Transgenic Mice with −6A Haplotype of the Human Angiotensinogen Gene Have Increased Blood Pressure Compared with −6G Haplotype

Abstract

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at ؊6, and the ؊6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with ؊6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with ؊6G. We have found that the hAGT gene has three additional SNPs (A/G at ؊1670, C/G at ؊1562, and T/G at ؊1561). Variants ؊1670A, ؊1562C, and ؊1561T almost always occur with ؊6A, and variants ؊1670G, ؊1562G, and ؊1561G almost always occur with ؊6G. Therefore, the hAGT gene may be subdivided into either ؊6A or ؊6G haplotypes. We show that these polymorphisms affect the binding of HNF-1␣ and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with ؊6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with ؊6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the ؊6A or ؊6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1␣ and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing ؊6A haplotype. Our studies also show that transgenic mice containing ؊6A haplotype have increased plasma AGT level and increased blood pressure as compared with ؊6G haplotype. Our studies explain the molecular mechanism involved in association of the ؊6A allele of the hAGT gene with hypertension.

A Haplotype of Angiotensin Receptor Type 1 Associated with Human Hypertension Increases Blood Pressure in Transgenic Mice

“…Nucleotide sequencing of the BAC revealed that it contains haplotype I of the hAT 1 R gene. The 1.2-kb promoter of the hAT 1 R gene in this BAC was modified to haplotype II by recombineering using the 1.2-kb promoter fragment from a human subject containing haplotype II of the hAT 1 R gene as described previously for hAGT gene (18). The nucleotide sequence of the resulting BAC was confirmed by sequence analysis.…”

“…BP was measured in the conscious state by radio telemetry as described previously (20). After 4 days of basal blood pressure measurement, animals were treated with the AT 1 R blocker losartan (Sigma-Aldrich; 30 mg/kg/day) in drinking water as described previously (18). The blood pressure was then measured 24 h after losartan treatment.…”

Dexamethasone Promotes Hypertension by Allele-specific Regulation of the Human Angiotensinogen Gene

“…The TG mice utilized in this study were generated in our laboratory as described previously by Jain et al (3). Genotyping analysis of the tail snips, followed by sequencing, was performed to confirm the genetic lineage of these TG mice.…”

“…The angiotensin II concentration in the samples was determined directly from the standard curve according to the manufacturer's protocol as described previously (3).…”

“…Differential gene regulation by endogenous or exogenous glucocorticoids, via activation of the glucocorticoid receptors (GRs), is an area of intense current research. Importantly, we have shown that the polymorphisms in Hap Ϫ6A positively affect the binding of transcriptional regulators, such as the glucocorticoid receptors, to the promoter region of the hAGT gene (3). In this study, we tested the hypothesis that glucocorticoids via activation of GR will preferentially induce the hAGT gene in Hap Ϫ6A TG mice with, consequentially, an increase in blood pressure.…”