The Val66Met brain-derived neurotrophic factor gene variant interacts with early pain exposure to predict cortisol dysregulation in 7-year-old children born very preterm: Implications for cognition.

Early stress in the form of repetitive neonatal pain, in infants born very preterm, is associated with long-term dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and with poorer cognitive performance. Brain-derived neurotrophic factor (BDNF) which is important in synaptic plasticity and cognitive functions is reduced by stress. Therefore the BDNF Val66Met variant, which affects secretion of BDNF, may interact with early exposure to pain-related stress in children born very preterm, to differentially affect HPA regulation that in turn may be associated with altered cognitive performance. The aims of this study were to investigate whether in children born very preterm, the BDNF Val66Met variant modulates the association between neonatal pain-related stress and cortisol levels at age 7years, and if cortisol levels were related to cognitive function. Furthermore, we examined whether these relationships were sex-specific. Using a longitudinal cohort design, N=90 children born very preterm (24-32weeks gestation) were followed from birth to age 7years. Cortisol was assayed from hair as an index of cumulative stress and from saliva to measure reactivity to a cognitive challenge. BDNF Val66Met variant was genotyped at 7years using real-time polymerase chain reaction (PCR). Using generalized linear modeling, in boys with the Met allele, greater neonatal pain-related stress (adjusted for clinical risk factors) predicted lower hair cortisol (p=0.006) and higher reactivity salivary cortisol (p=0.002). In both boys and girls with the Met allele, higher salivary cortisol reactivity was correlated with lower IQ (r=-0.60; p=0.001) and poorer visual-motor integration (r=-0.48; p=0.008). Our findings show associations between lower BDNF availability (presence of the Met allele) and vulnerability to neonatal pain/stress in boys, but not girls. This exploratory study suggests new directions for research into possible mechanisms underlying how neonatal pain/stress is related to cognitive performance in children born very preterm.

Interaction between the Val158Met catechol-O-methyltransferase gene variant and second-generation antipsychotic treatment on blood pressure in children.

Second-generation antipsychotic (SGA) medications are associated with cardiometabolic risk factors such as obesity and elevated blood pressure (BP) in some individuals. The goal of this study is to determine whether the Val158Met variant (rs4680) in the catechol-O-methyltransferase (COMT) gene, associated with BP in adults, is associated with elevated BP in SGA-treated children. A cross-sectional population of SGA-treated (n=134) and SGA-naive (n=168) children, ⩽18 years of age, were genotyped and assessed for markers of cardiometabolic health. An interaction was found between SGA treatment and COMT genotype for BP. After adjusting for covariates, SGA-treated children with the Met allele had higher systolic and diastolic BP (P=0.014 and P=0.034, respectively), and higher fasting glucose concentrations (P=0.030) compared with children with the Val/Val genotype. This was not observed in SGA-naive children. The Met allele of the COMT Val158Met variant may identify SGA-treated children at risk for elevated BP and fasting blood glucose concentrations.

Antidepressant use during pregnancy and serotonin transporter genotype (SLC6A4) affect newborn serum reelin levels.

This study was undertaken to determine whether altered early serotonin signaling either via gestational serotonin reuptake inhibitor (SRI) exposure or genetic variations in the serotonin transporter promoter region (SLC6A4) alters levels of reelin, an important glycoprotein in neurodevelopment, in mothers and their neonates. Serum reelin protein expression was quantified by immunoblot from maternal and neonatal blood collected at delivery from women taking either an SRI during gestation or controls. SRI-exposed mothers had higher levels of one reelin fragment, while SRI-exposed neonates had lower total reelin levels, particularly in females and reelin levels differed with SLC6A4 genotype. Lower neonatal reelin levels predicted less time spent sleeping and more irritability during neonatal behavioral assessment on Day 6 of life. Our results suggest that prenatal SRI exposure and the SLC6A4 genotype influences reelin protein expression in both the mother and newborn and that this may be reflected in neonatal behavior.

Cardiometabolic risk and the MTHFR C677T variant in children treated with second-generation antipsychotics.

Second-generation antipsychotics (SGAs) are increasingly being used to treat children with a variety of psychiatric illnesses. Metabolic syndrome (MetS), a risk factor for cardiovascular disease, is a side-effect of SGA-treatment. We conducted a cross-sectional study and assessed the association of the methylenetetrahydrofolate reductase (MTHFR) C677T variant with features of MetS in SGA-treated (n=105) and SGA-naïve (n=112) children. We targeted the MTHFR C677T variant, because it is associated with risk for cardiovascular disease, and features of MetS in adults without psychiatric illness. MetS in children is based on the presence of any three of the following: waist circumference ≥ 90th percentile for age and sex; plasma triglyceride ≥ 1.24 mmol l(-1); plasma high-density lipoprotein-cholesterol ≤ 1.03 mmol l(-1); systolic or diastolic blood pressure ≥ 90th percentile for age, sex, and height; and fasting glucose ≥ 5.6 mmol l(-1). We found that 15% of SGA-treated children had MetS compared with 2% of SGA-naïve children (OR 8.113, P<0.05). No effect of the MTHFR C677T variant on psychiatric diagnosis was observed. The MTHFR 677T allele was associated (P<0.05) with MetS (OR 5.75, 95% CI= 1.18-28.12) in SGA-treated children. Models adjusted for duration of SGA treatment, ethnicity, sex, age and use of other medications revealed a positive relationship between the MTHFR 677T allele and diastolic blood pressure Z-scores (P=0.001) and fasting plasma glucose (P<0.05) in SGA-treated children. These findings illustrate the high prevalence of MetS in SGA-treated children and suggest metabolic alterations associated with the MTHFR C677T variant may have a role in the development of MetS features in SGA-treated children.

Cerebral edema associated with betaine treatment in classical homocystinuria.

A child with cystathionine beta-synthase deficiency developed cerebral edema 4 to 6 weeks after starting betaine therapy. There was no evidence of intracranial thrombosis, but there was widespread edema of the white matter. He recovered fully after emergency decompressive craniotomy and withdrawal of betaine.

Clinical outcomes of hemispherectomy for epilepsy in childhood and adolescence.

Hemispherectomy has been performed in the treatment of epilepsy in association with hemiplegia for over 50 years. However, the optimal timing of surgery with respect to age at presentation and the influence of underlying pathology on outcome is only slowly emerging. This study reports on the clinical course and outcomes of 33 children who underwent hemispherectomy at Great Ormond Street Hospital, London, between 1991 and 1997. Age at surgery was 0.33-17 years (median 4.25) with 1-8 years follow-up (median 3.4). The underlying pathology was developmental in 16 (10 hemimegalencephaly, two polymicrogyria, two focal cortical dysplasia, one diffuse cortical dysplasia and one microdysgenesis), acquired in 11 (six middle cerebral artery infarct, three post encephalitis/trauma, and one each of hemiconvulsion-hemiplegia epilepsy and perinatal ischaemic insult) and progressive in six children (four Rasmussen encephalitis, two Sturge-Weber syndrome). At follow-up, 52% were seizure free, 9% experienced rare seizures, 30% showed >75% reduction in seizures and 9% showed <75% seizure reduction or no improvement. Seizure freedom was highest in those with acquired pathology (82%), followed by those with progressive pathology (50%) and those with developmental pathology (31%). However, seizure freedom, rare seizures or >75% reduction in seizures occurred in 100% of those with progressive pathology, 91% of those with acquired and 88% of those with developmental pathology, indicating a worthwhile seizure outcome in all groups. Hemiplegia remained unchanged following surgery in 22 out of 33 children, improved in five and was worse in six. No significant cognitive deterioration or loss of language occurred, and four children showed significant cognitive improvement. Behavioural improvement was reported in 92% of those who had behaviour problems pre-operatively.

Is estradiol cardioprotection a nitric oxide-mediated effect?

BACKGROUND: Estradiol exerts a number of biological effects that support extensive observational data suggesting a protective role for estrogen in cardiovascular disease prevention. These include effects on lipid and carbohydrate metabolism, coagulation/fibrinolysis as well as a possible effect on vascular reactivity. It has been proposed that this might be mediated by vascular endothelial nitric oxide (NO) production. Accordingly, we designed complementary in-vivo and in-vitro studies to investigate this hypothesis further. METHODS: Firstly, in a group of 10 healthy post-menopausal women, bilateral venous occlusion plethysmography was used to examine forearm vasoconstrictor responses to intrabrachial N(G)-monomethyl-l-arginine (l-NMMA; a substrate inhibitor of nitric oxide synthase) both before and after 4 weeks of treatment with transdermal 17beta-estradiol (E(2)) (80 microg/day). Secondly, we examined the direct effects of acute (24 h) and chronic (7 days) treatment with E(2) (10 pmol/l and 10 nmol/l) on endothelial nitric oxide synthase (eNOS) gene expression in cultured human aortic endothelial cells. RESULTS: No significant differences were observed between the vasoconstrictor responses to l-NMMA (2, 4, 8 micromol/min) before and after E(2) treatment. Comparison of E(2)-treated endothelial cells with control cells showed no significant increase in eNOS mRNA expression following either acute or chronic estradiol treatment. CONCLUSIONS: The present studies do not provide evidence for an eNOS-mediated cardioprotective response to estrogen and therefore suggest that additional mechanisms other than the endothelial NO system may have an important role in the cardiovascular effects of estrogen.

Reduced folate carrier: tissue distribution and effects of chronic ethanol intake in the micropig.

BACKGROUND: Folate deficiency is common in alcoholic patients, in part due to abnormal transport across membranes relevant to folate homeostasis. The reduced folate carrier (RFC) transports monoglutamyl folates across tissue membranes and could be affected by chronic exposure to ethanol. The micropig model is suitable to study the effect of alcoholism on RFC and folate transport across membranes. METHODS: The membrane transport of [3H]-folic acid was measured by a vacuum filtration method in jejunal brush border (JBB), liver plasma membrane (LPM), and kidney brush border (KBB) membranes vesicles from micropigs fed control or 40% ethanol diets for 12 months. RFC transcripts were analyzed by reverse transcription polymerase chain reaction in jejunal mucosa, liver, and kidney from the same animals. RESULTS: When we compared results from three relevant membranes in control animals, the transport of [3H]-folic acid was highest in LPM, 3-fold lower in KBB (p < 0.001), and 6-fold lower in JBB (p < 0.001). The concentration of RFC transcripts per total RNA was greatest in liver, followed by kidney and jejunum. The transport of [3H]-folic acid by JBB vesicles from chronic ethanol-fed animals exhibited 2-fold lower Km and Vmax (p < 0.05), whereas there was no ethanol effect on the Vmax of [3H]-folic acid transport by LPM or KBB. RFC transcript levels were 10-fold lower in jejunal mucosa from ethanol-fed animals than in control-fed animals (p < 0.005). CONCLUSIONS: Although our findings demonstrate different RFC transcript amounts and transport efficiencies among tissues, the present studies suggest that chronic ethanol exposure decreases the intestinal absorption of folic acid by altering the expression of RFC and consequently its transport kinetics in JBB. These findings provide a mechanism for the clinical finding of reduced folic acid absorption in chronic alcoholics.

A simple method for assessing the regression or progression of ventricular hypertrophy in the growing child and adult: the value of left ventricular wall-to-cavity ratios.

AIMS: To establish the normal range of diastolic and systolic left ventricular wall-to-cavity ratios. METHODS AND RESULTS: Two hundred and sixty-two normal subjects (age 0-40 years), 15 children with valvar aortic stenosis and 11 childhood athletes were studied with M-mode echocardiography. Values of diastolic septum-to-cavity ratio and diastolic left ventricular wall-to-cavity ratio were not influenced by sex nor, in adults, by height, weight or body surface area. There were slight age variations from 0-15 years of age, but not in adults from 15-40 years of age. Values of diastolic left ventricular wall-to-cavity ratio in neonates were 0.18 (95% confidence limits 0.17-0.19); in 3-5 year olds 0.16 (0.15-0.16); and in adults 0.18 (0.17-0.19). In valvar aortic stenosis there is a positive correlation between the Doppler-estimated pressure gradient and the degree of left ventricular hypertrophy, as expressed by both diastolic and systolic left ventricular wall-to-cavity ratio (r = 0.90;P < 0.00001 and r = 0.85;P = 0.00006 respectively). CONCLUSIONS: Diastolic septum and left ventricular wall-to-cavity ratios accurately differentiate physiological from pathological left ventricular hypertrophy in the growing child. Because these ratios are independent of sex and body size, they may also be more sensitive than absolute wall thickness in the detection of abnormal left ventricular hypertrophy in adults.

Glutamate carboxypeptidase II: a polymorphism associated with lower levels of serum folate and hyperhomocysteinemia.

Low blood folate levels result in hyperhomocysteinemia, which has been associated with increased risk for cardiovascular disease, neural tube defects and cognitive deficits. Intake of dietary folates is the chief determinant of blood folate levels. Molecular defects in the intestinal absorption of dietary folates that precipitate low blood folate levels and hyperhomocysteinemia have not been investigated previously. Dietary folates are a mixture of polyglutamylated folates which are digested to monoglutamyl folates by the action of folylpoly-gamma-glutamate carboxypeptidase (FGCP), an enzyme that is anchored to the intestinal brush border membrane and is expressed by the glutamate carboxypepidase II (GCPII) gene. We cloned GCPII cDNA from human intestine and identified both a full-length transcript and a 93 bp shorter transcript lacking exon 18, consistent with the presence of a splice variant. In addition, we identified an H475Y polymorphism in GCPII in DNA samples from a healthy Caucasian population (n = 75). We found that membranes of transfected COS-7 cells expressing the H475Y variant GCPII cDNA had 53% less FGCP activity than did cells expressing wild-type GCPII. The presence of the H475Y GCPII allele was significantly associated with lower folate and higher homocysteine levels in this population. These data suggest that the presence of the H475Y GCPII allele impairs the intestinal absorption of dietary folates, resulting in relatively low blood folate levels and consequent hyperhomocysteinemia.

Diagnosis of hypertrophic cardiomyopathy and screening for the phenotype suggestive of gene carriage in familial disease: a simple echocardiographic procedure.

OBJECTIVES: To design a new echocardiographic method of screening for hypertrophic cardiomyopathy applicable to children and adults, with a low false positive rate in athletes. SETTING: Regional centre of cardiology, Oxford, UK. METHODS: Forty one patients with hypertrophic cardiomyopathy, 66 first degree relatives from families with familial hypertrophic cardiomyopathy, 262 normal subjects, and 32 athletes were studied by long axis M mode and cross sectional echocardiography to determine the frequency distribution of diastolic and systolic ratios of cardiac wall thickness to cavity diameter. RESULTS: The best screening measure for hypertrophic cardiomyopathy is diastolic septum to cavity ratio, where a value of > 0.26 yielded a 100% disease detection rate at all ages with 0% false positives in the ordinary population. In comparison, the conventional screening tool of diastolic septum to posterior left ventricular wall ratio of > 1.5 yielded a detection rate of only 75%, for a false positive rate of 2%. In first degree relatives, a septum to cavity ratio > 0.26 yielded a 100% detection rate for an abnormal phenotype suggestive of carriage of a mutation for hypertrophic cardiomyopathy with no obvious false positives. Conventional screening showed a detection rate of only 44%. Athletes with physiological cardiac hypertrophy showed only a 6% false positive rate with diastolic septum to cavity ratio, and could be differentiated from subjects with hypertrophic cardiomyopathy by the absence of hypercontractility shown by a normal systolic left ventricular wall to cavity ratio (cut off < 0.63; 0% false positives). CONCLUSIONS: M mode echocardiographic measurement of the septum to cavity ratio provides a good screening test for hypertrophic cardiomyopathy at all ages. Combining this measurement with systolic left ventricular wall to cavity ratio improves the accuracy further.

HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation.

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

DNA synthesis and apoptosis in smooth muscle cells from a model of genetic hypertension.

The present study was designed to assess vascular smooth muscle cell (VSMC) proliferation and apoptosis in primary cultured VSMCs prepared from the aortic tunica media of adult (4 to 5 months old) age- and gender-matched groups of stroke-prone spontaneously hypertensive rats (SHRSP) and the normotensive reference strain, Wistar-Kyoto (WKY) rats. In the present study, VSMC proliferation was assessed with measurement of DNA synthesis in response to stimulation of G(0)/G(1) arrested VSMCs with 10% serum, whereas apoptosis was measured in response to serum deprivation. Apoptosis in aortic VSMCs was assessed in vitro with the technique of Annexin V binding in combination with propidium iodide exclusion with bivariate flow cytometric analysis. The percentage of necrotic VSMCs in the cell populations was assessed simultaneously. The light-scattering properties of the cells were assessed to provide further information on cell shrinkage and chromatin condensation. Results of the present study have shown enhanced DNA synthesis in VSMCs from SHRSP (n=10; 5.2+/-0.9 cpmx10(3)/mg protein) compared with WKY (n=12; 2.4+/-0.7 cpmx10(3) /mg protein; P<0.05, 95% CI, -5271 to -296). In addition, the results of the present study have demonstrated the role of serum in the survival of VSMCs in vitro, because SHRSP VSMCs underwent significantly more apoptosis in response to insult by serum deprivation (n=13; 10.21+/-1.8%) than WKY VSMCs (n=7; 3.44+/-1.4%; P<0.01, 95% CI, -11.5 to -2.0). Thus, it appears that both proliferation and apoptosis are enhanced in synthetic phenotype aortic medial VSMCs from the SHRSP in vitro.

Increased Na(+)/H(+) exchanger isoform 1 activity in spontaneously hypertensive rats: lack of mutations within the coding region of NHE1.

Enhanced Na(+)/H(+) exchange, measured as amiloride derivative-sensitive Na(+) and H(+) fluxes in cells with a preliminary acidified cytoplasm (Deltamu(H+)-induced Na(+)/H(+) exchange), is one of the most prominent intermediate phenotypes of altered vascular smooth muscle cell (VSMC) function in spontaneously hypertensive rats (SHR). Analysis of Na(+)/H(+) exchange in F(2) hybrids of SHR and normotensive rats seems to be the most appropriate approach in the search for the genetic determinants of abnormal activity of this carrier. However, the measurement of Deltamu(H+)-induced Na(+)/H(+) exchange is hardly appropriate for precise analysis of the carrier's activity in VSMC derived from several hundred F(2) hybrids. To overcome this problem, we compared the rate of (22)Na influx under baseline conditions and in Na(+)-loaded (ouabain-treated) VSMC. The dose-dependency of the rate of Deltamu(H+)-induced H(+) efflux as well as of (22)Na influx in control and ouabain-treated cells on ethylisopropylamiloride (EIPA) concentration were not different (K(0.5) approximately 0.3 microM), suggesting that these ion transport pathways are mediated by the same carrier. EIPA-sensitive (22)Na influx in Na(+)-loaded cells was approximately 6-fold higher than in ouabain-untreated VSMC and was increased by 50-70% in two different substrains of SHR. About the same increment of EIPA-sensitive (22)Na influx in Na(+)-loaded VSMC was observed in 5- to 6-week-old SHR (an age at which hypertension has not yet developed) as well as in stroke-prone SHR (SHRSP) with severe hypertension, indicating that the heightened activity of Na(+)/H(+) exchange is not a consequence of long-term blood pressure elevation. To examine whether or not the augmented activity of Na(+)/H(+) exchange in SHR is caused by mutation of NHE1, i.e. the only isoform of this carrier expressed in VSMC, we undertook single-stranded conformational polymorphism analysis of 23 NHE1 cDNA fragments from SHR and SHRSP and sequencing of the 456-2421 NHE1 cDNA fragment. This study did not reveal any mutation in the entire coding region of NHE1. The lack of mutation in the coding region of NHE1 indicates that the augmented activity of the ubiquitous Na(+)/H(+) exchanger in primary hypertension is caused by altered regulation of carrier turnover number or/and its plasma membrane content.

Different effects of antihypertensive agents on cardiac and vascular hypertrophy in the transgenic rat line TGR(mRen2)27.

The hypertensive transgenic rat model TGR(mRen2)27 has been used to investigate the development of cardiac and vascular hypertrophy in response to two different drug regimes. Cardiac hypertrophy was shown to be related to age and gender with the copy number of mouse renin transgenes having an additive effect. A similar observation was noted for hypertrophy in the vasculature, which was assessed using flow cytometry cell cycle DNA analysis of aortic vascular smooth muscle cells. Chronic treatment from weaning with equihypotensive doses of perindopril (2 mg/kg/day) or hydralazine and hydrochlorothiazide (4 mg/day of each) prevented the development of cardiac hypertrophy. Perindopril treatment also effectively prevented the development of vascular hypertrophy; however, treatment with hydralazine and hydrochlorothiazide was not as effective despite equivalent blood pressure reduction. These studies have demonstrated the presence of marked vascular and cardiac hypertrophy in the hypertensive transgenic TGR(mRen2)27 model of hypertension. Furthermore, these results provide new evidence to support the role of a locally activated renin angiotensin system in the blood vessel wall, which is involved in the pathogenesis of vascular hypertrophy in this transgenic rat model.

A comparison of MRI and echocardiography in hypertrophic cardiomyopathy.

This study compares MRI and echocardiography as imaging modalities in hypertrophic cardiomyopathy, with particular reference to measurement of left ventricular wall thickness and mass. 10 subjects underwent echocardiography and MRI. Contiguous 10 mm short axis 35 degrees flip angle cine gradient recalled echo MR images were acquired from the apex to the base of the left ventricle at 1.5 tesla. Standard M-mode and cross-sectional echocardiographic views of the left ventricle were obtained. Excellent agreement between measurements occurred with MRI and M-mode echocardiographic assessment of the thickness of the anterior interventricular septum (95% limits of agreement -1.5 to +1.5 mm). Other comparisons of MRI vs M-mode echocardiographic measurements had the following limits of agreement: posterior free wall -3.3 to +2.9 mm; end-diastolic dimension -5 to +8 mm, left ventricular mass -291 to +55.5 g. Comparing MRI with cross-sectional echocardiographic measurements, the limits of agreement were: anterior interventricular septum -2.4 to +1.7 mm, posterior interventricular septum -2.4 to +2.9 mm, posterior free wall -3.4 to +2.5 mm, anterior free wall -2.4 to +1.7 mm, end-diastolic dimension -4.1 to +8 mm. MRI estimates of LVM in systole vs diastole showed good agreement with 95% limits of agreement of -20 to +17 g, with excellent interobserver variability in diastole (-9 to +5 g) and in systole (-7 to +12 g). In conclusion, MRI is superior to echocardiography for the quantification of ventricular mass in the abnormal left ventricle because it does not make invalid geometrical assumptions. Comparisons of wall thickness show greater discrepancy with increasing distance from the echocardiographic transducer. This study suggests that sequential echocardiography could rationalize the need for MRI in left ventricular hypertrophy. A change in anterior septal thickness of > or = 3 mm on echocardiography merits a further MRI study.

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.

Dietary phospholipid alters biliary lipid composition in formula-fed piglets.

Plasma cholesterol, arachidonic acid (AA, 20:4n-6), and docosahexaenoic acid (DHA, 22:6n-3) are higher in breast-fed infants than in infants fed formula without cholesterol, AA, or DHA. This study investigated differences in plasma, hepatic, and bile lipids and phospholipid fatty acids, and expression of hepatic proteins involved in sterol metabolism that result from feeding formula with cholesterol with egg phospholipid to provide AA and DHA. For this study, three groups of piglets were evaluated: piglets fed formula with 0.65 mmol/L cholesterol, the same formula with 0.8% AA and 0.2% DHA from egg phospholipid, and piglets fed sow milk. Piglets fed the formula with phospholipid AA and DHA had higher plasma high density lipoprotein, but not apoprotein (apo) B cholesterol or triglyceride; higher bile acid and phospholipid concentrations in bile; and higher liver and bile phospholipid AA and DHA than piglets fed formula without AA and DHA (P < 0.05). Hydroxy methylglutaryl (HMG)-CoA reductase and 7-alpha-hydroxylase, the rate-limiting enzymes of cholesterol and bile acid synthesis, respectively, and low density lipoprotein receptor mRNA levels were not different between piglets fed formula without and with phospholipid AA and DHA, but HMG-CoA reductase and 7alpha-hydroxylase mRNA were higher, and plasma apo B containing lipoprotein cholesterol was lower in all piglets fed formula than in piglets fed milk. These studies show that supplementing formula with AA and DHA from egg phospholipid alters bile metabolism by increasing the bile AA and DHA, and bile acid and phospholipid.

Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model.

OBJECTIVE: We have shown previously that there is a relative nitric oxide deficiency at the level of vascular endothelium in the stroke-prone spontaneously hypertensive rat (SHRSP), a model of human essential hypertension, as compared to its normotensive reference strain Wistar Kyoto (WKY) rat. The aim of the current study was to investigate whether adenoviral-mediated gene transfer of an endothelial nitric oxide synthase (eNOS) cDNA (AdCMVeNOS) into carotid arteries of the SHRSP may improve endothelial function. METHODS: Enzyme activity of the recombinant eNOS protein encoded by AdCMVeNOS was tested using a Griess assay in endothelial cells in culture. Left carotid arteries of SHRSP were surgically isolated and exposed to either the AdCMVeNOS or control beta-galactosidase-containing virus, (2 x 10(9) pfu/ml) ex vivo and in vivo. The vessels were harvested 24 h after surgery and analysed by Western blotting, immunohistochemistry and by examining endothelial function ex vivo. RESULTS: Cultured endothelial cells showed almost 100% transduction with both viruses and a dose response of eNOS expression showed a five-fold increase in nitrite production for AdCMVeNOS with no change for beta-galactosidase-containing virus. Western blotting demonstrated a significant increase of eNOS expression in vessels infused with AdCMVeNOS when compared to controls. Immunohistochemistry showed highly positive staining with monoclonal antibodies against eNOS in the intact endothelial cells of the AdCMVeNOS infused vessels. The areas under the curve of the concentration responses to phenylephrine (10(-9) to 3 x 10(-6) M) in the absence and presence of NG-nitroarginine methyl ester (100 microM) showed increased basal nitric oxide bioavailability in the carotid arteries infused with AdCMVeNOS compared to the control (n = 6 for each; P = 0.0069; 95% CI, 0.864 to 3.277). CONCLUSIONS: Our results show that AdCMVeNOS is an effective tool for vascular gene transfer and that it can improve endothelial NO availability in the SHRSP, a genetic model of essential hypertension and endothelial dysfunction.