Delyth Graham

Superoxide Excess in Hypertension and Aging: A Common Cause of Endothelial Dysfunction

There is evidence in humans that hypertension and aging similarly impair endothelial function, although the mechanism remains unclear. Superoxide anion (O2−) is a major determinant of nitric oxide (NO) bioavailability and thus endothelial function. We sought to determine the relationship between endothelial function, O2−, and age in normotensive Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). Aortic rings were removed from female WKY and SHRSP at 3 to 4 months (young) and 9 to 12 months (old). O2− generation by aortic rings was measured before and after removal of the endothelium or incubation with NG nitro-l-arginine methyl ester, diphenyleneiodonium, or apocynin. Levels of p22phox were studied with immunohistochemistry and used as a marker of NAD(P)H oxidase expression. NO bioavailability was significantly lower in old WKY compared with young WKY (P =0.0009) and in old SHRSP compared with young SHRSP (P =0.005). O2− generation was significantly greater in old WKY compared with young WKY (P =0.0001). Removal of the endothelium and NG nitro-l-arginine methyl ester treatment resulted in a significant reduction in O2− generation in old SHRSP (P =0.009 and 0.001, respectively). Diphenyleneiodonium significantly reduced O2− generation in 12-month WKY (P =0.008) and 12-month SHRSP (P =0.009). Apocynin attenuated O2− generation by older WKY (P =0.038) and SHRSP (P =0.028). p22phox was increased in older animals compared with young. We conclude that NO bioavailability decreases with age in female WKY and SHRSP. O2− generation increases with age in WKY and is higher in SHRSP and may contribute to the reduced NO by scavenging. NAD(P)H oxidase may contribute to the age-related increase in O2−.

Gender-Linked Hypertension in Offspring of Lard-Fed Pregnant Rats

Abstract—Epidemiological studies suggest an association between maternal nutrition and offspring cardiovascular disease. We previously demonstrated endothelial dysfunction and abnormal aortic fatty acid composition in adult female offspring of rats fed animal lard during pregnancy. We have now further investigated this model. Female Sprague-Dawley rats were fed a control breeding diet (5.3% fat) or a diet rich in lard (25.7% fat) 10 days before and throughout pregnancy and lactation. Male and female offspring were implanted with radiotelemeters for recording of blood pressure, heart rate, and activity at 80, 180, and 360 days of age. Reactivity to acetylcholine and to nitric oxide were assessed in isolated small mesenteric arteries from 80- and 180-day-old littermates. Systolic blood pressure (awake phase) was raised in female offspring (180 days: offspring of control, 130.7±1.6 mm Hg, n=5, versus offspring of lard-fed, 138.1±2.9, n=5, P =0.029; 360 days: offspring of control, 129.7±3.7 mm Hg, n=6, versus offspring of lard-fed, 142.1±3.2, n=6, P =0.005). Diastolic blood pressure was also raised at 180 days (offspring of control, 87.6±1.0 mm Hg, n=5, versus offspring of lard-fed, 94.7±2.6, n=5, P =0.011). Blood pressure was not raised in male offspring. Endothelium-dependent relaxation to acetylcholine was blunted in male and female offspring of lard-fed dams (80 and 180 days). Feeding a diet rich in lard to pregnant rats leads to gender-related cardiovascular dysfunction in normally fed offspring.

Genome-wide association study of blood pressure extremes identifies variant near UMOD associated with hypertension

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.

Mitochondria-Targeted Antioxidant MitoQ10 Improves Endothelial Function and Attenuates Cardiac Hypertrophy

Mitochondria are a major site of reactive oxygen species production, which may contribute to the development of cardiovascular disease. Protecting mitochondria from oxidative damage should be an effective therapeutic strategy; however, conventional antioxidants are ineffective, because they cannot penetrate the mitochondria. This study investigated the role of mitochondrial oxidative stress during development of hypertension in the stroke-prone spontaneously hypertensive rat, using the mitochondria-targeted antioxidant, MitoQ10. Eight-week-old male stroke-prone spontaneously hypertensive rats were treated with MitoQ10 (500 &mgr;mol/L; n=16), control compound decyltriphenylphosphonium (decylTPP; 500 &mgr;mol/L; n=8), or vehicle (n=9) in drinking water for 8 weeks. Systolic blood pressure was significantly reduced by ≈25 mm Hg over the 8-week MitoQ10 treatment period compared with decylTPP (F=5.94; P=0.029) or untreated controls (F=65.6; P=0.0001). MitoQ10 treatment significantly improved thoracic aorta NO bioavailability (1.16±0.03 g/g; P=0.002, area under the curve) compared with both untreated controls (0.68±0.02 g/g) and decylTPP-treated rats (0.60±0.06 g/g). Cardiac hypertrophy was significantly reduced by MitoQ10 treatment compared with untreated control and decylTPP treatment (MitoQ10: 4.01±0.05 mg/g; control: 4.42±0.11 mg/g; and decylTPP: 4.40±0.09 mg/g; ANOVA P=0.002). Total MitoQ10 content was measured in liver, heart, carotid artery, and kidney harvested from MitoQ10-treated rats by liquid chromatography-tandem mass spectrometry. All of the organs analyzed demonstrated detectable levels of MitoQ10, with comparable accumulation in vascular and cardiac tissues. Administration of the mitochondria-targeted antioxidant MitoQ10 protects against the development of hypertension, improves endothelial function, and reduces cardiac hypertrophy in young stroke-prone spontaneously hypertensive rats. MitoQ10 provides a novel approach to attenuate mitochondrial-specific oxidative damage with the potential to become a new therapeutic intervention in human cardiovascular disease.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats

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.

Microarray Analysis of Rat Chromosome 2 Congenic Strains

Abstract—Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of human essential hypertension, and a number of reproducible blood pressure regulation quantitative trait loci have been found to map to rat chromosome 2. The SP.WKYGla2c* congenic strain was produced by introgressing a region of rat chromosome 2 from the normotensive Wistar Kyoto (WKY) strain into the genetic background of the SHRSP. Systolic and diastolic blood pressures were significantly reduced in the SP.WKYGla2c* compared with the SHRSP parental strain (198/134±6.1/3.3 versus 172/120±3.8/3.4 mm Hg; F=15.8/8.1, P =0.0009/0.013). Genome-wide microarray expression profiling was undertaken to identify differentially expressed genes among the parental SHRSP, WKY, and congenic strain. We identified a significant reduction in expression of glutathione S-transferase &mgr;-type 2, a gene involved in the defense against oxidative stress. Quantitative reverse transcription–polymerase chain reaction relative to a &bgr;-actin standard confirmed the microarray results with SHRSP mRNA at 8.56×10−4 ±1.6×10−4 compared with SP.WKYGla2c* 3.67×10−3±2.8×10−4 (95% CI −3.9×10−3 to −1.8×10−3;P =0.0034) and WKY 4.03×10−3±5.1×10−4; (95% CI −5.4×10−3 to −8.9×10−4;P =0.027). We also identified regions of conserved synteny, each containing the Gstm2 gene, on mouse chromosome 3 and human chromosome 1.

Interleukin-1 regulates multiple atherogenic mechanisms in response to fat feeding

Background Atherosclerosis is an inflammatory process that develops in individuals with known risk factors that include hypertension and hyperlipidaemia, influenced by diet. However, the interplay between diet, inflammatory mechanisms and vascular risk factors requires further research. We hypothesised that interleukin-1 (IL-1) signaling in the vessel wall would raise arterial blood pressure and promote atheroma. Methodology/Principal Findings Apoe−/− and Apoe−/−/IL-1R1−/− mice were fed high fat diets for 8 weeks, and their blood pressure and atherosclerosis development measured. Apoe−/−/IL-R1−/− mice had a reduced blood pressure and significantly less atheroma than Apoe−/− mice. Selective loss of IL-1 signaling in the vessel wall by bone marrow transplantation also reduced plaque burden (p<0.05). This was associated with an IL-1 mediated loss of endothelium-dependent relaxation and an increase in vessel wall Nox 4. Inhibition of IL-1 restored endothelium-dependent vasodilatation and reduced levels of arterial oxidative stress. Conclusions/Significance The IL-1 cytokine system links atherogenic environmental stimuli with arterial inflammation, oxidative stress, increased blood pressure and atherosclerosis. This is the first demonstration that inhibition of a single cytokine can block the rise in blood pressure in response to an environmental stimulus. IL-1 inhibition may have profound beneficial effects on atherogenesis in man.

Telemetry for Cardiovascular Monitoring in a Pharmacological Study: New Approaches to Data Analysis

Radio-telemetry systems offer the ability to measure blood pressure and heart rate in experimental models of hypertension without the stress artifacts induced by some other methods. We therefore aimed to develop improved, nonparametric regression methods for radio-telemetry data and to use these to assess the effects of pharmacological interventions on cardiac and vascular hypertrophy in the stroke-prone spontaneously hypertensive rat. One control group and 5 groups treated either with losartan (alone or in combination with NG-nitro-L-arginine methyl ester [ L-NAME]), perindopril (also alone or in combination with L-NAME), or hydralazine plus hydrochlorothiazide were monitored for 4 weeks. Cardiac hypertrophy was assessed by the left ventricle plus septum weight to body weight ratio and vascular hypertrophy by flow-cytometry analysis of vascular smooth muscle cell polyploidy. Hemodynamic series were split into trend and cyclic components by the seasonal and trend decomposition procedure based on Loess and compared between groups by Loess regression modeling. Systolic and diastolic blood pressures were reduced systematically by losartan and perindopril (P<10(-10)) but to a lesser extent by hydralazine plus hydrochlorothiazide (P<10(-8)), and diurnal variation was reduced in the latter group (P<10(-6)). L-NAME significantly reduced the hypotensive effect only of losartan. Vascular and cardiac hypertrophy were significantly attenuated with losartan or perindopril, but were unchanged with other treatments. The new analysis proposed here identifies differential effects on trends and cyclic variation and associations with regression of end-organ damage for losartan and perindopril compared with hydralazine plus hydrochlorothiazide. The method offers a powerful tool for detailed investigation of radio-telemetry data.

A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: The NIH heterogeneous stock

The laboratory rat (Rattus norvegicus) is a key tool for the study of medicine and pharmacology for human health. A large database of phenotypes for integrated fields such as cardiovascular, neuroscience, and exercise physiology exists in the literature. However, the molecular characterization of the genetic loci that give rise to variation in these traits has proven to be difficult. Here we show how one obstacle to progress, the fine-mapping of quantitative trait loci (QTL), can be overcome by using an outbred population of rats. By use of a genetically heterogeneous stock of rats, we map a locus contributing to variation in a fear-related measure (two-way active avoidance in the shuttle box) to a region on chromosome 5 containing nine genes. By establishing a protocol measuring multiple phenotypes including immunology, neuroinflammation, and hematology, as well as cardiovascular, metabolic, and behavioral traits, we establish the rat HS as a new resource for the fine-mapping of QTLs contributing to variation in complex traits of biomedical relevance.

Genetic and Gender Influences on Sensitivity to Focal Cerebral Ischemia in the Stroke-Prone Spontaneously Hypertensive Rat

We have investigated genetic transmission of increased sensitivity to focal cerebral ischemia and the influence of gender in the stroke-prone spontaneously hypertensive rat (SHRSP). Halothane-anesthetized, 3- to 5-month-old male and female Wistar-Kyoto rats (WKY), SHRSP, and the first filial generation rats (F1 crosses 1 and 2) underwent distal (2 mm) permanent middle cerebral artery occlusion (MCAO) by electrocoagulation. Infarct volume was measured by using hematoxylin-eosin-stained sections and image analysis 24 hours after ischemia and expressed as a percentage of the volume of the ipsilateral hemisphere. Infarct volume in males and females grouped together were significantly larger in SHRSP, F1 cross 1 (SHRSP father), and F1 cross 2 (WKY father), at 36.6+/-2.3% (mean+/-SEM, P<0.001, n=15), 25.4+/-2.4% (P<0.01, n=14), and 33. 9+/-1.6% (P<0.001, n=18), respectively, compared with WKY (14+/-2%, n=17). Male F1 cross 1 (18.9+/-2.4%, n=6) developed significantly smaller infarcts than male F1 cross 2 (32.8+/-2%, n=8, P<0.005). Females, which underwent ischemia during metestrus, developed larger infarcts than respective males. A group of females in which the cycle was not controlled for developed significantly smaller infarcts than females in metestrus. Thus, the increased sensitivity to MCAO in SHRSP is retained in both F1 cross 1 and cross 2 hybrids, suggesting a dominant or codominant trait; response to cerebral ischemia appears to be affected by gender and stage in the estrous cycle. In addition, the male progenitor of the cross (ie, SHRSP versus WKY) influences stroke sensitivity in male F1 cohorts.