Integrated computational and experimental analysis of the neuroendocrine transcriptome in genetic hypertension identifies novel control points for the cardiometabolic syndrome.
ABSTRACT: Essential hypertension, a common complex disease, displays substantial genetic influence. Contemporary methods to dissect the genetic basis of complex diseases such as the genomewide association study are powerful, yet a large gap exists between the fraction of population trait variance explained by such associations and total disease heritability.We developed a novel, integrative method (combining animal models, transcriptomics, bioinformatics, molecular biology, and trait-extreme phenotypes) to identify candidate genes for essential hypertension and the metabolic syndrome. We first undertook transcriptome profiling on adrenal glands from blood pressure extreme mouse strains: the hypertensive BPH (blood pressure high) and hypotensive BPL (blood pressure low). Microarray data clustering revealed a striking pattern of global underexpression of intermediary metabolism transcripts in BPH. The MITRA algorithm identified a conserved motif in the transcriptional regulatory regions of the underexpressed metabolic genes, and we then hypothesized that regulation through this motif contributed to the global underexpression. Luciferase reporter assays demonstrated transcriptional activity of the motif through transcription factors HOXA3, SRY, and YY1. We finally hypothesized that genetic variation at HOXA3, SRY, and YY1 might predict blood pressure and other metabolic syndrome traits in humans. Tagging variants for each locus were associated with blood pressure in a human population blood pressure extreme sample with the most extensive associations for YY1 tagging single nucleotide polymorphism rs11625658 on systolic blood pressure, diastolic blood pressure, body mass index, and fasting glucose. Meta-analysis extended the YY1 results into 2 additional large population samples with significant effects preserved on diastolic blood pressure, body mass index, and fasting glucose.The results outline an innovative, systematic approach to the genetic pathogenesis of complex cardiovascular disease traits and point to transcription factor YY1 as a potential candidate gene involved in essential hypertension and the cardiometabolic syndrome.
Project description:The purpose of this study is to understand whether naturally occurring genetic variation in the promoter of chromogranin B (CHGB), a major constituent of catecholamine storage vesicles, is functional and confers risk for cardiovascular disease.CHGB plays a necessary (catalytic) role in catecholamine storage vesicle biogenesis. Previously, we found that genetic variation at CHGB influenced autonomic function, with association maximal toward the 5' region.Here we explored transcriptional mechanisms of such effects, characterizing 2 common variants in the proximal promoter, A-296C and A-261T, using transfection/cotransfection, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP). We then tested the effects of promoter variation on cardiovascular traits.The A-296C disrupted a c-FOS motif, exhibiting differential mobility shifting to chromaffin cell nuclear proteins during EMSA, binding of endogenous c-FOS on ChIP, and differential response to exogenous c-FOS. The A-261T disrupted motifs for SRY and YY1, with similar consequences for EMSA, endogenous factor binding, and responses to exogenous factors. The 2-SNP CHGB promoter haplotypes had a profound (p=3.16E-20) effect on blood pressure (BP) in the European ancestry population, with a rank order of CT<AA<<CA<AT on both systolic blood pressure (SBP) and diastolic blood pressure (DBP), accounting for approximately 2.3% to approximately 3.4% of SBP/DBP variance; the haplotype effects on BP in vivo paralleled those on promoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly nonadditive effects on SBP/DBP. The CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In normotensive twins, parallel effects were noted for a pre-hypertensive phenotype, BP response to environmental stress.The common CHGB promoter variants A-296C and A-261T, and their consequent haplotypes, alter binding of specific transcription factors to influence gene expression in cella as well as BP in vivo. Such variation contributes substantially to risk for human hypertension. Involvement of the sex-specific factor SRY suggests a novel mechanism for development of sexual dimorphism in BP.
Project description:Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in the SHR rat and in WKY rats that had the SHR Y chromosome locus crossed into their genome (SHR/y rat). A potential candidate for this Y chromosome hypertension locus is Sry, a gene that encodes a transcription factor that is responsible for testes development and the Sry protein may affect other target genes.The following study examined if exogenous Sry would elevate adrenal Th, adrenal catecholamines, plasma catecholamines and blood pressure. We delivered 10 mug of either the expression construct, Sry1/pcDNA 3.1, or control vector into the adrenal medulla of WKY rats by electroporation. Blood pressure was measured by the tail cuff technique and Th and catecholamines by HPLC with electrochemical detection.In the animals receiving Sry there were significant increases after 3 weeks in resting plasma NE (57%) and adrenal Th content (49%) compared to vector controls. BP was 30 mmHg higher in Sry injected animals (160 mmHg, p < .05) compared to vector controls (130 mmHg) after 2-3 weeks. Histological analysis showed that the electroporation procedure did not produce morphological damage.These results provide continued support that Sry is a candidate gene for hypertension. Also, these results are consistent with a role for Sry in increasing BP by directly or indirectly activating sympathetic nervous system activity.
Project description:3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5'-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.
Project description:Aims: Hypertension poses a significant challenge to vasculature homeostasis and stands as the most common cardiovascular disease in the world. Its effects are especially profound on vasculature-lining endothelial cells that are directly exposed to the effects of excess pressure. Here, we characterize the in vivo transcriptomic response of cardiac endothelial cells to hypertension using the spontaneous hypertension mouse model BPH/2J. Methods and results: Verification of defective endothelial function in the BPH/2J hypertensive mouse strain was followed by acute isolation of cardiac endothelial cells and transcriptional profiling using RNA sequencing. Gene profiles from normotensive BPN/3J mice were compared to hypertensive animals. We observed over 3000 transcriptional differences between groups including pathways consistent with the cardiac fibrosis found in hypertensive animals. Importantly, many of the fibrosis-linked genes also differ between juvenile pre-hypertensive and adult hypertensive BPH/2J mice, suggesting that these transcriptional differences are hypertension-related. We also show that blood pressure normalization with amlodipine resulted in a subset of genes reversing their expression pattern, supporting the hypertension-dependency of altered gene expression. Yet, other transcripts were recalcitrant to therapeutic intervention illuminating the possibility that hypertension may irreversibly alter some endothelial transcriptional patterns. Conclusions: Hypertension has a profound effect on both function and transcription of endothelial cells, the latter of which was only partially restored with normalization of blood pressure. This study represents one of the first to quantify how endothelial cells are reprogrammed at the molecular level in cardiovascular pathology and advances our understanding of the transcriptional events associated with endothelial dysfunction. Overall design: Endothelium from hypertensive mice were acutely extracted at two different ages (4 weeks and 22 weeks) and compared to endothelium from 22 week old normotensive mice.
Project description:BACKGROUND AND OBJECTIVE:We demonstrated that the Sry gene complex on the spontaneously hypertensive rat (SHR) Y chromosome is a candidate locus for hypertension that accounts for the SHR Y chromosome blood pressure effect. All rat strains examined to date share six Sry loci, and a seventh Sry locus (Sry3) appears to be unique to SHR male rats. Previously, we showed that Sry1 increased activity of the tyrosine hydroxylase promoter in transfected PC12 cells, and Sry1 delivered to adrenal gland of Wistar-Kyoto (WKY) rats increased blood pressure and sympathetic nervous system activity. The objective of this study was to determine whether renin-angiotensin system genes participate in Sry-mediated effects. METHOD:Sry expression vectors were co-transfected into CHO cells with luciferase reporter constructs containing promoters of angiotensinogen (Agt -1430/+22), renin (Ren -1050/-1), angiotensin-converting enzyme (ACE) (ACE -1677/+21) and ACE2 (ACE2 -1091/+83). RESULTS:Sry1, Sry2 and Sry3 differentially upregulated activity of the promoters of angiotensinogen, renin and ACE genes and downregulated ACE2 promoter activity. The largest effect was seen with Sry3, which increased activity of angiotensinogen promoter by 1.7-fold, renin promoter by 1.3-fold, ACE promoter by 2.6-fold and decreased activity of ACE2 promoter by 0.5-fold. The effect of Sry1 on promoter activity was significantly less than that of Sry3. Sry2 activated promoters at a significantly lower level than Sry1 did. The result of either an additive effect of Sry regulation of multiple genes in the renin-angiotensin system or alterations in expression of a single gene could favor increased levels of Ang II and decreased levels of Ang-(1-7). CONCLUSION:These actions of Sry could result in increased blood pressure in males and contribute to sex differences in blood pressure.
Project description:Essential hypertension is a common multifactorial heritable condition in which increased sympathetic outflow from the central nervous system is involved in the elevation in blood pressure (BP), as well as the exaggerated morning surge in BP that is a risk factor for myocardial infarction and stroke in hypertensive patients. The Schlager BPH/2J mouse is a genetic model of hypertension in which increased sympathetic outflow from the hypothalamus has an important etiological role in the elevation of BP. Schlager hypertensive mice exhibit a large variation in BP between the active and inactive periods of the day, and also show a morning surge in BP. To investigate the genes responsible for the circadian variation in BP in hypertension, hypothalamic tissue was collected from BPH/2J and normotensive BPN/3J mice at the 'peak' (n?=?12) and 'trough' (n?=?6) of diurnal BP. Using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays, validation by quantitative real-time PCR and a statistical method that adjusted for clock genes, we identified 212 hypothalamic genes whose expression differed between 'peak' and 'trough' BP in the hypertensive strain. These included genes with known roles in BP regulation, such as vasopressin, oxytocin and thyrotropin releasing hormone, as well as genes not recognized previously as regulators of BP, including chemokine (C-C motif) ligand 19, hypocretin and zinc finger and BTB domain containing 16. Gene ontology analysis showed an enrichment of terms for inflammatory response, mitochondrial proton-transporting ATP synthase complex, structural constituent of ribosome, amongst others. In conclusion, we have identified genes whose expression differs between the peak and trough of 24-hour circadian BP in BPH/2J mice, pointing to mechanisms responsible for diurnal variation in BP. The findings may assist in the elucidation of the mechanism for the morning surge in BP in essential hypertension.
Project description:Hypertension poses a significant challenge to vasculature homeostasis and stands as the most common cardiovascular disease in the world. Its effects are especially profound on endothelial cells that form the inner lining of the vasculature and are directly exposed to the effects of excess pressure. Here, we characterize the in vivo transcriptomic response of cardiac endothelial cells to hypertension by rapidly isolating these cells from the spontaneous hypertension mouse model BPH/2J and its normotensive BPN/3J control strain and performing and RNA sequencing on both. Comparison of transcriptional differences between these groups reveals statistically significant changes in cellular pathways consistent with cardiac fibrosis found in hypertensive animals. Importantly, many of the fibrosis-linked genes identified also differ significantly between juvenile prehypertensive and adult hypertensive BPH/2J mice, suggesting that these transcriptional differences are hypertension related. We examined the dynamic nature of these transcriptional changes by testing whether blood pressure normalization using either a calcium channel blocker (amlodipine) or a angiotensin II receptor blocker (losartan) is able to reverse these expression patterns associated with hypertension. We find that blood pressure reduction is capable of reversing some gene-expression patterns, while other transcripts are recalcitrant to therapeutic intervention. This illuminates the possibility that unmanaged hypertension may irreversibly alter some endothelial transcriptional patterns despite later intervention. This study quantifies how endothelial cells are remodeled at the molecular level in cardiovascular pathology and advances our understanding of the transcriptional events associated with endothelial response to hypertensive challenge.
Project description:Hypertension is a condition with major cardiovascular and renal complications, affecting nearly a billion patients worldwide. Few validated gene targets are available for pharmacological intervention, so there is a need to identify new biological pathways regulating blood pressure and containing novel targets for treatment. The genetically hypertensive “blood pressure high” (BPH), normotensive “blood pressure normal” (BPN), and hypotensive "blood pressure low" (BPL) inbred mouse strains are an ideal system to study differences in gene expression patterns that may represent such biological pathways. We profiled gene expression in liver, heart, kidney, and aorta from BPH, BPN, and BPL mice and determined which biological processes are enriched in observed organ-specific gene signatures. As a result, we identified multiple biological pathways linked to blood pressure phenotype that could serve as a source of candidate genes causal for hypertension. In order to distinguish causal genes from responsive genes in the kidney gene signature we integrated phenotype associated genes into Genetic Bayesian networks, identifying several novel candidate genes causal for hypertension. The integration of data from gene expression profiling and genetics networks is a valuable approach to identify novel potential targets for the pharmacological treatment of hypertension. Overall design: Animals: BPH/2J, BPL/1J and BPN/3J (142 ± 5 mmHg, 69 ± 1.7 mmHg, 94 ± 6 mmHg, SBP respectively) male mice (Jackson Laboratory) were maintained on a 12:12-h light-dark cycle and fed with standard chow ad libitum in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All procedures were in conformance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Tissue collection: Mice (12 weeks old) were euthanized with CO2. Liver, heart, and kidney were flash frozen in liquid nitrogen. Aorta from the aortic root to the left renal artery was rinsed with PBS, immersed in 300ul of RNeasy Lysis buffer (Qiagen), and flash frozen in liquid nitrogen. RNA preparation: Tissues (~100mg, 5 mice per strain) were collected independently and homogenized in 2ml of Trizol (Invitrogen). After extraction with 0.4 ml of chloroform, RNA was extracted with SV Total RNA extraction kit (Promega) followed by DNase I treatment and purification using the RNeasy Kit (Qiagen). RNA was assayed for quality (Agilent Bioanalyzer) and yield (Ribogreen). Kidney, heart and liver RNA was amplified and labeled using a custom automated version of the RT/IVT protocol and reagents provided by Affymetrix. Aorta RNA was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol (NuGEN). Hybridization, labeling and scanning were according to Affymetrix. All samples were processed independently. Microarray analysis: Merck/Affymetrix mouse 1.0 custom arrays monitoring 38384 individual transcripts (25846 Entrez genes) were used. Raw intensity was normalized using the RMA algorithm.
Project description:Identification of hypothalamic genes whose expression differs between active (peak of blood pressure) and inactive periods in the high blood pressure (BPH/2J) Schlager mouse, adjusted by their age- and activity-matched normal blood pressure (BPN/3J) controls using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays. Overall design: The whole hypothalamus was removed from BPH/2J and age-matched BPN/2J (n=3/group, 19 week old, ‘trough’ blood pressure) in the inactive period, when the blood pressure levels of the BPH/2J and BPN/3J models are similar. Hypothalamus of BPH/2J and age-matched BPN/2J (n=6/group, 26 week old, ‘peak’ blood pressure) were collected on the same way at the peak of the circadian variation, when there blood pressure difference between the strains was maximal. No pooling was performed. After extraction of RNA, cRNA was prepared and arrays performed using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays performed at the Ramaciotti Gene Function Analysis facility, University of New South Wales in Sydney, Australia.
Project description:Identification of hypothalamic genes whose expression differs between active (peak of blood pressure) and inactive periods in the high blood pressure (BPH/2J) Schlager mouse, adjusted by their age- and activity-matched normal blood pressure (BPN/3J) controls using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays. The whole hypothalamus was removed from BPH/2J and age-matched BPN/2J (n=3/group, 19 week old, ‘trough’ blood pressure) in the inactive period, when the blood pressure levels of the BPH/2J and BPN/3J models are similar. Hypothalamus of BPH/2J and age-matched BPN/2J (n=6/group, 26 week old, ‘peak’ blood pressure) were collected on the same way at the peak of the circadian variation, when there blood pressure difference between the strains was maximal. No pooling was performed. After extraction of RNA, cRNA was prepared and arrays performed using Affymetrix GeneChip® Mouse Gene 1.0 ST Arrays performed at the Ramaciotti Gene Function Analysis facility, University of New South Wales in Sydney, Australia.