Overexpression of Vascular Endothelial Growth Factor in the Avian Embryo Induces Hypervascularization and Increased Vascular Permeability without Alterations of Embryonic Pattern Formation

Abstract

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.

Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells

“…The plasmid constructs used are v-P3k, p85⌬iSH2, Myr-Akt, and quail VEGF expressed by the avian retrovirus vector RCAS as previously described (2,3,10,11). PTEN (12,13) and Myr-P3k, c-P3k fused with the amino-terminal sequences of c-Src, were subcloned into an adaptor vector pBSFI and then inserted into a modified avian retrovirus vector RCAS.Sfi (10).…”

“…Aliquots of 0.5 g poly(A) ϩ RNA were fractionated by formaldehyde gel electrophoresis and transferred to a nylon membrane (Schleicher & Schuell). The blots were hybridized with probes of quail VEGF cDNA (11) or actin cDNA, and analyzed by autoradiography.…”

Myc regulates VEGF production in B cells by stimulating initiation of VEGF mRNA translation

“…For ELISA detection of avian VEGF, a rabbit antibody was raised against chicken VEGF 165. The mRNA encoding the 165-amino-acid chicken VEGF isoform (Flamme et al, 1995) was reverse-transcribed using primers 5 0 -GAGCGGAAGCC-CAATGAAG and 3 0 -CGCTGCTCACCGTCTCGG, and then expressed in a pET expression plasmid (Novagen, Madison, WI, USA) in BL21-DE3 Escherichia coli (Stratagene, La Jolla, CA, USA). The resulting 6xHis-tagged VEGF protein was purified from cell pellets by Nickel-NTA chromatography (Qiagen, Valencia, CA, USA), renatured by dialysis to remove urea (Christinger et al, 1996), and concentrated using Centricon-10k filters (Millipore, Billerica, MA, USA).…”

Neovascularization of the Xenopus embryo

“…Drake and Little (1995) injected human recombinant VEGF protein into early stage quail embryos, between the endoderm and the splanchnic mesoderm, and observed a significant perturbation of the embryonic vasculature, especially in the formation of abnormal blood vessels with large lumens. In contrast, Flamme et al (1995b) injected retrovirus infected VEGF-secreting cells into the wingbuds of quail embryos resulting in a significant increase in the complexity of the capillary network and in lumen diameter, but without detectable reorganization of the vascular architecture. Therefore, although absolutely required for vascular development, the precise role of VEGF is vascular patterning remains unclear.…”