Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms

Abstract

The essential role of VEGF in blood vessel formation in the embryo and in the adult is well established (1, 2).A single VEGF gene gives rise, by alternative splicing, to multiple isoforms (VEGF 121 , VEGF 165 , and VEGF 189 in humans versus VEGF 120 , VEGF 164 , and VEGF 188 in the mouse) that differ in molecular mass, solubility, and receptor binding (3).VEGF 120 lacks exons 6 and 7, encoding extracellular matrix binding structures, and is therefore the most soluble.VEGF 188 contains all exons and avidly binds to the cell surface and extracellular matrix.VEGF 164 lacks only exon 6 and has intermediate properties.The VEGF isoforms bind to several receptors: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1), and neuropilin-1 (NP-1) (3).NP-1 is a semaphorin receptor involved in neuron guidance.As a VEGF 164 -specific coreceptor for VEGFR-2, NP-1 also affects angiogenesis (4) and enhances the angiogenic activity of VEGF 164 (5).To define the differential role of the VEGF isoforms in vivo, mice expressing single VEGF isoforms were gen-erated.Impaired myocardial angiogenesis has been demonstrated in VEGF 120/120 mice (expressing VEGF 120 ) (6).Here we report that loss of VEGF 164 (in VEGF 120/120 and VEGF 188/188 mice) impaired retinal arterial development, whereas loss of VEGF 164 and VEGF 188 (in VEGF 120/120 mice) led to dysregulated vessel outgrowth and patterning in the retina.These observations suggest possible mechanisms for the distinct roles of the VEGF isoforms in retinal vascular patterning and arterial endothelial cell specification. Methods Generation of transgenic mice.Targeted mutagenesis was achieved by homologous and Cre/lox P-mediated sitespecific recombination in embryonic stem (ES) cells.The strategy to generate VEGF 120/120 mice (deletion of exons 6 and 7) has been described previously (6).Targeting vectors used to generate VEGF 164/164 mice and VEGF 188/188 mice were constructed by replacing the genomic sequence with a cDNA containing the fused