Expression of Genes Encoding Vascular Endothelial Growth Factor and its Flk-1 Receptor in the Chick Embryonic Heart

Abstract

Y. Sugishita, T. Takahashi, T. Shimizu, A. Yao, K.-i. Kinugawa, K. Sugishita, K. Harada, H. Matsui and R. Nagai. Expression of Genes Encoding Vascular Endothelial Growth Factor and its Flk-1 Receptor in the Chick Embryonic Heart.Journal of Molecular and Cellular Cardiology (2000) 32, 1039–1051. Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chick VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of 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 μ m), or phorbol 12-myristate, 13-acetate (200 n m) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 μ m), but not by H89 (100 μ m) or bisindolylmaleimide (75 μ m). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression 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 hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves.