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

Summary

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-, midand hindbrain of VEGFlox/lox/nestin-cre mouse embryos homozygous for a VEGF mutation in the neural tube. These observations demonstrate thatVEGF, 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.

Keywords

Angiogenesis - brain development - microcephaly - retina - VEGF

• References

• 1 Risau W. Mechanisms of angiogenesis. Nature 1997; 386: 671-4.
• 2 Veikkola T, Alitalo K. VEGFs, receptors and angiogenesis. Semin Cancer Biol 1999; 09 (03) 211-20.
• 3 Adams RH, Klein R. Eph receptors and ephrin ligandsessential mediators of vascular development. Trends Cardiovasc Med 2000; 10 (05) 183-8.
• 4 Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000; 06: 389-95.
• 5 Ferrara N. Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am J Physiol Cell Physiol 2001; 280 (06) C1358-66.
• 6 Carmeliet P, Storkebaum E. Vascular and neuronal effects of VEGF in the nervous system: implications for neurological disorders. Semin Cell Dev Biol 2002; 13 (01) 39-53.
• 7 Neufeld G, Cohen T, Gengrinovitch S. et al. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999; 13 (01) 9-22.
• 8 Kawasaki T, Kitsukawa T, Bekku Y. et al. A requirement for neuropilin-1 in embryonic vessel formation. Development 1999; 126 (21) 4895-902.
• 9 Risau W. Development of the vascular system of organs and tissues. In: Schaper W, Schaper J. editors. Collateral circulation. Kluwer Academic Publishers; 1993: 17-28.
• 10 Breier G, Albrecht U, Sterrer S. et al. Expression of vascular endothelial growth factor during embryonic angiogenesis and endothelial cell differentiation. Development 1992; 114 (02) 521-32.
• 11 Breier G, Clauss M, Risau W. Coordinate expression of vascular endothelial growth factor receptor-1 (flt-1) and its ligand suggests a paracrine regulation of murine vascular development. Dev Dyn 1995; 204 (03) 228-39.
• 12 Yang XJ, Cepko CL. Flk-1, a receptor for vascular endothelial growth factor (VEGF), is expressed by retinal progenitor cells. J Neurosci 1996; 16: 6089-99.
• 13 Oosthuyse B, Moons L, Storkebaum E. et al. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet 2001; 28 (02) 131-8.
• 14 Gerber HP, Hillan KJ, Ryan AM. et al. VEGF is required for growth and survival in neonatal mice. Development 1999; 126 (06) 1149-59.
• 15 Vajkoczy P, Farhadi M, Gaumann A. et al. Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2. J Clin Invest 2002; 109 (06) 777-85.
• 16 Beck H, Acker T, Puschel AW. et al. Cell type-specific expression of neuropilins in an MCA-occlusion model in mice suggests a potential role in post-ischemic brain remodeling. J Neuropathol Exp Neurol 2002; 61 (04) 339-50.
• 17 Kappel A, Ronicke V, Damert A. et al. Identification of vascular endothelial growth factor (VEGF) receptor-2 (Flk-1) promoter/ enhancer sequences sufficient for angioblast and endothelial cell-specific transcription in transgenic mice. Blood 1999; 93 (12) 4284-92.
• 18 Hammes HP, Lin J, Bretzel RG. et al. Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat. Diabetes 1998; 47 (03) 401-6.
• 19 Hammes HP, Brownlee M, Jonczyk A. et al. Subcutaneous injection of a cyclic peptide antagonist of vitronectin receptor-type integrins inhibits retinal neovascularization. Nat Med 1996; 02 (05) 529-33.
• 20 Tronche F, Kellendonk C, Kretz O. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet 1999; 23 (01) 99-103.
• 21 Akagi K, Sandig V, Vooijs M. et al. Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res 1997; 25: 1766-73.
• 22 Aiello LP, Pierce EA, Foley ED. et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGFreceptor chimeric proteins. Proc Natl Acad Sci USA 1995; 92 (23) 10457-61.
• 23 Ozaki H, Seo MS, Ozaki K. et al. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol 2000; 156 (02) 697-707.
• 24 Carmeliet P, Ferreira V, Breier G. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380 6573 435-9.
• 25 Ferrara N, Carver-Moore K, Chen H. et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996; 380 6573 439-42.
• 26 Alon T, Hemo I, Itin A. et al. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat Med 1995; 01 (10) 1024-8.
• 27 Ruhrberg C, Gerhardt H, Golding M. et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 2002; 16 (20) 2684-98.
• 28 Wang GX, Li GR, Wang YD. et al. Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia. Life Sci 2001; 69 (23) 2801-10.
• 29 Silverman WF, Krum JM, Mani N. et al. Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures. Neuroscience 1999; 90 (04) 1529-41.
• 30 Sondell M, Sundler F, Kanje M. Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur J Neurosci 2000; 12 (12) 4243-54.
• 31 Ogunshola OO, Antic A, Donoghue MJ. et al. Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system. J Biol Chem 2002; 277 (13) 11410-5.
• 32 Bagnard D, Vaillant C, Khuth ST. et al. Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor. J Neurosci 2001; 21 (10) 3332-41.
• 33 Jin K, Zhu Y, Sun Y. et al. Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci U S A 2002; 99 (18) 11946-50.
• 34 Mochida GH, Walsh CA. Molecular genetics of human microcephaly. Curr Opin Neurol 2001; 14 (02) 151-6.
• 35 Pardanaud L, Yassine F, Dieterlen-Lievre F. Relationship between vasculogenesis, angiogenesis and haemopoiesis during avian ontogeny. Development 1989; 105: 473-85.
• 36 Louissaint A, Rao S, Leventhal C. et al. Coordinated interaction of neurogenesis and angiogenesis in the adult songbird brain. Neuron 2002; 34: 945-60.
• 37 Lammert E, Cleaver O, Melton D. Induction of pancreatic differentiation by signals from blood vessels. Science 2001; 294 5542 564-7.
• 38 Mukouyama YS, Shin D, Britsch S. et al. Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin. Cell 2002; 109 (06) 693-705.