New insights into the role of angiotensin-converting enzyme obtained from the analysis of genetically modified mice

Abstract

Angiotensin-converting enzyme (ACE) has been well-recognized for its role in blood pressure regulation. ACE is made by many tissues, though it is most abundantly expressed on the luminal surface of vascular endothelium. ACE knockout mice show a profound phenotype with low blood pressure, but also with hemopoietic and developmental defects, which complicates understanding the biological functions of ACE in individual tissue types. Using a promoter-swapping strategy, several mouse lines with unique ACE tissue expression patterns were studied. These include mice with ACE expression in the liver (ACE 3/3), the heart (ACE 8/8), and macrophages (ACE 10/10). We also investigated mice with a selective inactivation of either the N- or C-terminal ACE catalytic domain. Our studies indicate that ACE plays a role in many other physiologic processes beyond simple blood pressure control.

Fig. 1

Generation of ACE 7/7 mice lacking N-terminal ACE catalytic activity. Arrows indicate both the somatic and testis ACE promoters. Black boxes represent exons 1–25 of sACE. A testis-specific exon is indicated in the gray box located between exons 12 and 13. Exons 8 and 20 (patterned boxes) include the N- and C-catalytic domains of sACE, respectively. Site-directed mutagenesis was used to convert the zinc-binding domain within the eighth exon (N-domain) from HEMGH to KEMGK. A neomycin resistance cassette (neo^R), used for positive selection, was inserted into the seventh intron of the ACE gene. A thymidine kinase (TK) cassette, used for negative selection, was inserted at the 5’-end of the construction. The resulting allele contains both mutated N-domain of ACE and neo gene (“targeted allele”). Excision of the neo gene by Cre recombinase resulted in the final mutated allele with a residual 34-base pair loxP sequence present in the seventh intron. To create ACE 13/13 mice, the same approach was used except for this line, we targeted that C-terminal catalytic site present in exon 20. ACE 13/13 mice have an N-terminal domain equivalent to WT mice

Fig. 2

Promoter manipulation as a means of selective expression of ACE. In the ACE 3/3 gene, homologous recombination was used to position a neomycin resistance cassette (neo^R) and an albumin promoter cassette between the somatic ACE promoter and exon 1. The neo^R cassette works for both positive selection and to block the somatic ACE promoter, such that the structural portion of the ACE gene is now under the control of the albumin promoter