Development of novel mouse models deficient in vasoactive substances-Clinical implication of the natriuretic peptide family and its application to gene therapy-

点击翻译按钮获取中文摘要

The mouse BNP and CNP genes were isolated from a 129Sv mouse genomic library. Targeting vectors for the disruption of BNP or CNP were constructed, in wihch the 2nd and 3rd exons of the BNP gene or the 1st exon of the CNP gene were replaced by the neomycin resistance gene. Several chimeric mice were obtained from the targeted ES cell lines. We are currently mating heterozygotes to obtain mice that are homozygous for the disrupted allele.We characterized a genomic DNA fragment containing the ANP and BNP genes in mice and humans. In mice, the BNP gene was located about 12kb upstream of the ANP gene. An 11-kb human genomic DNA fragment was isolated, which contained the 3rd exon of the BNP gene and the 1st and 2nd exons of the ANP gene, approximately 8kb apart. Therefore, ANP and BNP genes are organized in tandem in mice and humans.We examined BNP gene expression in cultured neonatal rat ventricular cardiocytes. During ET-1-induced cardiocyte hypertrophy, BNP mRNA was induced more rapidly t … More han ANP mRNA.BNP secretion was also stimulated more rapidly than ANP secretion. Furthermore, BNP mRNA turnover was significantly earlier than ANP mRNA turnover. These results demonstrate that BNP gene expression is distinctly regulated from ANP gene expression at transcriptional and posttranscriptional levels, suggesting the possible role of BNP as an "emergency" cardiac hormone against ventricular overload.We examined the interaction of endothelial cells (ECs) and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth. The data indicate augmented production of CNP with the intracellular cGMP accumulation in the EC/SMC coculture. Biologically active TGF-beta in the coculture with direct contact of ECs and SMCs stimulated endothelial productin of CNP.Furthermore, the culture medium from ECs stimulated by TGF-beta had a growth-inhibitory effect on SMCs. These results indicate that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-beta, suggesting the pathophysiological significance of CNP as a regulator of vascular growth in the interaction of ECs and SMCs. Less