The lineage of the Y chromosome accounts for up to 15 to 20 mm Hg in arterial pressure. Genes located on the Y chromosome from the spontaneously hypertensive rat (SHR) are associated with the renin-angiotensin system. Given the important role of the renin-angiotensin system in the renal regulation of fluid homeostasis and arterial pressure, we hypothesized that the origin of the Y chromosome influences arterial pressure via interaction between the intrarenal vasculature and the renin-angiotensin system. Sixteen-week-old normotensive rats (Wistar Kyoto [WKY]), spontaneously hypertensive stroke-prone rat (SHRSP), and 2 reciprocal Y consomic rat strains, 1 comprising the WKY autosomes and X chromosome with the Y chromosome from the hypertensive rat strain (WKY.SPGlaY) and vice versa (SP.WKYGlaY), were examined. SP.WKYGlaY had lower systolic blood pressure than SHRSP (195±5 versus 227±8 mm Hg; P<0.03), whereas WKY.SPGlaY had higher systolic blood pressure compared with WKY (157±3 versus 148±3 mm Hg; P<0.05), measured by radiotelemetry. Compared with WKY rats, SHRSP had higher plasma angiotensin(1-7) (Ang (1-7)):Ang II ratio (WKY: 0.13±0.01 versus SHRSP: 1.33±0.4; P<0.005), greater angiotensin II receptor type 2 and Mas receptor mRNA expression, and a blunted renal constrictor response to intrarenal Ang I and Ang(1-7) infusions. Introgression of the normotensive Y chromosome into the SHRSP background (SP.WKYGlaY) restored responses in the SHRSP to WKY levels, evidenced by a reduction in plasma Ang(1-7):Ang II ratio (SP.WKYGlaY: 0.24±0.02; P<0.01), angiotensin II receptor type 2, and Mas receptor mRNA expression and an increased vasoconstrictor response to intrarenal Ang I and Ang(1-7) infusion. This study demonstrates that the origin of the Y chromosome significantly impacts the renal vascular responsiveness and therefore may influence the long-term renal regulation of blood pressure.
The genetic contribution to salt-sensitivity in hypertension remains unclear. We have previously identified a quantitative trait locus on chromosome 2 in stroke-prone spontaneously hypertensive rats (SHRSPs) responsible for an increase in SBP in response to a salt challenge. This response is blunted in the congenic SHRSP strain with the Wistar-Kyoto (WKY) chromosome 2 region (10?cM) introgressed (SP.WKYGla2k). We aimed to discover the mechanisms that underlie the effects of this region on salt-handling in the SHRSP strain.
A recent genome-wide association study identified a locus on chromosome 16 in the promoter region of the uromodulin (UMOD) gene that is associated with hypertension. Here, we examined the hypertension signal with functional studies in Umod knockout (KO) mice. Systolic blood pressure was significantly lower in KO versus wild-type (WT) mice under basal conditions (KO: 116.6±0.3 mm Hg versus WT: 136.2±0.4 mm Hg; P<0.0001). Administration of 2% NaCl did not alter systolic blood pressure in KO mice, whereas it increased in WT mice by ?33%, P<0.001. The average 24-hour urinary sodium excretion in the KO was greater than that of WT mice (P<0.001). Chronic renal function curves demonstrate a leftward shift in KO mice, suggesting that the relationship between UMOD and blood pressure is affected by sodium. Creatinine clearance was increased during salt loading with 2% NaCl in the KO mice, leading to augmented filtered Na(+) excretion and further Na(+) loss. The difference in sodium uptake that exists between WT and KO strains was explored at the molecular level. Urinary tumor necrosis factor-? levels were significantly higher in KO mice compared with WT mice (P<0.0001). Stimulation of primary thick ascending limb of the loop of Henle cells with exogenous tumor necrosis factor-? caused a reduction in NKCC2A expression (P<0.001) with a concurrent rise in the levels of UMOD mRNA (P<0.001). Collectively, we demonstrate that UMOD regulates sodium uptake in the thick ascending limb of the loop of Henle by modulating the effect of tumor necrosis factor-? on NKCC2A expression, making UMOD an important determinant of blood pressure control.
Fibrosis pathophysiology is critically regulated by Smad 2- and Smad 3-mediated transforming growth factor-? (TGF-?) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-? stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-?-induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3 untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-?-mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-? signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis.
In an F2 cross between stroke-prone spontaneously hypertensive (SHRSP) and Wistar Kyoto (WKY) rats, we previously identified blood pressure quantitative trait loci (QTL) on rat chromosome (RNO) 2 and a pulse pressure QTL on RNO3. The aims of this study were to confirm the QTL on RNO3 and to investigate interaction between RNO2 and RNO3 loci through the generation and phenotypic assessment of single RNO3 congenic (SP.WKY(Gla)3a) and bicongenic (SP.WKY(Gla)2a/3a) strains. Hemodynamic profiling, vascular function, and renal histology were examined in these newly generated strains along with the previously reported RNO2 congenic strain (SP.WKY(Gla)2a). Our results demonstrate significant equivalent reduction in systolic, diastolic, and pulse pressure phenotypes in SP.WKY(Gla)3a and SP.WKY(Gla)2a rats, whereas greater reductions were observed with the SP.WKY(Gla)2a/3a bicongenic strain achieving blood pressure levels similar to normotensive WKY rats. Epistasis was observed between pulse pressure QTL on RNO2 and 3 at baseline and during 1% salt challenge. Vascular function and renal pathology studies indicate that QTL on RNO3 are responsible for salt-induced kidney pathology, whereas QTL on RNO2 seem to have greater impact on vascular function. RNO3 congenic and bicongenic strains have confirmed the importance of SHRSP alleles in the RNO3 congenic interval on pulse pressure variability and end-organ damage. These strains will allow interrogation of complex gene-gene and gene-environment interactions contributing to salt-sensitive hypertension and renal pathology in the SHRSP rat.
Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.
INTRODUCTION: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage. METHODS: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes. RESULTS: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC-EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation. CONCLUSION: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.
Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.
Astrocytes undergo major phenotypic changes in response to injury and disease that directly influence repair in the CNS, but the mechanisms involved are poorly understood. Previously, we have shown that neurosphere-derived rat astrocytes plated on poly-L-lysine (PLL-astrocytes) support myelination in dissociated rat spinal cord cultures (myelinating cultures). It is hypothesized that astrocyte reactivity can affect myelination, so we have exploited this culture system to ascertain how two distinct astrocyte phenotypes influence myelination. Astrocytes plated on tenascin C (TnC-astrocytes), a method to induce quiescence, resulted in less myelinated fibers in the myelinating cultures when compared with PLL-astrocytes. In contrast, treatment of myelinating cultures plated on PLL-astrocytes with ciliary neurotrophic factor (CNTF), a cytokine known to induce an activated astrocyte phenotype, promoted myelination. CNTF could also reverse the effect of quiescent astrocytes on myelination. A combination of microarray gene expression analysis and quantitative real-time PCR identified CXCL10 as a potential candidate for the reduction in myelination in cultures on TnC-astrocytes. The effect of TnC-astrocytes on myelination was eliminated by neutralizing CXCL10 antibodies. Conversely, CXCL10 protein inhibited myelination on PLL-astrocytes. Furthermore, CXCL10 treatment of purified oligodendrocyte precursor cells did not affect proliferation, differentiation, or process extension compared with untreated controls, suggesting a role in glial/axonal ensheathment. These data demonstrate a direct correlation of astrocyte phenotypes with their ability to support myelination. This observation has important implications with respect to the development of therapeutic strategies to promote CNS remyelination in demyelinating diseases.
Transforming growth factor (TGF)-? is one of the main fibrogenic cytokines that drives the pathophysiology of progressive renal scarring. MicroRNAs (miRNAs) are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. We examined the role of TGF-?-induced expression of miR-21, miRNAs in cell culture models and miRNA expression in relevant models of renal disease. In vitro, TGF-? changed expression of miR-21, miR-214, and miR-145 in rat mesangial cells (CRL-2753) and miR-214, miR-21, miR-30c, miR-200b, and miR-200c during induction of epithelial-mesenchymal transition in rat tubular epithelial cells (NRK52E). miR-214 expression was robustly modulated in both cell types, whereas in tubular epithelial cells miR-21 was increased and miR-200b and miR-200c were decreased by 58% and 48%, respectively, in response to TGF-?. TGF-? receptor-1 was found to be a target of miR-200b/c and was down-regulated after overexpression of miR-200c. To assess the differential expression of these miRNAs in vivo, we used the anti-Thy1.1 mesangial glomerulonephritis model and the unilateral ureteral obstruction model in which TGF-? plays a role and also a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat with and without salt loading. The expressions of miR-214 and miR-21 were significantly increased in all in vivo models, showing a possible miRNA signature of renal damage despite differing causes.
Sphingosine-1-phosphate (S1P), a multifunctional phospholipid, regulates vascular cell function. Whether S1P influences vascular inflammatory responses, particularly in hypertension, is unclear. We tested the hypothesis that S1P is a proinflammatory mediator signaling through receptor tyrosine kinase transactivation and that responses are amplified in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats (SHRSPs), a model in which we demonstrated Edg1 (S1P1 receptor) to be a candidate gene for salt-sensitive hypertension. Vascular smooth muscle cell from Wistar-Kyoto rats and SHRSPs were studied. S1P receptor subtypes, S1P1 and S1P2, were similarly expressed in Wistar-Kyoto rats and SHRSPs. S1P induced phosphorylation of epidermal growth factor receptor and platelet-derived growth factor and activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, with amplified effects in SHRSPs versus Wistar-Kyoto rats. Inhibition of epidermal growth factor receptor and platelet-derived growth factor (with AG1478 and AG1296, respectively) abolished S1P-induced phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase in Wistar-Kyoto rats with variable effects in SHRSPs. Vascular smooth muscle cell inflammation was evaluated by expression of adhesion molecules and functional responses assessed by monocyte adhesion. S1P stimulated expression of intercellular adhesion molecule 1 and vascular cell adhesion protein 1 and promoted monocyte adhesion, particularly in SHRSP cells. S1P-mediated inflammation was blunted by AG1478 and AG1296 in SHRSP cells. VPC23019, a S1P1 receptor antagonist, inhibited S1P-induced mitogen-activated protein kinase phosphorylation, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression, and monocyte adhesion. Our data indicate that molecular processes underlying vascular inflammation and cell adhesion in SHRSPs involve S1P/S1P1 receptors and phosphorylation of receptor tyrosine kinases. We identify a novel pathway linking S1P/S1P1 receptors to specific proinflammatory signaling pathways through epidermal growth factor receptor and platelet-derived growth factor transactivation, a process that is upregulated in SHRSPs. Such molecular events may contribute to vascular inflammation in hypertension.
This article describes and illustrates a novel method of microarray data analysis that couples model-based clustering and binary classification to form clusters of `response-relevant genes; that is, genes that are informative when discriminating between the different values of the response. Predictions are subsequently made using an appropriate statistical summary of each gene cluster, which we call the `meta-covariate representation of the cluster, in a probit regression model. We first illustrate this method by analysing a leukaemia expression dataset, before focusing closely on the meta-covariate analysis of a renal gene expression dataset in a rat model of salt-sensitive hypertension. We explore the biological insights provided by our analysis of these data. In particular, we identify a highly influential cluster of 13 genes--including three transcription factors (Arntl, Bhlhe41 and Npas2)-that is implicated as being protective against hypertension in response to increased dietary sodium. Functional and canonical pathway analysis of this cluster using Ingenuity Pathway Analysis implicated transcriptional activation and circadian rhythm signalling, respectively. Although we illustrate our method using only expression data, the method is applicable to any high-dimensional datasets. Expression data are available at ArrayExpress (accession number E-MEXP-2514) and code is available at http://www.dcs.gla.ac.uk/inference/metacovariateanalysis/.
Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2x10-16). In conclusion, using whole blood as a surrogate tissue in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease.
The use of policies and practices regarding surveillance, decolonization, and treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections and the formulary status of various antimicrobial agents used to treat MRSA were characterized.
Hypertension is a heritable and major contributor to the global burden of disease. The sum of rare and common genetic variants robustly identified so far explain only 1%-2% of the population variation in BP and hypertension. This suggests the existence of more undiscovered common variants. We conducted a genome-wide association study in 1,621 hypertensive cases and 1,699 controls and follow-up validation analyses in 19,845 cases and 16,541 controls using an extreme case-control design. We identified a locus on chromosome 16 in the 5 region of Uromodulin (UMOD; rs13333226, combined P value of 3.6 × 10?¹¹). The minor G allele is associated with a lower risk of hypertension (OR [95%CI]: 0.87 [0.84-0.91]), reduced urinary uromodulin excretion, better renal function; and each copy of the G allele is associated with a 7.7% reduction in risk of CVD events after adjusting for age, sex, BMI, and smoking status (H.R.?=?0.923, 95% CI 0.860-0.991; p?=?0.027). In a subset of 13,446 individuals with estimated glomerular filtration rate (eGFR) measurements, we show that rs13333226 is independently associated with hypertension (unadjusted for eGFR: 0.89 [0.83-0.96], p?=?0.004; after eGFR adjustment: 0.89 [0.83-0.96], p?=?0.003). In clinical functional studies, we also consistently show the minor G allele is associated with lower urinary uromodulin excretion. The exclusive expression of uromodulin in the thick portion of the ascending limb of Henle suggests a putative role of this variant in hypertension through an effect on sodium homeostasis. The newly discovered UMOD locus for hypertension has the potential to give new insights into the role of uromodulin in BP regulation and to identify novel drugable targets for reducing cardiovascular risk.
The structural integrity of cardiac cells is maintained by the Ca(2+)-dependent homophilic cell-cell adhesion of cadherins. N-cadherin is responsible for this adhesion under normal physiological conditions. The role of cadherins in adverse cardiac pathology is less clear. We studied the hearts of the stroke-prone spontaneously hypertensive (SHRSP) rat as a genetic model of cardiac hypertrophy and compared them to Wistar-Kyoto control animals. Western blotting of protein homogenates from 12-week old SHRSP animals indicated that similar levels of beta, gamma-, and alpha-catenin and T, N and R-cadherin were expressed in the control and SHRSP animals. However, dramatically higher levels of E-cadherin were detected in SHRSP animals compared to controls at 6, 12 and 18 weeks of age. This was confirmed by quantitative Taqman PCR and immunohistochemistry. E-cadherin was located at the intercalated disc of the myocytes in co-localisation with connexin 43. Adenoviral overexpression of E-cadherin in rat H9c2 cells and primary rabbit myocytes resulted in a significant reduction in myocyte cell diameter and breadth. E-cadherin overexpression resulted in re-localisation of beta-catenin to the cell surface particularly to cell-cell junctions. Subsequent immunohistochemistry of the hearts of WKY and SHRSP animals also revealed increased levels of beta-catenin in the intercalated disc in the SHRSP compared to WKY. Therefore, remodelling of the intercalated disc in the hearts of SHRSP animals may contribute to the altered function observed in these animals.
Essential hypertension affects 20 to 30% of the population worldwide and contributes significantly to cardiovascular mortality and morbidity. Heridability of blood pressure is around 15 to 40% but there are also substantial environmental factors affecting blood pressure variability. It is assumed that blood pressure is under the control of a large number of genes each of which has only relatively mild effects. It has therefore been difficult to discover the genes that contribute to blood pressure variation using traditional approaches including candidate gene studies and linkage studies. Animal models of hypertension, particularly in the rat, have led to the discovery of quantitative trait loci harbouring one or several hypertension related genes, but translation of these findings into human essential hypertension remains challenging. Recent development of genotyping technology made large scale genome-wide association studies possible. This approach and the study of monogenic forms of hypertension has led to the discovery of novel and robust candidate genes for human essential hypertension, many of which require functional analysis in experimental models.
Our previous studies demonstrated reduced rat glutathione S-transferase mu type 1 (Gstm1) expression in stroke-prone spontaneously hypertensive rats (SHRSPs), when compared with the normotensive Wistar-Kyoto rat.
Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis.
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