Transgenic mice with -6A haplotype of the human angiotensinogen gene have increased blood pressure compared with -6G haplotype

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-1α 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-1α 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.     

Physiological elevation in plasma angiotensinogen increases blood pressure

Hepatic angiotensinogen secretion is controlled by a complex pattern of physiological or pathophysiological mediators. Because plasma concentrations of angiotensinogen are close to the Michaelis-Menten constant, it was hypothesized that changes in circulating angiotensinogen affect the formation rate of ANG I and ANG II and, therefore, blood pressure. To further test this hypothesis, we injected purified rat angiotensinogen intravenously in Sprague-Dawley rats via the femoral vein and measured mean arterial blood pressure after arterial catheterization. In controls, mean arterial pressure was 131 +/- 2 mmHg before and after the injection of vehicle (sterile saline). The injection of 0.8, 1.2, and 2.9 mg/kg angiotensinogen caused a dose-dependent increase in mean arterial blood pressure of 8 +/- 0.4, 19.3 +/- 2.1, and 32 +/- 2.4 mmHg, respectively. In contrast, the injection of a purified rabbit anti-rat angiotensinogen antibody (1.4 mg/kg) resulted in a significant decrease in mean arterial pressure (-33 +/- 3.2 mmHg). Plasma angiotensinogen increased to 769 +/- 32, 953 +/- 42, and 1,289 +/- 79 pmol/ml, respectively, after substrate and decreased by 361 +/- 28 pmol/ml after antibody administration. Alterations in plasma angiotensinogen correlated well with changes in plasma renin activity. In summary, variations in circulating angiotensinogen can result in changes in blood pressure. In contrast to renin, which is known as a tonic regulator for the generation of ANG I, angiotensinogen may be a factor rather important for long-term control of the basal activity of the renin-angiotensin system.     

Glial-specific ablation of angiotensinogen lowers arterial pressure in renin and angiotensinogen transgenic mice

Angiotensinogen (AGT) is mainly expressed in glial cells in close proximity to renin-expressing neurons in the brain. We previously reported that glial-specific overexpression of ANG II results in mild hypertension. Here, we tested the hypothesis that glial-derived AGT plays an important role in blood pressure regulation in hypertensive mice carrying human renin (hREN) and human AGT transgenes under the control of their own endogenous promoters. To perform a glial-specific deletion of AGT, we used an AGT transgene containing loxP sites (hAGT(flox)), so the gene can be permanently ablated in the presence of cre-recombinase expression, driven by the glial fibrillary acidic protein (GFAP) promoter. Triple transgenic mice (RAC) containing a: 1) systemically expressed hREN transgene, 2) systemically expressed hAGT(flox) transgene, and 3) GFAP-cre-recombinase were generated and compared with double transgenic mice (RA) lacking cre-recombinase. Liver and kidney hAGT mRNA levels were unaltered in RAC and RA mice, as was the level of hAGT in the systemic circulation, consistent with the absence of cre-recombinase expression in those tissues. Whereas hAGT mRNA was present in the brain of RA mice (lacking cre-recombinase), it was absent from the brain of RAC mice expressing cre-recombinase, confirming brain-specific elimination of AGT. Immunohistochemistry revealed a loss of AGT immunostaining glial cells throughout the brain in RAC mice. Arterial pressure measured by radiotelemetry was significantly lower in RAC than RA mice and unchanged from nontransgenic control mice. These data suggest that there is a major contribution of glial-AGT to the hypertensive state in mice carrying systemically expressed hREN and hAGT genes and confirm the importance of a glial source of ANG II substrate in the brain.     

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Adipocyte-specific deficiency of angiotensinogen decreases plasma angiotensinogen concentration and systolic blood pressure in mice

Previous studies demonstrated that overexpression of angiotensinogen (AGT) in adipose tissue increased blood pressure. However, the contribution of endogenous AGT in adipocytes to the systemic renin-angiotensin system (RAS) and blood pressure control is undefined. To define a role of adipocyte-derived AGT, mice with loxP sites flanking exon 2 of the AGT gene (Agt(fl/fl)) were bred to transgenic mice expressing Cre recombinase under the control of an adipocyte fatty acid-binding protein 4 promoter (aP2) promoter to generate mice with adipocyte AGT deficiency (Agt(aP2)). AGT mRNA abundance in adipose tissue and AGT secretion from adipocytes were reduced markedly in adipose tissues of Agt(aP2) mice. To determine the contribution of adipocyte-derived AGT to the systemic RAS and blood pressure control, mice were fed normal laboratory diet for 2 or 12 mo. In males and females of each genotype, body weight and fat mass increased with age. However, there was no effect of adipocyte AGT deficiency on body weight, fat mass, or adipocyte size. At 2 and 12 mo of age, mice with deficiency of AGT in adipocytes had reduced plasma concentrations of AGT (by 24-28%) compared with controls. Moreover, mice lacking AGT in adipocytes exhibited reduced systolic blood pressures compared with controls (Agt(fl/fl), 117 ± 2; Agt(aP2), 110 ± 2 mmHg; P < 0.05). These results demonstrate that adipocyte-derived AGT contributes to the systemic RAS and blood pressure control.     

High-level regulated expression of the human G6PD gene in transgenic mice

The glucose-6-phosphate dehydrogenase-encoding gene (G6PD) belongs to a group with constitutive expression in all tissues. The regulation of these housekeeping genes is poorly understood, as compared to what is known about many genes whose expression is restricted to a particular tissue or stage of development, and which are often regulated by locus control regions (LCR) able to act over wide distances. In order to identify sequences in human G6PD which are necessary for its expression, we have generated transgenic mice carrying a 20-kb G6PD construct, including only 2.5 kb of upstream and 2.0 kb of downstream flanking sequence. All mice which carried the transgene (TG) expressed it, and the levels of expression detected in a range of tissues from three independent lines of mice were comparable to that of the endogenous murine G6PD. The variation in enzyme activity from tissue to tissue was remarkably similar for both the TG and the endogenous gene, and was shown to be due in both cases to variations in the steady-state mRNA levels.     

Expression of the human insulin gene in the gastric G cells of transgenic mice

The goal of this study was to engineer gastrin-producing G cells of the gastric antrum to produce insulin. A pGas-Ins chimeric gene in which the gastrin promoter drives expression of the human insulin gene was constructed and was validated by transient transfection of GH4 and AGS cells. RT-PCR analysis and sequencing revealed three forms of differentially spliced insulin mRNA in GH4 cells transiently transfected by pGas-Ins. Gas-Ins transgenic mice were generated utilizing this chimeric gene. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated expression of the human insulin gene specifically in antral G cells. Northern blot analysis demonstrated that the shortest of the insulin mRNA three forms is predominantly expressed in stomach tissue. RT-PCR analysis also showed expression of the transgene in colon, pancreas, and brain tissues that was undetectable by northern analysis. We conclude that gastrin promoter can be used for targeting expression of human insulin to antral G cells and that antral G cells can express human insulin. Further refining of the chimeric gene design is required to enhance expression.     

Expression of the human insulin gene in transgenic mice

Transgenic mice which specifically express the human insulin gene in the pancreatic beta cells have been obtained. Expression is demonstrated by the presence in the serum of human insulin (determined by a radioimmunoassay for the human C-peptide), the level of which increases upon glucose stimulus, and by the presence of human insulin mRNA in pancreas, but not in the other tissues tested. This result indicates that nucleotide sequences necessary for tissue-specific expression are within the transferred gene and/or in its vicinity. It also suggests that the regulatory molecules are quite similar if not identical in human and mouse.     

Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice.

Angiotensinogen is the precursor of the potent vasoactive peptide angiotensin II, and is therefore an important determinant of blood pressure and electrolyte homeostasis. In order to map the tissue-specific and inducible enhancer elements governing angiotensinogen gene expression in transgenic mice, we constructed minigenes containing either 0.75 kb or 4 kb or 5' flanking DNA from the BALB/c angiotensinogen gene. Sequences necessary and sufficient to mediate induction by glucocorticoids, oestrogen and bacterial endotoxin were contained on the minigene bearing 0.75 kb of DNA upstream of the capsite. This construct was also able to confer tissue specificity in the majority of organs producing angiotensinogen. In the testis and salivary gland, differences between the donor (BALB/c) and recipient (Swiss) strains were responsible for the apparently aberrant expression of the minigene constructs. The genetic lesion responsible for these expression polymorphisms has been characterized using recombinant inbred mice. An EcoRI restriction fragment length polymorphism which co-segregates with the angiotensinogen expression phenotypes into many inbred mouse strains is also described.     

The elevated blood pressure of human GRK4gamma A142V transgenic mice is not associated with increased ROS production

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the sensitivity of GPCRs, including dopamine receptors. The GRK4 locus is linked to, and some of its polymorphisms are associated with, human essential hypertension. Transgenic mice overexpressing human (h) GRK4gamma A142V on a mixed genetic background (C57BL/6J and SJL/J) have impaired renal D(1)-dopamine receptor (D(1)R) function and increased blood pressure. We now report that hGRK4gamma A142V transgenic mice, in C57BL/6J background, are hypertensive and have higher blood pressures than hGRK4gamma wild-type transgenic and nontransgenic mice. The hypertensive phenotype is stable because blood pressures in transgenic founders and F6 offspring are similarly increased. To determine whether the hypertension is associated with increased production of reactive oxygen species (ROS), we measured renal NADPH oxidase (Nox2 and Nox4) and heme oxygenase (HO-1 and HO-2) protein expressions and urinary excretion of 8-isoprostane and compared the effect of Tempol on blood pressure in hGRK4gamma A142V transgenic mice and D(5)R knockout (D(5)(-/-)) mice in which hypertension is mediated by increased ROS. The expressions of Nox isoforms and HO-2 and the urinary excretion of 8-isoprostane were similar in hGRK4gamma A142V transgenic mice and their controls. HO-1 expression was increased in hGRK4gamma A142V relative to hGRK4gamma wild-type transgenic mice. In contrast with the hypotensive effect of Tempol in D(5)(-/-) mice, it had no effect in hGRK4gamma A142V transgenic mice. We conclude that the elevated blood pressure of hGRK4gamma A142V transgenic mice is due mainly to the effect of hGRK4gamma A142V transgene acting via D(1)R and increased ROS production is not a contributor.     

Reduced cardiac hypertrophy and altered blood pressure control in transgenic rats with the human tissue kallikrein gene.

To evaluate the cardiovascular actions of kinins, we established a transgenic rat line harboring the human tissue kallikrein gene, TGR(hKLK1). Under the control of the zinc-inducible metallothionein promoter, the transgene was expressed in most tissues including the heart, kidney, lung, and brain, and human kallikrein was detected in the urine of transgenic animals. Transgenic rats had a lower 24-h mean arterial pressure in comparison with control rats, which was further decreased when their diet was supplemented with zinc. The day/night rhythm of blood pressure was significantly diminished in TGR(hKLK1) animals, whereas the circadian rhythms of heart rate and locomotor activity were unaffected. Induction of cardiac hypertrophy by isoproterenol treatment revealed a marked protective effect of the kallikrein transgene because the cardiac weight of TGR(hKLK1) increased significantly less, and the expression of atrial natriuretic peptide and collagen III as markers for hypertrophy and fibrosis, respectively, were less enhanced. The specific kinin-B2 receptor antagonist, icatibant, abolished this cardioprotective effect. In conclusion, the kallikrein-kinin system is an important determinant in the regulation of blood pressure and its circadian rhythmicity. It also exerts antihypertrophic and antifibrotic actions in the heart.     

Relations of blood pressure to angiotensinogen gene T174M polymorphism and alcohol intake

To clarify the interactive effects of alcohol intake and angiotensinogen gene codon 174 (T174M) polymorphisms on blood pressure in Japanese male workers. On the basis of data from health examinations, nutrition survey and T174M genotype analysis conducted for 185 Japanese male workers at 2000, the prevalence of high-normal blood pressure (HNBP) and hypertension were compared between the four subgroups crossed by two T174M genotype categories ('TT' type, and 'TM or MM' type) and two alcohol intake categories (less than 13.7 g per day, and 13.7 g or more per day). Furthermore, for 95 subjects who had been normotensive at 1998 among them, risk of development into HNBP or hypertension at 2000 were compared across the four subgroups. The findings showed that the HNBP prevalence adjusted for age, body mass index, smoking habits and sodium intake in 2000 was significantly (p=0.03) greater in 'TM or MM' type (57.9%) than in 'TT' type (24.9%) in subjects with 13.7 g or more of daily alcohol intake, whereas no difference in this parameter was found between the two genotypes in those with less than 13.7 g of daily alcohol intake (18.2% and 18.3%, respectively). The risk for development into HNBP at 2000 was also greatest in 'TM or MM' type with 13.7 g or more of daily alcohol intake among the four subgroups, although there were not significant differences between the four subgroups. The prevalence of hypertension or development risk for hypertension did not significantly differ between the four subgroups. Therefore, it can be seen that alcohol drinking might be specifically associated with the HNBP in M allele carriers of angiotensinogen gene T174M polymorphism.     

三种突变GRK4转基因小鼠的血压动态分析

目的建立GRK4γ野生型和三种GRK4γ基因突变的转基因小鼠,并对其血压进行动态分析,建立高血压模型。方法把人GRK4γ野生型、GRK4γR 65L、GRK4γA142V和GRK4γA486V三个突变基因分别插入鸡β-肌动蛋白启动子下游,构建转基因表达载体,显微注射法建立C57BL/6J GRK4野生型和突变的转基因小鼠,PCR鉴定转基因小鼠基因型。采用Western Blot鉴定GRK4在心脏、肾脏和肾上腺中的表达筛选高表达转基因品系。用无创血压测量仪分析转基因小鼠动态血压。结果建立了在心脏、肾脏和肾上腺均高表达GRK4基因的转基因小鼠。转基因表达人GRK4γ野生型和GRK4γR 65L突变的小鼠血压正常,转基因表达人GRK4γA142V突变的小鼠在正常的钠盐摄入情况下即可发生高血压,而转基因表达人GRK4γA486V突变的小鼠只有在增加钠盐摄入情况下才发生高血压。结论 GRK4γA142V转基因小鼠可作为自发性高血压动物模型,GRK4γA486V转基因小鼠可作为盐敏感性高血压动物模型。     

Tissue-specific expression of the human renin gene in transgenic mice

Transgenic mice carrying human renin gene were produced by microinjection of 15 kilobases (kb) DNA molecules with up to 3 kb of 5'-flanking sequence and 1.2 kb of 3'-flanking sequence. The transgenes have been shown to be stably transmitted to progeny. It was revealed by RNase protection assay that the human renin gene in a transgenic mouse is expressed preferentially in the kidney. The human renin RNA was also detected at a small level in a variety of tissues such as brain, heart, lung, pancreas, spleen, stomach, testis, and thymus. The direct radioimmunoassay using a monoclonal antibody specific for the active site of human renin demonstrated the synthesis of human active renin in the transgenic mouse kidney. These results suggest that the human renin gene in the transgenic mouse is regulated in a tissue-specific manner.     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.