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The article presents a study regarding the haplotype-dependent regulation of the human CYP11B2 gene. The study finds that the transcriptional regulation of the human CYP11B2 gene can cause salt-dependent high blood pressure. Details on the background of the study and the methods used by the researchers are given.

Hypertension is one of the co-morbid conditions for, and profoundly increases the incidence of stroke in humans. Hypertension is polygenic in nature and its familial inheritance is regulated by single nucleotide polymorphisms (SNPs) in the genes associated with renin angiotensin aldosterone system (RAAS). Angiotensin II (AngII) is shown to be at the center stage in driving the RAAS via activation of angiotensin 1 receptor (AT1R). This makes AT1R gene one of the prime candidates whose differential regulation leads to the predisposition to disorders associated with hypertension. A haplotype block of four SNPs is represented primarily by haplotype-I, or Hap-I (TTAA), and haplotype-II, or Hap-II (AGCG), in the promoter of human AT1R (hAT1R) gene. To better understand the physiological role of these haplotypes, transgenic (TG) mice containing haplotype-I and II of the hAT1R gene in a 166Kb BAC (bacterial artificial chromosome) were generated. Our results show that 8 weeks old male transgenic mice containing Hap-I have increased brain hAT1R expression (6.5 fold) and have increased systolic blood pressure (11mmHg) as compared to the mice with Hap-II. Also, the results post PMCAO (permanent middle cerebral artery occlusion) suggest that the mice containing Hap-I suffered from significantly higher (p<0.05) neurological deficits (30% higher than Hap-II) and larger brain infarcts (26% larger than Hap-II). Similarly the molecular analysis of oxidant/inflammatory markers post PMCAO show a significant increase (p<0.05) in NOX-1 (1.85 fold up), CRP (9 fold up) and IL6 (2.8 fold up) and a corresponding reduced expression of antioxidants SOD (60% down) and HO-1 (55% down) in Hap-I mice as compared to Hap-II mice. These results suggest that increased expression of hAT1R leads to the damage associated with endothelial dysfunction and vascular remodeling, eventually rendering Hap-I TG mice susceptible to stroke related pathology. [ABSTRACT FROM AUTHOR]

Human angiotensinogen gene (hAGT) gene has polymorphisms in its 2.5Kb promoter that forms two haplotype (Hap) blocks: -6A/G (-1670A/G, -1562C/T, -1561T/C) and -217A/G (-532T/C, -793A/G, -1074T/C, and -1178G/A). Hap -6A/-217A (Hap -6A) is associated with human hypertension whereas, Hap -6G/-217G (Hap -6G) reduces cardiovascular risk. In vitro, transient transfection shows that Hap -6A has increased promoter activity with a glucocorticoid receptor (GR) agonist (4.7 fold higher) when compared to the Hap -6G. Here, we examine the physiological significance of GR-mediated allele-specific regulation of the hAGT gene, in vivo, with consequential effects on the renin angiotensin system (RAS) in double transgenic (TG) mice engineered with human renin and either haplotype of the hAGT gene. TG mice were treated with and without low dose of a GR agonist, dexamethasone (DEX, 10μg/mL), for 72 hours. In untreated mice, ChIP assay shows greater chromatin-GR binding (Hap -6A- 0.46±0.02 vs. Hap -6G- 0.23±0.01 A.U., p<0.05) and immunoblot shows increased hAGT expression in liver of the Hap -6A-TG mice (Hap -6A- 0.90±0.04 vs. Hap -6G- 0.33±0.03 A.U., p<0.05). However, DEX treatment elicited increased chromatin binding (Hap -6A- 0.80±0.04 vs. Hap -6G- 0.26±0.06 A.U., p<0.05), higher hepatic hAGT expression (Hap -6A- 1.88±0.1 vs. Hap -6G- 0.38±0.09 A.U., p<0.05), increased plasma hAGT (Hap -6A- 1.40±0.11 vs. Hap -6G- 0.42±0.05 A.U., p<0.05) and elevated angiotensin II levels (Hap -6A- 870±45 vs. Hap -6G- 630±47 pg/mL, p<0.05) only in Hap -6A TG mice. No significant change was observed in the endogenous mAGT gene with or without DEX. Similar results were obtained from renal tissues of the two TG lines. Importantly, the GR agonist brought about greater increase in blood pressure (ΔSBP: Hap -6A- 14.5±1.2 vs. Hap -6G- 8.1±1.0 mm Hg, p<0.05) in mice with Hap -6A. Complementary experiments show upregulation of renal redox marker NOX1 (Hap -6A- 1.72±0.13 vs. Hap -6G- 0.45±0.06 A.U., p<0.05) in the -6A TG mice. Taken together, our results show that SNPs in the hAGT Hap -6A favor agonist-induced GR binding that lead to DEX-induced increased expression of the hAGT. This activates the RAS with pathophysiological implications including, increase in blood pressure and tissue oxidative stress. [ABSTRACT FROM AUTHOR]

Aldosterone, synthesized by the enzyme CYP11B2, induces positive sodium-balance and predisposes to hypertension. Various investigators, using genomic DNA analyses, have linked -344T polymorphism in the hCYP11B2 gene to human hypertension. We have identified three SNPs, in linkage disequilibrium, in the hCYP11B2 gene: T/A at -663, T/C at -470 and C/T at -344. Variants ACT occur together and form the haplotype I (Hap I) while variants TTC constitute haplotype II (Hap II). We hypothesize that these SNPs, when present together, will lead to haplotype-dependent transcription of the hCYP11B2 gene, differentially increase aldosterone and affect blood pressure. To this end, novel transgenic (TG) mice with the hCYP11B2 gene, targeted to the mHPRT locus, with either haplotype II or I variant are used in the study. ChIP assay, using anti-RNA pol II antibody, shows increased Pol II binding to the chromatin from Hap I TG mice in adrenal (2.8 fold higher, p<0.05) and renal tissues (1.3 fold higher, p<0.05) as compared to chromatin extracts from Hap II TG mice. Immunoblot analysis shows upregulation of the hCYP11B2 in adrenal (2.7 fold higher, p<0.05) and renal tissues (1.35 fold higher, p<0.05) of Hap I vs. Hap II-TG mice; no significant difference was observed in mCYP11B2 between the two haplotypes. Complementary ELISA shows higher circulating levels (p<0.05) of aldosterone in Hap I mice (1504±48.7 pg/mL) as compared to both, Hap II (778±142.8 pg/mL) and C57 mice (740±28.9 pg/mL). Importantly, we observed increased baseline blood pressure in Hap I TG mice (Hap I- 117±2.5 vs. Hap II- 109±1.9 mm Hg, p<0.05), an effect accentuated by high-salt diet (Hap I- 135±2.6 vs. Hap II- 122±2.2 mm Hg, p<0.05). Elevated aldosterone was accompanied by up-regulation (p<0.05) of proinflammatory markers in renal tissues from Hap I TG mice (IL1β, MCP1, ICAM). Thus, this study identifies -344T as a reporter polymorphism for Hap I of the hCYP11B2 gene. SNPs in Hap I promote increased transcription and expression of the gene, in multiple tissues, with resultant elevation of plasma aldosterone levels. Pathophysiological impact of this haplotype-dependent transcriptional regulation of the hCYP11B2 is highlighted by increased inflammation and blood pressure in TG mice with the Hap I of this transgene. [ABSTRACT FROM AUTHOR]

Age-associated inflammation and redox imbalance underlie etiopathogenesis of cardiovascular-renal diseases, including hypertension and end organ damage. Angiotensin II (Ang II), via activation of the AT1R, contributes to the development and progression of these pathophysiologies. We have identified two haplotype blocks of single nucleotide polymorphisms (SNPs) in the hAT1R gene: haplotype II (Hap II: -810A, -713G, -214C, -153G) and I (Hap I: -810T, -713T, -214A, -153A). In clinical studies, Hap I is linked to human hypertension. This study examines haplotype-dependent and age-associated transcriptional regulation of the hAT1R gene. In this regard, we have engineered transgenic (TG) mice with either haplotype of the hAT1R gene using a 166-kb bacterial artificial chromosome. ChIP assay shows increased RNA-Pol II binding (~1.6 fold higher) to the chromatin extracts from renal tissues of adult (4-6 months) male Hap I-TG mice with increased hAT1R expression (~6 fold higher). This was accompanied by higher baseline blood pressure in Hap I-TG mice (Hap I- 129±3 vs. Hap II- 116±4, p<0.05). Next, we examined the effects of age on this haplotype-dependent regulation of the hAT1R. Aged (>18 months), male mice were used for this part of the study. hAT1R expression increases with age in both haplotypes; however, this increase is significantly higher in Hap I-TG mice (3.17±0.5 to 5.5±0.75 fold) as opposed to mice with Hap II (1.1±0.2 to 1.8±0.1 fold). Age-associated change (Δ) in inflammatory and redox markers was significantly (p<0.05) greater in TG mice with Hap I including, IL1 (4.6±0.8 vs. 2.1±0.49 fold), IL6 (4.0±0.69 vs. 2.1±0.2 fold) and NOX1 (8.3±0.4 vs. 2.5±0.6 fold). This is accompanied by age-associated reduction in levels of antioxidant defenses (SOD1: 0.97±0.0 vs. 1.4±0.1 fold; HO1: 0.77±0.1 vs. 1.3±0.2 fold) and pro-survival genes including, NAMPTS (2.1 folds lower in Hap I vs. Hap II) and SIRT1 (1.8 fold lower in Hap I vs. Hap II). Thus, haplotype-dependent transcriptional regulation of the hAT1R gene causes increased hAT1R expression and blood pressure, in Hap I TG mice. Importantly, aging exacerbates this differential gene-expression regulation, further increasing hAT1R and promoting a prooxidant/inflammatory milieu in mice with Hap I. [ABSTRACT FROM AUTHOR]