Elisabeth Beattie

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.

Persistent hypertension following inhibition of nitric oxide formation in the young Wistar rat: role of renin and vascular hypertrophy.

Objective: To determine whether induction of arterial hypertension in young normotensive Wistar rats by chronic inhibition of nitric oxide production with NG-nitro-L-arginine methyl ester (L-NAME) produced a form of self-sustained hypertension, and to investigate the role of the renin—angiotensin system and vascular hypertrophy in the hypertensive process. Methods: Three-week-old Wistar rats were given 100 or 40mg/kg per day L-NAME or 40mg/kg per day L-NAME plus 100mg/kg per day captopril in their drinking water for between 4 and 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography both during treatment and after the treatment had been stopped. The effect of treatment on plasma renin was measured and the effect of treatment on mesenteric resistance artery structure was determined using a small-vessel myograph. Results: L-NAME produced a progressive and marked increase in blood pressure during the period of treatment. Hypertension was sustained for 14 weeks after stopping treatment. L-NAME resulted in a fourfold increase in plasma renin which remained elevated after treatment was stopped. Blood pressure was correlated with plasma renin levels. Treatment with L-NAME plus captopril markedly attenuated the rise in blood pressure and captopril also produced a marked fall in blood pressure in rats that developed persistent hypertension. Rats with self-sustained hypertension exhibited both cardiac and mesenteric resistance vessel hypertrophy. The induction of vascular hypertrophy with low-dose L-NAME did not result in the development of self-sustained hypertension. Conclusions: Chronic L-NAME treatment in young rats can produce a form of persistent hypertension which is renin-dependent and which does not seem to involve a vascular amplifier mechanism.

Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP

OBJECTIVE Previous studies from our group have shown a deficit in nitric oxide (NO) bioavailability and an excess production of the superoxide anion (O(2)(-)) in the stroke prone spontaneously hypertensive rat (SHRSP) compared to the normotensive Wistar Kyoto (WKY) strain. This present study has investigated whether adenoviral-mediated gene transfer of human eNOS or Cu/ZnSOD can alter the NO/O(2)(-) balance, thereby improving endothelial function. METHODS A recombinant adenovirus, Ad/Hu/eNOS, containing the human eNOS cDNA fragment was generated by homologous recombination in 293 cells. Ad/Hu/eNOS or Ad/Cu/ZnSOD was delivered into SHRSP carotid arteries in vivo, using a titre of 2x10(9)-2x10(10) plaque forming units (pfu)/ml, and the effect on gene expression was observed 24 h later. RESULTS Western blotting confirmed increased enzyme levels of eNOS and Cu/ZnSOD in the viral-infused vessels. Ex vivo, the pressor response to phenylephrine (PE) in the presence of L-NAME was increased in the eNOS-infused arteries relative to the contralateral controls, indicating restoration of basal NO availability to that observed in untreated control WKY rats. Infusion of the SOD virus produced a statistically insignificant increase in NO bioavailability. CONCLUSIONS Our results support our previous findings obtained using a bovine eNOS recombinant adenovirus, that recombinant adenoviral gene transfer of human eNOS has a significant effect on NO bioavailability. In contrast, AdCu/ZnSOD gene transfer does not elicit an effect in our model. These results indicate that short-term overexpression of a recombinant eNOS, but not Cu/ZnSOD gene, in carotid arteries of the SHRSP is an effective means of locally increasing NO bioavailability to improve endothelial function.

Candidate Genes That Determine Response to Salt in the Stroke-Prone Spontaneously Hypertensive Rat : Congenic Analysis

The existence of blood pressure quantitative trait loci exaggerated by salt on rat chromosome 2 has been confirmed previously using congenic strains derived from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. This study aimed to dissect the implicated chromosome 2 region and to identify candidate genes based on microarray expression profiling and real-time PCR. A marker-assisted breeding strategy generated congenic strains SP.WKYGla2a (D2Rat13-D2Rat157), SP.WKYGla2c* (D2Wox9-D2Mgh12), and SP.WKYGla2k (D2Mit21-D2Rat157) using SHRSP as the recipient and WKY as the donor strain. The SP.WKYGla2k strain contains a 10-cM congenic interval, which is encompassed within the larger (64-cM) SP.WKYGla2a congenic region. Salt-loaded systolic blood pressure, measured by radiotelemetry, was significantly lower in the SP.WKYGla2a and SP.WKYGla2k strains compared with SHRSP. Salt sensitivity in SP.WKYGla2c* was not significantly different from SHRSP. Exclusion mapping identified a 6-Mbp region harboring genes responsible for salt-sensitive blood pressure regulation. Microarray expression profiling was carried out in whole homogenized kidneys from parental and SP.WKYGla2a strains. Examination of expression data within the minimal congenic interval identified the positional candidates Edg1 and Vcam1, demonstrating significantly elevated renal RNA expression levels in the SHRSP compared with WKY and SP.WKYGla2a congenic strains. These results were confirmed by quantitative real-time PCR. DNA sequencing identified SNPs in both Edg1 and Vcam1 between SHRSP and WKY rats. In conclusion, we have identified a suggestive minimal interval encompassing a 6-Mbp region on rat chromosome 2. This region contains several physiological candidate genes for salt-sensitive hypertension in the SHRSP, including Edg1 and Vcam1, which are differentially expressed and lie on common and functionally important pathways.

Combined therapeutic benefit of mitochondria-targeted antioxidant, MitoQ10, and angiotensin receptor blocker, losartan, on cardiovascular function

Objective: Mitochondria-derived reactive oxygen species (ROS) play important roles in the development of cardiovascular disease highlighting the need for novel targeted therapies. This study assessed the potential therapeutic benefit of combining the mitochondria-specific antioxidant, MitoQ10, with the low-dose angiotensin receptor blocker (ARB), losartan, on attenuation of hypertension and left ventricular hypertrophy. In parallel, we investigated the impact of MitoQ10 on cardiac hypertrophy in a neonatal cardiomyocyte cell line. Methods and results: Eight-week-old male stroke-prone spontaneously hypertensive rats (SHRSPs, n = 8–11) were treated with low-dose losartan (2.5 mg/kg per day); MitoQ10 (500 &mgr;mol/l); a combination of MitoQ10 and losartan (M + L); or vehicle for 8 weeks. Systolic pressure and pulse pressure were significantly lower in M + L rats (167.1 ± 2.9 mmHg; 50.2 ± 2.05 mmHg) than in untreated SHRSP (206.6 ± 9 mmHg, P < 0.001; 63.7 ± 2.7 mmHg, P = 0.001) and demonstrated greater improvement than MitoQ10 or low-dose losartan alone, as measured by radiotelemetry. Left ventricular mass index was significantly reduced from 22.8 ± 0.74 to 20.1 ± 0.61 mg/mm in the combination group (P < 0.05). Picrosirius red staining showed significantly reduced cardiac fibrosis in M + L rats (0.82 ± 0.22 A.U.) compared with control (5.94 ± 1.35 A.U., P < 0.01). In H9c2 neonatal rat cardiomyocytes, MitoQ10 significantly inhibited angiotensin II mediated hypertrophy in a dose-dependent manner (500 nmol/l MitoQ10 153.7 ± 3.1 microns vs. angiotensin II 200.1 ± 3.6 microns, P < 0.001). Conclusion: Combining MitoQ10 and low-dose losartan provides additive therapeutic benefit, significantly attenuating development of hypertension and reducing left ventricular hypertrophy. In addition, MitoQ10 mediates a direct antihypertrophic effect on rat cardiomyocytes in vitro. MitoQ10 has potential as a novel therapeutic intervention in conjunction with current antihypertensive drugs.

Hormone and electrolyte changes in post-deoxycorticosterone salt hypertension in rats

Male Sprague-Dawley rats were uninephrectomized and given either deoxycorticosterone (DOC) pivalate (12.5 mg three times weekly) and 1% NaCl/0.2% KCl to drink for 4 weeks (DOC-treated), after which DOC was stopped and tap water substituted (post-DOC), or tap water to drink throughout (controls), DOC treatment increased blood pressure, serum sodium, plasma atrial natriuretic peptide (P-ANP) and plasma deoxycorticosterone (P-DOC) (P less than 0.05), while serum potassium, plasma renin and plasma angiotensin II were lower (P less than 0.05) than in control animals. Plasma vasopressin (P-AVP) was also raised but not significantly. These changes persisted for up to 4 weeks post-DOC and, in the case of plasma renin, plasma angiotensin II, P-AVP and P-ANP, for up to 12 weeks. Total body sodium was also increased at 2 weeks post-DOC (P less than 0.05). Rats which were adrenalectomized after 4 weeks of DOC treatment in which DOC injections were stopped, then drank either NaCl/KCl or tap water; blood pressure and P-DOC remained elevated while plasma renin remained suppressed. There were more deaths in rats given NaCl/KCl (five of six) than in the group given water (one of six). Rats treated with a subcutaneous DOC silastic implant had a comparable rise in blood pressure to rats given DOC injections. However, after removal of the implant, while blood pressure remained elevated, P-DOC levels were not raised and plasma renin rose to control levels after 4 weeks. These findings indicate that, in rats given DOC injections, post-DOC hypertension results from sodium and fluid retention as a consequence of chronic hangover of exogenously administered DOC.

Interaction Between Chromosome 2 and 3 Regulates Pulse Pressure in the Stroke-Prone Spontaneously Hypertensive Rat

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.WKYGla3a) and bicongenic (SP.WKYGla2a/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.WKYGla2a). Our results demonstrate significant equivalent reduction in systolic, diastolic, and pulse pressure phenotypes in SP.WKYGla3a and SP.WKYGla2a rats, whereas greater reductions were observed with the SP.WKYGla2a/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.

Role of Tumor Necrosis Factor-α and Natural Killer Cells in Uterine Artery Function and Pregnancy Outcome in the Stroke-Prone Spontaneously Hypertensive Rat

Women with chronic hypertension are at increased risk of maternal and fetal morbidity and mortality. We have previously characterized the stroke-prone spontaneously hypertensive rat (SHRSP) as a model of deficient uterine artery function and adverse pregnancy outcome compared with the control Wistar–Kyoto. The activation of the immune system plays a role in hypertension and adverse pregnancy outcome. Therefore, we investigated the role of tumor necrosis factor-&agr; in the SHRSP phenotype in an intervention study using etanercept (0.8 mg/kg SC) at gestational days 0, 6, 12, and 18 in pregnant SHRSP compared with vehicle-treated controls (n=6). Etanercept treatment significantly lowered systolic blood pressure after gestational day 12 and increased litter size in SHRSP. At gestational day 18, etanercept improved the function of uterine arteries from pregnant SHRSP normalizing the contractile response and increasing endothelium-dependent relaxation, resulting in increased pregnancy-dependent diastolic blood flow in the uterine arteries. We identified that the source of excess tumor necrosis factor-&agr; in the SHRSP was a pregnancy-dependent increase in peripheral and placental CD3– CD161+ natural killer cells. Etanercept treatment also had effects on placental CD161+ cells by reducing the expression of CD161 receptor, which was associated with a decrease in cytotoxic granzyme B expression. Etanercept treatment improves maternal blood pressure, pregnancy outcome, and uterine artery function in SHRSP by antagonizing signaling from excess tumor necrosis factor-&agr; production and the reduction of granzyme B expression in CD161+ natural killer cells in SHRSP.

Abnormal uterine artery remodelling in the stroke prone spontaneously hypertensive rat

Introduction The stroke prone spontaneously hypertensive rat (SHRSP) is an established model of human cardiovascular risk. We sought to characterise the uteroplacental vascular response to pregnancy in this model and determine whether this is affected by the pre-existing maternal hypertension. Methods Doppler ultrasound and myography were utilised to assess uterine artery functional and structural changes pre-pregnancy and at gestational day 18 in SHRSP (untreated and nifedipine treated) and in the normotensive Wistar-Kyoto (WKY) rat. Maternal adaptations to pregnancy were also assessed along with histology and expression of genes involved in oxidative stress in the placenta. Results SHRSP uterine arteries had a pulsatile blood flow and were significantly smaller (70906 ± 3903 μm2 vs. 95656 ± 8524 μm2 cross-sectional area; p < 0.01), had a significant increase in contractile response (57.3 ± 10.5 kPa vs 27.7 ± 1.9 kPa; p < 0.01) and exhibited impaired endothelium-dependent vasorelaxation (58.0 ± 5.9% vs 13.9 ± 4.6%; p < 0.01) compared to WKY. Despite significant blood pressure lowering, nifedipine did not improve uterine artery remodelling, function or blood flow in SHRSP. Maternal plasma sFLT-1/PlGF ratio (5.3 ± 0.3 vs 4.6 ± 0.1; p < 0.01) and the urinary albumin/creatinine ratio (1.9 ± 0.2 vs 0.6 ± 0.1; p < 0.01) was increased in SHRSP vs WKY. The SHRSP placenta had a significant reduction in glycogen cell content and an increase in Hif1α, Sod1 and Vegf. Discussion We conclude that the SHRSP exhibits a number of promising characteristics as a model of spontaneous deficient uteroplacental remodelling that adversely affect pregnancy outcome, independent of pre-existing hypertension.