P312	Altered embryonic coronary arterial and venous differentiation in a mouse model with abnormal VEGF expression.
1N.van den Akker, 2D.Molin, 1H.Lie-Venema, 1M.DeRuiter, 1R.Poelmann, 3P.Carmeliet, 1A.Gittenberger-de Groot
1Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, NL; 2Department of Vascular Physiology, Maastricht University, Maastricht, NL; 3Center for Transgene Technology & Gene Therapy, University Leuven, Leuven, BE.

One of the key factors of vascular development in general is VEGF. In the VEGF120/120 mouse model, exons 6 and 7 of the Vegf-gene are deleted. Therefore, the VEGF164 and the VEGF188 isoforms are absent but the VEGF120 isoform can still be produced. Embryos ranging from embryonic day (E) 10.5 until E19.5 were used to investigate the role of VEGF specifically in coronary development. Coronary development starts with the formation of a subepicardially located primitive plexus of vessels. After the formation of the coronary orifices in the aorta, the differentiation between arteries and veins is triggered and the network will give rise to the final coronary system. In the VEGF120/120 mouse model, we observed a high incidence of developmental coronary abnormalities. Until E14.5, the developing coronary system showed normal morphology, although the number of vessels was increased. After connection to the aorta at E14, the coronary veins became abnormally enlarged and were increased in number, whereas the arteries were small both in size and number. Often, 1 or 3 instead of 2 coronary orifices were seen. To investigate the role of changed VEGF levels in these processes, we investigated the expression patterns of Vegf mRNA in thoraces of wild type and VEGF120/120 mouse embryos between E10.5 and E18.5 using in situ hybridisation. Cardiac Vegf mRNA expression was tightly spatio-temporally regulated, both in normal and VEGF120/120 mouse embryos. At E14.5, the highest expression was observed in the septal myocardium and outflow tract myocardium, whereas at E18.5, expression was mainly seen in the compact myocardium. The level of Vegf mRNA in all investigated hearts of VEGF120/120 embryos was increased compared to wild type controls. This increased expression was also observed in other thoracic structures such as lung or bone tissue. We conclude that increased (venous) endothelial proliferation, especially in the older stages, may be caused by increased VEGF expression. The lack of VEGF164 may account for the decrease in arterial differentiation, since endothelial arterial identity is partly dependent on stimulation of the Notch-pathway via combined stimulation of VEGFR2 and neuropilin-1 by VEGF164. This project is supported by the Netherlands Heart Foundation Grant no. 2001B57 and 2001B15.

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