A Haplotype of Angiotensin Receptor Associated with Human Hypertension Increases Blood Pressure in Transgenic Mice Running Title: Angiotensin Receptor Haplotype and Hypertension

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, links haplotype-I of the 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 & A7r5) transfected with reporter construct containing haplotype-I. We also show increased binding of the transcription factor, USF-2, to oligonucleotide containing nucleoside -214A as opposed to -214C. Recombineering of a 166 kb BAC 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 hAT1R mRNA level is increased in the kidney and heart of transgenic mice containing haplotype-I as compared to haplotype-II; (b) USF-2 binds more strongly to the chromatin obtained from the kidney of transgenic mice containing haplotype-I as compared to haplotype-II; (c) blood pressure and oxidative stress is increased in transgenic mice containing haplotype-I as compared to haplotype-II. Hypertension is a risk factor for myocardial infarction, heart failure, vascular disease, stroke, and renal failure (1). Reninangiotensin system is central to the regulation of blood pressure and plays an important role in patho-physiology of hypertension (2). The octapeptide, angiotensin-II (AngII), is synthesized from its precursor molecule, angiotensinogen by sequential proteolytic action of renin and angiotensin converting enzyme (ACE). Ang II is a potent vasoactive peptide that promotes adrenal aldosterone secretion and regulates the renal excretion of salt and water (3,4). These biological responses of Ang II are mediated by its interaction with the high-affinity G-protein coupled receptors (GPCRs) localized on the surface of target cells (5). Two main Ang II receptor subtypes – the AT1 and the AT2 receptor – have been identified (6). AT1 receptor (AT1R) is the principal mediator of the aforementioned physiological effects of Ang II in mammalian adults and mounting evidence, unequivocally, shows the contributions of AT1R in cardiovascular pathologies (3). Ito et al have shown a 50% reduction in AT1a receptor expression and a significantly reduced systolic blood pressure in heterozygous AT1aR knock-out mice (7). On the other hand, Le et al (8) have generated transgenic mice over-expressing 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. Additionally, transgenic mice overexpressing AT1R in the cardiac myocytes demonstrate cardiac enlargement, heart failure, and early death (9,10). Spontaneously hypertensive rats (SHR) have been shown to have dys-functional AT1R activation in the brain (11) and increased ATRs are present in the brain of DOCA-salt sensitive rats (12). by gest on M arch 4, 2020 hp://w w w . jb.org/ D ow nladed from