Angiotensinogen Promoter Research Articles

Analysis of the combined effect of rs699 and rs5051 on angiotensinogen expression and hypertension.

Hypertension(HTN) involves genetic variability in the renin-angiotensin system and influences antihypertensive response. We previously reported that angiotensinogen (AGT) messenger RNA (mRNA) is endogenously bound by miR-122-5p and rs699 A > G decreases reporter mRNA in the microRNA functional-assay PASSPORT-seq. The AGT promoter variant rs5051 C > T is in linkage disequilibrium (LD) with rs699 A > G and increases AGT transcription. The independent effect of these variants is understudied due to their LD therefore we aimed to test the hypothesis that increased AGT by rs5051 C > T counterbalances AGT decreased by rs699 A > G, and when these variants occur independently, it translates to HTN-related phenotypes. We used in silico, in vitro, in vivo, and retrospective models to test this hypothesis. In silico, rs699 A > G is predicted to increase miR-122-5p binding affinity by 3%. Mir-eCLIP results show rs699 is 40-45 nucleotides from the strongest microRNA-binding site in the AGT mRNA. Unexpectedly, rs699 A > G increases AGT mRNA in an AGT-plasmid-cDNA HepG2 expression model. Genotype-Tissue Expression (GTEx) and UK Biobank analyses demonstrate liver AGT expression and HTN phenotypes are not different when rs699 A > G occurs independently from rs5051 C > T. However, GTEx and the in vitro experiments suggest rs699 A > G confers cell-type-specific effects on AGT mRNA abundance, and suggest paracrine renal renin-angiotensin-system perturbations could mediate the rs699 A > G associations with HTN. We found that rs5051 C > T and rs699 A > G significantly associate with systolic blood pressure in Black participants in the UK Biobank, demonstrating a fourfold larger effect than in White participants. Further studies are warranted to determine if altered antihypertensive response in Black individuals might be due to rs5051 C > T or rs699 A > G. Studies like this will help clinicians move beyond the use of race as a surrogate for genotype.

Abstract 430: Epigenome Editing Of The AGT Gene As A Therapeutic Approach For Cardiovascular Disorders

Overactivation of the renin-angiotensin system contributes to many common and rare cardiovascular diseases such as hypertension, atherosclerosis, and genetic thoracic aortopathies. Current pharmacologic therapies that target this pathway have been proven safe and effective, yet they require individuals with these conditions to adhere to daily medications. CRISPR-Cas9-mediated hepatic inactivation of the angiotensinogen ( AGT ) gene, the precursor of all angiotensin peptides, could be a promising “one and done” therapeutic approach for these disorders. However, disruption of AGT by Cas9 nuclease would result in irreversible changes to the genome and would likely have negative consequences if targeted to organs outside of the liver. CRISPRoff is a CRISPR-based epigenome editing tool consisting of catalytically inactive Cas9 fused to a DNA methyltransferase, as well as a guide RNA (gRNA) designed to target a genomic promoter of interest. Targeting to a gene promoter can result in DNA methylation and subsequent silencing of gene expression, a phenomenon that has the potential to be reversed by CRISPRon, a catalytically inactive Cas9 fused to a demethylase that could be targeted to the same site using the same gRNA. We hypothesized that CRISPRoff can be used to efficiently and durably silence AGT expression by methylation of the AGT promoter. To test this, we screened HuH-7 hepatoma cells with CRISPRoff and either individual gRNAs or dual combinations of gRNAs targeting the AGT promoter. We measured AGT mRNA expression by RT-qPCR and selected the two combinations of gRNAs that resulted in the greatest decrease in AGT expression for methylation analysis. We found that AGT expression was decreased by ≈50% in treated cells, and this correlated with substantially increased methylation throughout the AGT promoter. A long-term in vitro study to assess the durability of this methylation is underway. We are also generating a humanized AGT mouse model to test whether these CRISPRoff gRNAs can silence the human AGT gene in vivo. These results provide a proof of concept of CRISPRoff for silencing of AGT expression as a therapeutic approach for common and rare cardiovascular disorders.

Prorenin Induces Intracellular Signaling And Reactive Oxygen Species In The Brainstem

The importance of the brain renin angiotensin system (RAS) in blood pressure (BP) regulation and water homeostasis is well accepted. However, it remains unclear whether renin and/or its precursor, prorenin, are expressed in the brain and whether they trigger signaling pathways in specific brain regions. We previously used double transgenic mice expressing enhanced green fluorescent protein controlled by renin promoter and beta‐galactosidase controlled by the human angiotensinogen (AGT) promoter to localize their expression in the brain. We reported that neurons expressing renin are localized in close proximity to AGT‐expressing cells in the rostral ventrolateral medulla (RVLM), a brainstem nucleus involved in sympathetic activity and BP control. Here, we hypothesized that prorenin induces phosphorylation of intracellular cascades such as extracellular signal‐regulated kinases (ERK1/2) and activation of nicotinamide adenine dinucleotide phosphate oxidase (NADPHox) in the brainstem. Neonatal brainstem cells were cultured from C57BL/6J mice at postnatal day 1–3 and maintained for 7–10 days in B27‐supplemented neurobasal medium. Immunofluorescence experiments demonstrated the cultures contain both MAP2+ neurons and GFAP+ astrocytes. Expression of type 1 angiotensin receptor (AT1R), renin, and prorenin receptor (PRR) was confirmed by qPCR. To quantify ERK1/2 phosphorylation, cells were stimulated with recombinant prorenin (rProrenin) at 100 nM for 0, 30, and 60 min. Total and phosphorylated ERK1/2 were detected by Western Blot. The effect of rProrenin on NADPHox activity was measured by lucigenin chemiluminescence assay. Specific activity of NADPHox was expressed as chemiluminescence units per min per microgram of protein. Vehicle and phorbol myristate acetate were used as negative and positive controls, respectively. Incubation with rProrenin increased the ratio of phosphorylated‐to‐total ERK1/2 from 0.65±0.07 A.U. at baseline to 1.01±0.13 A.U. at 30 min (54% increase; p=0.0035; n=7) and 1.29±0.09 A.U. at 60 min (102% increase; p<0.0001; n=7). We generated preliminary data showing that both PRO20, a PRR blocker, and Ro‐31, a protein kinase C inhibitor, abrogated prorenin‐induced ERK1/2 phosphorylation. In an additional experiment, we observed that Angiotensin II (Ang II) at 100 nM induced ERK1/2 phosphorylation from 0.98 A.U. to 1.52 A.U. at 15 min (55 % increase), 1.61 A.U. at 30 min (65% increase), and 1.35 A.U. at 60 min (38% increase). Incubation of cells with 100 nM rProrenin for 6 hours resulted in 47.2±9.5 % induction of NADPHox activity compared to vehicle control (p<0.05, n=5). Finally, fluorescent in situ hybridization (RNAscope) data suggests that PRR is expressed in the RVLM neurons and RVLM‐targeted stereotactic microinjection of glutamate, rProrenin, or Ang II, but not vehicle, induced an acute BP elevation in isoflurane‐anesthetized mice. We conclude that prorenin in the brainstem induces downstream signaling involving ERK/2 phosphorylation and generation of ROS, which might contribute to BP elevation and sympathoexcitation. Future studies will determine whether prorenin‐elicited signaling in the brainstem requires binding to PRR, generation of Ang II, and/or AT1R.Support or Funding InformationP01 HL084207 to Sigmund and 5T32HL134643 to Nakagawa

Angiotensinogen promoter methylation predicts bevacizumab treatment response of patients with recurrent glioblastoma.

Patients with recurrent glioblastoma achieving response to bevacizumab combined with chemotherapy have clinical improvement and prolonged survival. High gene expression of angiotensinogen (AGT) is associated with a poor bevacizumab response. Because AGT expression is epigenetically regulated, we aimed to investigate whether AGT promoter methylation in tumor tissue predicts response to bevacizumab combination therapy in patients with recurrent glioblastoma. The study included 159 patients with recurrent glioblastoma, treated with bevacizumab combination treatment (training cohort, n = 77; validation cohort, n = 82). All patients could be evaluated for treatment response and biomarkers. DNA methylation of 4 CpG sites in the AGT promoter was measured using pyrosequencing. A model for nonresponse was established using logistic regression analysis. In the training cohort, lower methylation of each of the four CpG sites in the AGT promoter was significantly associated with nonresponse (all P < 0.05). Moreover, the mean methylation level of all four CpG sites was associated with an increased likelihood of not achieving response to bevacizumab combination therapy (twofold decrease: odds ratio = 3.01; 95% confidence interval: 1.41–6.44; P = 0.004). We developed a model for nonresponse in the training cohort, where a threshold of mean AGT promoter methylation levels was set to below 12%. The model could predict bevacizumab nonresponse with 96% specificity. Importantly, this predictor was also significantly associated with nonresponse in the validation cohort (P = 0.037). Taken together, our findings suggest that low AGT promoter methylation in tumor tissue predicts nonresponse to bevacizumab combination treatment in patients with recurrent glioblastoma. We have, thus, established and successfully validated a predictor for nonresponse that can be used to identify patients who will not benefit from bevacizumab combination therapy.

PATH-28. ANGIOTENSINOGEN PROMOTER METHYLATION TO PREDICT BEVACIZUMAB RESPONSE IN RECURRENT GLIOBLASTOMA PATIENTS

Abstract Recurrent glioblastoma patients achieving response to bevacizumab combination therapy have clinical improvement and prolonged survival. High gene-expression of angiotensinogen (AGT) is associated with a poor response to bevacizumab combination therapy. Because AGT gene-expression is epigenetically regulated, we investigate if lower AGT promoter methylation in tumor tissue predicts a poor response to bevacizumab combination therapy in recurrent glioblastoma patients. Methods: Patients were assessed for eligibility using our clinical database comprising all recurrent glioblastoma patients consecutively treated with bevacizumab combination therapy at our center. The study included 159 response and biomarker evaluable patients: A training cohort of 77 patients and a validation cohort of 82 patients treated in the period between year 2005–2011 and 2012–2015. DNA methylation of 4 CpG sites in the AGT promoter was measured using pyrosequencing. Using logistic regression analysis, a predictive model for non-response was established. Results: In the training cohort, lower methylation of each of the four CpG sites was significantly associated with non-response (p &lt; 0.05). Lower mean methylation of the AGT promoter was significantly associated with non-response (2-fold decrease: OR &amp;eq; 3.01; 95% CI:1.41–6.44; p &amp;eq; 0.004). A predictive model able to predict bevacizumab non-response in clinical practice was established. This predictor was significantly associated with non-response in the validation cohort (p &amp;eq; 0.037). Conclusion: AGT promoter methylation is lower in tumor tissue from non-responsive recurrent glioblastoma patients treated with bevacizumab combination therapy. A predictive model for non-response was established and successfully validated. This model can be used to identify patients who will not benefit from bevacizumab combination therapy.

Abstract P2048: Epigenetic Modification By Angiotensin II Mediates Augmentation Of Intrarenal Angiotensinogen Expression

Inappropriately activated intrarenal renin-angiotensin system, including proximal tubular angiotensinogen (AGT) augmentation, contributes to the progression of hypertension and associated kidney injury. It has been demonstrated that interleukin 6 (IL-6) is required for angiotensin II (Ang II)-induced AGT augmentation in renal proximal tubular cells (PTC). However, molecular mechanisms remain unelucidated. Recent studies suggest that Ang II downregulates cardiac class II histone deacetylases (HDAC), which exhibit protective effects against hypertension related tissue injury. Furthermore, HDAC9 (a class II HDAC) has been shown to act as an epigenetic repressor of proximal tubular AGT. Accordingly, the present study investigated the hypothesis that Ang II decreases HDAC9 levels, thereby enabling IL-6 to promote AGT expression in PTC. Ang II (400 ng/kg/min) was chronically perfused to male mice (N=7) for 14 days to investigate the regulation of intrarenal HDAC9. Moreover, cultured human PTC (hPTC) were used to create wild-type, HDAC9 overexpression, and HDAC9 knockdown by shRNA models and were treated with 100 nM Ang II, 10 ng/ml IL-6, or both for 24 hours. Chronic Ang II infusion elevated blood pressure and suppressed renal cortical HDAC9 levels. A decrease in HDAC9 was also observed in Ang II-treated hPTC. Treatment of hPTC with both Ang II and IL-6 increased AGT mRNA levels (1.50±0.10, ratio to the control, N=6), whereas individual treatments with Ang II or IL-6 did not alter AGT expression levels as previously demonstrated. This upregulation of AGT expression by Ang II and IL-6 co-stimulation was attenuated by the overexpression of HDAC9 in hPTC (1.10±0.08, ratio to the control). Importantly, AGT expression was enhanced by IL-6 without Ang II (2.31±0.08 by IL-6 and 2.27±0.15 by Ang II+IL-6, ratio to the control) in cells receiving the HDAC9 gene knockdown, suggesting that HDAC9 down-regulation mediates IL-6-induced AGT upregulation in hPTC. These results indicate that Ang II-induced epigenetic modification, such as the suppression of intrarenal HDAC9, results in increased accessibility of IL-6-derived transcription factors to the AGT promoter and consequent augmentation of intrarenal AGT expression in Ang II-dependent hypertension.

Identification of platelet-derived growth factor C as a mediator of both renal fibrosis and hypertension

Platelet-derived growth factors (PDGF) have been implicated in kidney disease progression. We previously found that PDGF-C is upregulated at sites of renal fibrosis and that antagonism of PDGF-C reduces fibrosis in the unilateral ureteral obstruction model. We studied the role of PDGF-C in collagen 4A3-/- ("Alport") mice, a model of progressive renal fibrosis with greater relevance to human kidney disease. Alport mice were crossbred with PDGF-C-/- mice or administered a neutralizing PDGF-C antibody. Both PDGF-C deficiency and neutralization reduced serum creatinine and blood urea nitrogen levels and mitigated glomerular injury, renal fibrosis, and renal inflammation. Unexpectedly, systolic blood pressure was also reduced in both Alport and wild-type mice treated with a neutralizing PDGF-C antibody. Neutralization of PDGF-C reduced arterial wall thickness in the renal cortex of Alport mice. Aortic rings isolated from anti-PDGF-C-treated wildtype mice exhibited reduced tension and faster relaxation than those of untreated mice. Invitro, PDGF-C upregulated angiotensinogen in aortic tissue and in primary hepatocytes and induced nuclear factor κB (NFκB)/p65-binding to the angiotensinogen promoter in hepatocytes. Neutralization of PDGF-C suppressed transcript expression of angiotensinogen in Alport mice and angiotensin II receptor type 1 in Alport and wildtype mice. Finally, administration of neutralizing PDGF-C antibodies ameliorated angiotensin II-induced hypertension in healthy mice. Thus, in addition to its key role in mediating renal fibrosis, we identified PDGF-C as a mediator of hypertension via effects on renal vasculature and on the renin-angiotensin system. The contribution to both renal fibrosis and hypertension render PDGF-C an attractive target in progressive kidney disease.

The Higher Frequency of G Allele Found in the -152 Site of Agt Gene But was Not Aligned with the Angiotensinogen Levels

Pathogenesis of essential hypertension involves an interaction between genetic and environmental factors. Genetic variant G-152A of angiotensinogen (AGT) promoter is thought to affect AGT gene transcription and angiotensinogen levels. Angiotensinogen is an important substrate for renin in the RAS that is finally converted into angiotensin II that plays a key role in the control of blood pressure. However, the studies that discuss the single nucleotide polymorphism (SNP) G-152A of AGT gene are still limited, especially in Indonesian hypertensive patients. Therefore, this study was designed to detect the SNP G-152A of AGT and reported its serum angiotensinogen levels. The variants were identified in 62 patients in Malang, Indonesia with essential hypertension by PCR and further identified by automated sequencing. Serum samples were collected to analyze the angiotensinogen levels by a sandwich ELISA. The data showed that the AGT promoter in our patients had genetic variants -152G (G allele/GG genotype) and -152A (A allele/AG genotype) with a frequency of 0.92 : 0.08, respectively. There were no patients with AA genotype. In the analysis of serum angiotensinogen levels (mean ± SD), it was found that A allele had an angiotensinogen level of 371.30 ± 69.92 (ng/mL) and G allele was 343.53 ± 74.95 (ng/mL). The A allele had slightly higher angiotensinogen level than G allele with no significant difference (P=0.437). Thus, in our study is found a genetic variant G-152A of AGT gene represented the GG, AG genotypes in the (-152) site and it has higher frequency of G allele. Further research is still needed to determine that G/A allele directly affects on the occurrence of essential hypertension.

High glucose augments angiotensinogen in human renal proximal tubular cells through hepatocyte nuclear factor-5.

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5’-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy.

HDAC9 is an epigenetic repressor of kidney angiotensinogen establishing a sex difference

BackgroundSexual difference has been shown in the pathogenesis of chronic kidney disease induced by hypertension. Females are protected from hypertension and related end-organ damage. Augmentation of renal proximal tubular angiotensinogen (AGT) expression can promote intrarenal angiotensin formation and the development of associated hypertension and kidney injury. Female rodents exhibit lower intrarenal AGT levels than males under normal conditions, suggesting that the suppressed intrarenal AGT production by programmed mechanisms in females may provide protection from these diseases. This study was performed to examine whether epigenetic mechanisms serve as repressors of AGT.MethodsMale and female Sprague Dawley rats were used to investigate sex differences of systemic, hepatic, and intrarenal AGT levels. All histone deacetylase (HDAC) mRNA levels in the kidneys were determined using a PCR array. HDAC9 protein expression in the kidneys and cultured renal proximal tubular cells (PTC) was analyzed by Western blot analysis and immunohistochemistry. The effects of HDAC9 on AGT expression were evaluated by using an inhibitor and siRNA. ChIP assay was performed to investigate the interaction between the AGT promoter and HDAC9.ResultsPlasma and liver AGT levels did not show differences between male and female Sprague-Dawley rats. In contrast, females exhibited lower AGT levels than males in the renal cortex and urine. In the absence of supplemented sex hormones, primary cultured renal cortical cells isolated from female rats sustained lower AGT levels than those from males, suggesting that the kidneys have a unique mechanism of AGT regulation controlled by epigenetic factors rather than sex hormones. HDAC9 mRNA and protein levels were higher in the renal cortex of female rats versus male rats (7.09 ± 0.88, ratio to male) while other HDACs did not exhibit a sex difference. HDAC9 expression was localized in PTC which are the primary source of intrarenal AGT. Importantly, HDAC9 knockdown augmented AGT mRNA (1.92 ± 0.35-fold) and protein (2.25 ± 0.50-fold) levels, similar to an HDAC9 inhibitor. Furthermore, an interaction between HDAC9 and a distal 5’ flanking region of AGT via a histone complex containing H3 and H4 was demonstrated.ConclusionsThese results indicate that HDAC9 is a novel suppressing factor involved in AGT regulation in PTC, leading to low levels of intrarenal AGT in females. These findings will help to delineate mechanisms underlying sex differences in the development of hypertension and renin-angiotensin system (RAS) associated kidney injury.

Dynamic regulation of the angiotensinogen gene by DNA methylation, which is influenced by various stimuli experienced in daily life.

Angiotensinogen (AGT) has a central role in maintaining blood pressure and fluid balance. DNA methylation is an epigenomic modification maintaining a steady pattern in somatic cells. Herein we summarize the link between AGT regulation and DNA methylation. DNA methylation negatively regulates AGT expression and dynamically changes in response to continuous AGT promoter stimulation. High-salt intake and excess circulating aldosterone cause DNA demethylation around the CCAAT enhancer-binding protein-binding sites, thereby converting the phenotype of AGT expression from an inactive to an active state in visceral adipose tissue. Salt-dependent hypertension may be partially affected by increased adipose AGT expression. Because angiotensin II is a well-established aldosterone-releasing hormone, stimulation of adipose AGT by aldosterone creates a positive feedback loop. This effect is pathologically associated with obesity-related hypertension, although it would be physiologically favorable for humans to efficiently retain their body fluid. The clear difference in DNA demethylation patterns between aldosterone and cortisol indicates a difference in the respective target DNA-binding sites between mineralocorticoid and glucocorticoid receptors in the AGT promoter. Stimulation-induced interactions between transcription factors and target DNA-binding sites trigger DNA demethylation. Dynamic changes in DNA methylation occur in relaxed chromatin regions both where transcription factors actively interact and where transcription is initiated. In contrast to rapid histone modifications, DNA demethylation and remethylation will progress relatively slowly over days or years. A wide variety of stimuli in daily life will continue to slowly and dynamically change DNA methylation patterns throughout life. Wise choices of beneficial stimuli will improve health.

Novel mechanism of blood pressure regulation by forkhead box class O1-mediated transcriptional control of hepatic angiotensinogen.

The renin-angiotensin system is a major determinant of blood pressure regulation. It consists of a cascade of enzymatic reactions involving 3 components: angiotensinogen, renin, and angiotensin-converting enzyme, which generate angiotensin II as a biologically active product. Angiotensinogen is largely produced in the liver, acting as a major determinant of the circulating renin-angiotensin system, which exerts acute hemodynamic effects on blood pressure regulation. How the expression of angiotensinogen is regulated is not completely understood. Here, we hypothesize that angiotensinogen is regulated by forkhead transcription factor forkhead box class O1 (Foxo1), an insulin-suppressed transcription factor, and thereby controls blood pressure in mice. We generated liver-specific Foxo1 knockout mice, which exhibited a reduction in plasma angiotensinogen and angiotensin II levels and a significant decrease in blood pressure. Using hepatocyte cultures, we demonstrated that overexpression of Foxo1 increased angiotensinogen expression, whereas hepatocytes lacking Foxo1 demonstrated a reduction of angiotensinogen gene expression and partially impaired insulin inhibition on angiotensinogen gene expression. Furthermore, mouse angiotensinogen prompter analysis demonstrated that the angiotensinogen promoter region contains a functional Foxo1-binding site, which is responsible for both Foxo1 stimulation and insulin suppression on the promoter activity. Together, these data demonstrate that Foxo1 regulates hepatic angiotensinogen gene expression and controls plasma angiotensinogen and angiotensin II levels, modulating blood pressure control in mice.

Angiotensinogen promoter and angiotensinogen II receptor type 1 gene polymorphisms and incidence of ischemic stroke and neurologic phenotype in Fabry disease

Context: Stroke and/or white matter lesions (WMLs) are significant in Fabry disease. Polymorphisms of angiotensinogen (AGT), AGT Promoter and angiotensinogen II receptor type 1 (AGTR1) are correlated with WMLs.Objectives: We compared AGT. AGT Promoter and AGTR1 genotypes to stroke incidence, Fabry-specific [Mainz Severity Score Index (MSSI)] severity score, and neurologic sub-score (n-MSSI).Methods: Sixty-three Fabry patients and 49 matched controls plus historic controls were genotyped. Institutional Review Board approval was received.Results. C and/or CC alleles of AGT Promoter and AGTR1 were significantly correlated with stroke and n-MSSI.Discussion/conclusion: Findings are suggestive of role of AGT Promoter and AGTR1 genotypes in Fabry phenotypes.

Clinical Implications

HomeHypertensionVol. 62, No. 1Clinical Implications Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBClinical Implications Originally published1 Jul 2013https://doi.org/10.1161/HYP.0b013e31829d4408Hypertension. 2013;62:2Angiotensinogen Expression in Human Adipose Tissue (page 41)Blood pressure is a heritable trait; the genetic causes of essential hypertension remain unclear. Despite being the first gene linked to hypertension, the importance of polymorphisms in angiotensinogen, the precursor to angiotensin-II, remains controversial. The renin–angiotensin system has both endocrine and intracrine functions controlling arterial pressure. The mechanisms regulating angiotensinogen expression is complicated. Because sequence variants in the promoter may differentially affect transcription factor binding, the mechanisms regulating angiotensinogen may differ depending on the polymorphism(s) carried. The current issue of Hypertension reports an effective strategy to examine whether variation in the angiotensinogen promoter effects its expression through differential binding of transcription factors in adipose tissue. The investigators report increased expression of angiotensinogen mRNA and binding of the transcription factor USF2 in chromatin specifically from the −20C allele in subcutaneous adipose tissue from subjects heterozygous for the −20A/C alleles of angiotensinogen. Thus, subjects carrying high-expressing alleles may have increased levels of angiotensinogen and angiotensin-II in some fat depots. This may be clinically important because circulating angiotensinogen derived from adipose tissue is increased in obesity and evidence implicates a role for angiotensin-II derived from adipose angiotensinogen in the regulation of metabolism and blood pressure. Consequently, further studies will need to assess whether this and other polymorphisms control the level of angiotensin-II in hypertensive and obese subjects.Download figureDownload PowerPointCD8+ T Cells and Chemokines in Human Hypertension (page 126)The pathogenic role of T cells in the development of hypertension has been well documented in animal studies. However, the existence of T-cell–driven inflammation in human hypertension has not been demonstrated. In this issue of Hypertension, Youn et al reported an increased fraction of immunosenescent, proinflammatory, cytotoxic CD8+ T cell, and increased circulating levels of T cell chemokines (MIG, IP-10, and I-TAC) in patients with hypertension. In patients with hypertensive nephrosclerosis, I-TAC was expressed more abundantly in the proximal and distal tubules. This evidence, along with an increased level of serum granzyme B, supports the increased activity of cytotoxic T cells in human hypertension. The authors provide evidence for the first time in favor of the existence of T-cell–driven inflammation in human hypertension.Download figureDownload PowerPointCardiac Effects of Hyperaldosteronism (page 62)Affecting >11% of the patients referred to specialized hypertension centers, primary aldosteronism (PA) is the most common endocrine cause of high blood pressure. Furthermore, it induces cardiovascular damage in excess of that expected based on the degree of blood pressure elevation. PA is potentially curable with a targeted treatment, but whether normalization of the blood pressure and regression of associated cardiovascular damage could be achieved in the long term remained uncertain. Rossi et al prospectively assigned 180 PA patients to either adrenalectomy (n=110) or medical therapy (n=70), based on whether there was lateralization of aldosterone excess at adrenal vein sampling. Long-term follow-up study with serial echocardiography evaluations showed for the first time in a prospective study that atrial fibrillation was ≈6-fold higher in PA than in essential hypertensive patients. After treatment blood pressure fell comparably in adrenalectomized and medically treated PA, as it did in optimally treated essential hypertensives. Left ventricular (LV) mass index and the rate of LV hypertrophy also fell mainly through LV inward remodeling in PA patients. Notwithstanding this, the rate of LV mass that was disproportionally elevated for cardiac workload and sex in PA subjects unexpectedly increased. While highlighting the cardiovascular consequences of PA patients, these findings underscore the importance of a timely identification of PA to enable reversal of the LV abnormalities and lowering cardiovascular risk.Download figureDownload PowerPoint Previous Back to top Next FiguresReferencesRelatedDetails July 2013Vol 62, Issue 1 Advertisement Article InformationMetrics © 2013 American Heart Association, Inc.https://doi.org/10.1161/HYP.0b013e31829d4408 Originally publishedJuly 1, 2013 PDF download Advertisement

Angiotensinogen Promoter Polymorphisms Predict Low Diffusing Capacity in U.S. and Spanish IPF Cohorts

Single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT) at positions -20 and -6 are associated with increased severity and progression of various fibrotic diseases. Our earlier work demonstrated that the progression of idiopathic pulmonary fibrosis (IPF) was associated with the A-6 allele. This study examined the hypothesis that the homozygous CC genotype at -20 and the AA genotype at -6 would confer worse measures of pulmonary function (measured by pulmonary function tests) in IPF. Multiple logistic regression analysis was applied to a NIH Lung Tissue Research Consortium cohort and a Spanish cohort, while also adjusting for covariates to determine the effects of these SNPs on measures of pulmonary function. Analysis demonstrated that the CC genotype at -20 was strongly associated with reduced diffusing capacity in males in both cohorts (p=0.0028 for LTRC and p=0.017 for the Spanish cohort). In females, the AA genotype was significantly associated with lower FVC (p=0.0082) and V alv (p=0.022). In males, the haplotype CA at -20 and -6 in AGT was also strongly associated with reduced diffusing capacity in both cohorts. This study is the first to demonstrate an association of AGT polymorphisms (-20A>C and -6G>A) with lower measures of pulmonary function in IPF. It is also the first to relate the effect of gender in lung fibrosis with polymorphisms in AGT.

Allele-Specific Expression of Angiotensinogen in Human Subcutaneous Adipose Tissue

The angiotensinogen gene is genetically linked with hypertension, but the mechanistic basis for association of sequence variants in the promoter and coding region of the gene remains unclear. An E-box at position -20 has been hypothesized to control the level of angiotensinogen expression, but its mechanistic importance for angiotensinogen expression in human tissues is uncertain. We developed an allele-specific polymerase chain reaction-based assay to distinguish between angiotensinogen mRNA derived from variants at the -20 position (rs5050) in the angiotensinogen promoter in adipose tissues obtained during surgery. The assay takes advantage of linkage disequilibrium between the rs5050 (located in the promoter) and rs4762 (located in the coding region) single nucleotide polymorphisms. This strategy allowed us to assess the level of allele-specific expression in A-20C heterozygous subjects comparing the relative proportion of each allele with the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that angiotensinogen mRNA derived from the -20C allele is expressed significantly higher than that derived from the -20A allele in subcutaneous adipose tissue, and increased expression correlates with enriched chromatin binding of upstream stimulatory factor-2 to the -20C E-box compared with -20A. This may be depot selective because we were unable to detect these differences in omental adipose. This provides the first data directly comparing expression of angiotensinogen mRNA and differential transcription factor binding derived from 2 variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.

Abstract 162: Increased Allele-Specific Expression of Angiotensinogen Derived From the -20C vs. -20A In Subcutaneous Adipose Tissue From Anonymous Human Donors

The mechanism by which single nucleotide polymorphisms (SNP) regulate angiotensinogen (AGT) expression in humans has been extensively studied but remains unclear. The E box ( C ACGTG) at position -20 in the human AGT promoter binds USF1 and USF2 in vitr o. Recently, we published that USF1 and USF2 are both essential for human AGT expression in double transgenic mice carrying a -20C SNP in human AGT and a gene trap USF1 knockdown. Therefore, we hypothesize that selective binding of USF1/2 to -20C versus -20A (the rs5050 SNP) in the promoter region underlies the enhanced expression of AGT in humans containing the rs5050 SNP. We genotyped subcutaneous adipose tissue from 89 anonymous human donors for both the A-20C SNP and a coding region SNP known to be in linkage disequilibrium with the A-20C (C620T; rs4762). Based on sequencing and TOPO TA cloning, 22% of the samples were heterozygous for A-20C and linkage disequilibrium between -20A with 620C and -20C with 620T was confirmed in 80% of the samples. AGT and USF2 mRNA was elevated significantly (2.0±0.3 and 2.2±0.4 fold, n=8, respectively) in the male -20AC compared to the -20AA group, whereas expression of fatty acid synthase, a known USF target gene, was unchanged. These changes in expression were not observed in females, suggesting sexual dimorphism in the regulation of both USF1/2 and AGT. We developed and validated a quantitative PCR assay using Taqman probes selective for the mRNA separately corresponding to 620T and 620C. We used this assay on RNA derived from 16 heterozygous (-20AC and 620CT) subcutaneous adipose samples. Expression derived from the -20C promoter (620T mRNA) was 4.1±0.6 fold higher compared to the -20A promoter (620C mRNA) (n=16). We have also developed a quantitative allele-specific assay to measure USF1/2 binding to -20AC for use in ChIP. We conclude that USF1/2 and the -20C SNP play physiological roles in AGT regulation in human adipose tissue which we hypothesize is due to increased USF recruitment at -20C.

Gene Trapping Uncovers Sex-Specific Mechanisms for Upstream Stimulatory Factors 1 and 2 in Angiotensinogen Expression

A single-nucleotide polymorphism (C/A) located within an E-box at the -20 position of the human angiotensinogen (AGT) promoter may regulate transcriptional activation through differential recruitment of the transcription factors upstream stimulatory factor (USF) 1 and 2. To study the contribution of USF1 on AGT gene expression, mice carrying a (-20C) human AGT (hAGT) transgene were bred with mice harboring a USF1 gene trap allele designed to knock down USF1 expression. USF1 mRNA was reduced relative to controls in liver (9 ± 1%), perigenital adipose (16 ± 3%), kidney (17 ± 1%), and brain (34 ± 2%) in double-transgenic mice. This decrease was confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation analyses revealed a decrease in USF1, with retention of USF2 binding at the hAGT promoter in the liver of male mice. hAGT expression was reduced in the liver and other tissues of female but not male mice. The decrease in endogenous AGT expression was insufficient to alter systolic blood pressure at baseline but caused reduced systolic blood pressure in female USF1 gene trap mice fed a high-fat diet. Treatment of USF1 knockdown males with intravenous adenoviral short hairpin RNA targeting USF2 resulted in reduced expression of USF1, USF2, and hAGT protein. Our data from chromatin immunoprecipitation assays suggests that this decrease in hAGT is attributed to decreased USF2 binding to the hAGT promoter. In conclusion, both USF1 and USF2 are essential for AGT transcriptional regulation, and distinct sex-specific and tissue-specific mechanisms are involved in the activities of these transcription factors in vivo.

Promoter Polymorphism G-6A, which Modulates Angiotensinogen Gene Expression, Is Associated with Non-Familial Sick Sinus Syndrome

BackgroundIt is well known that familial sick sinus syndrome (SSS) is caused by functional alterations of ion channels and gap junction. Limited information is available on the mechanism of age-related non-familial SSS. Although evidence shows a close link between arrhythmia and the renin-angiotensin system (RAS), it remains to be determined whether the RAS is involved in the pathogenesis of non-familial SSS.MethodsIn this study, 113 patients with documented non-familial SSS and 125 controls were screened for angiotensinogen (AGT) and gap junction protein-connexin 40 (Cx40) promoter polymorphisms by gene sequencing, followed by an association study. A luciferase assay was used to determine the transcriptional activity of the promoter polymorphism. The interaction between nuclear factors and the promoter polymorphism was characterized by an electrophoretic mobility shift assay (EMSA).ResultsAssociation study showed the Cx40 -44/+71 polymorphisms are not associated with non-familial SSS; however, it indicated that four polymorphic sites at positions -6, -20, -152, and -217 in the AGT promoter are linked to non-familial SSS. Compared to controls, SSS patients had a lower frequency of the G-6A AA genotype (OR 2.88, 95% CI 1.58–5.22, P = 0.001) and a higher frequency of the G allele at -6 position (OR 2.65, 95% CI 1.54–4.57, P = 0.0003). EMSA and luciferase assays confirmed that nucleotide G at position -6 modulates the binding affinity with nuclear factors and yields a lower transcriptional activity than nucleotide A (P<0.01).ConclusionG-6A polymorphism, which modulates the transcriptional activity of the AGT promoter, may contribute to non-familial SSS susceptibility.

A-6G and A-20C Polymorphisms in the Angiotensinogen Promoter and Hypertension Risk in Chinese: A Meta-Analysis

BackgroundNumerous studies in Chinese populations have evaluated the association between the A-6G and A-20C polymorphisms in the promoter region of angiotensinogen gene and hypertension. However, the results remain conflicting. We carried out a meta-analysis for these associations.Methods and ResultsCase–control studies in Chinese and English publications were identified by searching the MEDLINE, EMBASE, CNKI, Wanfang, CBM, and VIP databases. The random-effects model was applied for dichotomous outcomes to combine the results of the individual studies. We finally selected 24 studies containing 5932 hypertensive patients and 5231 normotensive controls. Overall, we found significant association between the A-6G polymorphism and the decreased risk of hypertension in the dominant genetic model (AA+AG vs. GG: P = 0.001, OR = 0.71, 95%CI 0.57–0.87, Pheterogeneity = 0.96). The A-20C polymorphism was significantly associated with the increased risk for hypertension in the allele comparison (C vs. A: P = 0.03, OR = 1.14, 95%CI 1.02–1.27, Pheterogeneity = 0.92) and recessive genetic model (CC vs. CA+AA: P = 0.005, OR = 1.71, 95%CI 1.18–2.48, Pheterogeneity = 0.99). In the subgroup analysis by ethnicity, significant association was also found among Han Chinese for both A-6G and A-20C polymorphisms. A borderline significantly decreased risk of hypertension between A-6G and Chinese Mongolian was seen in the allele comparison (A vs. G: P = 0.05, OR = 0.79, 95%CI 0.62–1.00, Pheterogeneity = 0.84).ConclusionOur meta-analysis indicated significant association between angiotensinogen promoter polymorphisms and hypertension in the Chinese populations, especially in Han Chinese.