic status in cells lacking both uPA and tPA. However, in contrast with tumors induced by PymT itself, the tumorigenic potential of mutant and wild-type End. cell lines was found to be highly dependent on the proteolytic status of both the tumor cells and the host. Thus, genetic alterations in the PA/plasmin system affect vascular tumor development, indicating that this system is a causal component in PymTmediated oncogenesis.

Embryonic mouse kidneys induce angiogenesis when transplanted on the quail chorioallantoic membrane (Ekblom, P., H. Sariola, M. Karkinen, and L. Saxén, 1982, Cell Differ., 11:35-39). In these experiments all blood vessels were derived from the quail host, suggesting that kidney endothelium is derived from outside blood vessels. We have now analyzed whether kidney angiogenesis is regulated by kidney-derived soluble factors that stimulate the growth of new blood vessels. In the rabbit cornea, 11-d embryonic kidneys induced angiogenesis, whereas uninduced 11-d kidney mesenchymes did not. To characterize and purify this activity from an embryonic organ, we dissected between 600 and 1,000 14-17-d-old embryonic mouse kidneys for each purification experiment. Growth factor activity for capillary endothelial cells was found to bind to heparin-Sepharose and eluted at 0.9-1.1 M sodium chloride. Gel filtration revealed a molecular weight of 16,000-20,000 of this factor. A major 18,000-mol-wt band was seen after gel electrophoresis and silver staining of partially purified growth factor material. The chromatographed factor is mitogenic for endothelial cells but not for smooth muscle cells and stimulates angiogenesis in vivo in the rabbit cornea. Adult kidneys contained two heparin-binding endothelial cell growth factors. The differentiation-dependent production of an angiogenesis factor by the embryonic kidney suggests an important role of angiogenesis in organogenesis.