Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart

Vascular endothelial growth factor (VEGF) is known to play an essential rol e in embryonic vascular development. The heart is one of the main organs th at produce VEGF, but it is still unknown how expression of VEGF gene is reg ulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF a nd its receptor (a KDR/flk-1 or Quek-1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymera se chain reaction revealed that the chide VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 1 22 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 12 2 amino acids were dominant. Northern blot analysis detected an abundance o f VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin ( 100 mu M), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 mu M), but not by H89 (100 mu M) or bisindolylmaleimide (75 mu M). Northern blot analysis also detected the chick flk-1 mRNA in abu ndance in the embryonic heart, and to a much lesser extent in CEVM. The exp ression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic he arts, in situ hybridization confirmed that mRNA encoding VEGF was mainly ex pressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected i n the microvascular endothelial cells, and to a lesser extent in the ventri cular myocytes. These data suggest that VEGF is produced in embryonic ventr icular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by t he chick embryonic heart may play important roles in embryonic cardiovascul ar development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves. (C) 2000 Academic Press.