Abstract
The renin-angiotensin system plays an important role in the regulation of blood pressure via angiotensin II and the angiotensin II receptor type 1 (AT1R). Human AT1R gene promoter has four SNPs: T/A at -777, T/G at -680, A/C at -214, and A/G at -119, that are in linkage disequilibrium. Variants -777T, -680T, -214A, and -119A almost always occur together (named haplotype I), and variants -777A, -680G, -214C, and -119G almost always occur together (named haplotype II) in Caucasian subjects. Genomic DNA analyses, from 388 normotensive and 374 hypertensive subjects, link haplotype I of the human AT1R (hAT1R) gene with hypertension in Caucasians (p = 0.004, χ(2) = 8.46). Our results show increased basal promoter activity of the hAT1R gene in cells (H295R and A7r5) transfected with reporter construct containing haplotype I. We also show increased binding of the transcription factor, USF2, to oligonucleotide containing nucleoside -214A as opposed to -214C. Recombineering of a 166-kb bacterial artificial chromosome containing 68 kb of the 5'-flanking region, 45 kb of the coding sequence, and 53 kb of the 3'-flanking region of the hAT1R gene was employed to generate transgenic mice with either haplotype. We show that (a) hAT1R mRNA level is increased in the kidney and heart of transgenic mice containing haplotype I as compared with haplotype II; (b) USF2 binds more strongly to the chromatin obtained from the kidney of transgenic mice containing haplotype I as compared with haplotype II; and (c) blood pressure and oxidative stress are increased in transgenic mice containing haplotype I as compared with haplotype II.
Highlights
AT1 receptor (AT1R) activation induces oxidative stress, promotes inflammation, and increases blood pressure
We show that (a) human AT1R (hAT1R) mRNA level is increased in the kidney and heart of transgenic mice containing haplotype I as compared with haplotype II; (b) upstream stimulatory factor-2 (USF2) binds more strongly to the chromatin obtained from the kidney of transgenic mice containing haplotype I as compared with haplotype II; and (c) blood pressure and oxidative stress are increased in transgenic mice containing haplotype I as compared with haplotype II
Ϫ680T Allele of the hAT1R Gene Is Associated with Increased Risk of Hypertension in Caucasians—Because of linkage disequilibrium (LD) of the SNPs in haplotypes, Ϫ680T or Ϫ680G was used as reporter polymorphism for haplotype I and II, respectively (Fig. 1)
Summary
AT1R activation induces oxidative stress, promotes inflammation, and increases blood pressure. Transgenic mice with haplotype I have USF2-dependent AT1R overexpression, increased oxidative stress, and increased blood pressure. Le et al [8] have generated transgenic mice overexpressing the AT1R gene and have shown that increasing the gene copy number from 2 to 4 leads to doubling of AT1R mRNA expression in the vasculature and the heart of these mice This resulted in hypertension, especially in female transgenic mice. In Caucasian hypertensive subjects we identified four novel SNPs, in linkage disequilibrium (LD), in the promoter region of the human AT1R (hAT1R) gene. We generated transgenic (TG) mice with the either haplotype and tested the hypothesis that haplotype-dependent, differential regulation of the hAT1R gene will increase hAT1R expression and predispose to AT1R-induced hypertension. Complementary experiments revealed significantly higher blood pressure; pro-inflammatory markers, including IL-6, TNF␣, and CRP; and oxidative stress markers, such as NADPH oxidase, in TG mice containing haplotype I of the hAT1R gene as compared with TG mice with the haplotype II
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