bluuebunny/crossref_metadata_2025 huggingface dataset:
Homocysteine, folate, methylene tetrahydrofolate reductase genotype and vascular morbidity in diabetic subjects
Authors: KAYE Joey M. , STANTON Kim G. , MCCANN Vincent J. , VASIKARAN Samuel D. , BURKE Valerie , TAYLOR Roger R. , VAN BOCKXMEER Frank M.
Published: August 2015

In the present study, the determinants of fasting plasma homocysteine in diabetic subjects were examined; whether plasma homocysteine and vascular disease are related and the influence of the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene on serum and erythrocyte folate, plasma homocysteine and vascular disease. Diabetic clinic subjects (Type I, n = 354; Type II, n = 392) were recalled for a cross-sectional survey. Standard methods were used to measure biochemical variables and to characterize vascular disease and MTHFR genotype. Plasma homocysteine was significantly and directly related to age, male sex and serum urea, and inversely related to serum folate and vitamin B12, independently in stepwise regression. When corrected for age and sex, homocysteine was significantly related to hard end points of coronary artery disease and stroke (each P<0.01), remaining significant when additionally adjusted for serum folate (P = 0.043 and P = 0.019 respectively). Serum folate was not clearly related to these events, although there was a trend to associate with the lower quintile of serum folate. The MTHFR genotype was not a determinant of plasma homocysteine, even in those in the lowest quintile of serum folate, nor of vascular disease. TT homozygosity at residue 677 was associated with elevation of total erythrocyte folate compared with both other genotypes (P<0.0001), almost certainly due to the diversion of 5,10-methylenetetrahydrofolate into derivates subsequent to the partial metabolic block that results from the MTHFR enzyme defect. In conclusion, in this clinic cohort of people with diabetes, vascular disease is related to plasma homocysteine, which is correlated with serum folate. The MTHFR genotype does not significantly influence either plasma homocysteine or vascular disease, despite it being a determinant of erythrocyte folate, which reflects its effect on folate metabolism.
Serum from healthy pregnant women reduces oxidative stress in human umbilical vein endothelial cells
Authors: SCALERA Fortunato , FISCHER Tina , SCHLEMBACH Dietmar , BEINDER Ernst
Published: August 2015

This study was conducted to compare the effects of serum from healthy pregnant women and that from pregnant women with pre-eclampsia on oxidative stress in endothelial cells in culture. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from 18 pre-eclamptic, 18 healthy pregnant and 18 healthy non-pregnant women for 24h. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs) were measured in endothelial cell lysates. Measurement of malondialdehyde in combination with 4-hydroxyalkenals has been used as an indicator of LPOs. Serum from healthy pregnant women decreased significantly the LPO content in HUVECs in comparison with serum from pre-eclamptic women and healthy non-pregnant women (30.7±6.6 compared with 39.3±10.9 and 41.0±12.7pmol/mg of protein respectively; P<0.003 and P<0.01 respectively). No differences in GSH content between the three groups (18.3±2.1nmol/mg of protein for healthy pregnant, 19.2±3.3nmol/mg for pre-eclamptic and 18.3±2.0nmol/mg for healthy non-pregnant women) were found. Thus serum from normal pregnant women contains a factor(s) that decreases oxidative stress in human endothelial cells. This mechanism might be altered in pre-eclampsia.
Differential mechanisms of endothelium-dependent vasodilator responses in human myometrial small arteries in normal pregnancy and pre-eclampsia
Authors: KENNY Louise C. , BAKER Philip N. , KENDALL David A. , RANDALL Michael D. , DUNN William R.
Published: August 2015

Pre-eclampsia is a pregnancy-specific disorder associated with hypertension and proteinuria, characterized by alterations in endothelial cell function. In the present study we have compared responses to the endothelium-dependent vasodilator, bradykinin, in small myometrial arteries from normal pregnant and non-pregnant women and women with pre-eclampsia, in order to assess the relative contributions of nitric oxide, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in mediating endothelium-dependent vasodilatation. Bradykinin-induced concentration-dependent relaxation in arteries isolated from the three subject groups did not differ with regard to sensitivity or maximum response. Responses to bradykinin in all three groups were unaffected by cyclo-oxygenase inhibition alone, and were similarly unaffected by partial depolarization. The nitric oxide synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester, significantly attenuated the responses to bradykinin in arteries from non-pregnant women and almost abolished responses in arteries from women with pre-eclampsia. However, in arteries from normal pregnant women, bradykinin-induced responses were maintained in the presence of NOS inhibition. Inhibition of NOS combined with partial depolarization abolished responses to bradykinin in these vessels. These results support the suggestion that, in the absence of NO, an EDHF can mediate vasodilator responses to bradykinin during normal pregnancy, an effect not apparent in arteries from non-pregnant women or women with pre-eclampsia. The up-regulation of EDHF-type function may represent a vascular adaptation to normal pregnancy that is absent in pre-eclampsia, and this might contribute to the clinical features of the disease.
Peripheral vascular disease is associated with abnormal arteriolar diameter relationships at bifurcations in the human retina
Authors: CHAPMAN N. , DELL'OMO G. , SARTINI M.S. , WITT N. , HUGHES A. , THOM S. , PEDRINELLI R.
Published: August 2015

Arterial diameters at branch points are believed to conform to design principles that optimize circulatory efficiency and maintain constant shear stress across arterial networks. The objective of this study was to examine whether optimality at bifurcations is affected in individuals with atherosclerosis. Retinal images were analysed in normotensive men with abnormal ankle brachial index (n = 13) and healthy controls (n = 8), matched for age and clinic blood pressure. Compared with controls, men with peripheral vascular disease had adverse metabolic profiles (relative insulin resistance and greater total cholesterol levels). In healthy men, retinal arterial diameters at bifurcations conformed to predicted optimal values but in men with peripheral vascular disease, junction exponents deviated significantly from the optimum. Retinal arteriolar bifurcation angles did not differ significantly between the groups. Atherosclerosis is associated with abnormalities in the arteriolar network of the retina. In view of the importance of the endothelium in maintaining network co-ordination of branch diameters this is suggestive of a generalized abnormality of endothelial function in atherosclerosis.
Correlation of the adipocyte-derived protein adiponectin with insulin resistance index and serum high-density lipoprotein-cholesterol, independent of body mass index, in the Japanese population
Authors: YAMAMOTO Yukihiro , HIROSE Hiroshi , SAITO Ikuo , TOMITA Motowo , TANIYAMA Matsuo , MATSUBARA Koichi , OKAZAKI Yasunori , ISHII Tatsuya , NISHIKAI Kanako , SARUTA Takao
Published: August 2015

Adiponectin, which is secreted specifically by adipose tissue, has been shown to act as an anti-atherosclerotic protein by direct effects on endothelial cells. Clinical studies have shown that adiponectin levels are lower in individuals with obesity, diabetes and coronary artery disease. The present study investigated relationships between serum adiponectin levels and body mass index (BMI), blood pressure, insulin resistance index, lipid profile, uric acid and high-sensitivity C-reactive protein levels in a large number of Japanese subjects not taking any medication for metabolic disease and without severe illness (705 men and 262 women; age 30–65 years; BMI 22.5±2.9kg/m2). The serum adiponectin concentration was measured by ELISA, without a protein-denaturing step. The insulin resistance index was assessed by homoeostasis model assessment (HOMA-IR). The serum concentration of adiponectin in women (13.5±7.9μg/ml) was significantly higher than that in men (7.2±4.6μg/ml). The serum adiponectin level was negatively correlated with BMI, systolic blood pressure, diastolic blood pressure, fasting plasma glucose, insulin, HOMA-IR, total cholesterol, triacylglycerols, low-density lipoprotein (LDL)-cholesterol and uric acid, and positively correlated with high-density lipoprotein (HDL)-cholesterol. The correlations between serum adiponectin level and insulin, HOMA-IR, triacylglycerols, HDL-cholesterol, LDL-cholesterol and uric acid were significant even after adjustment for age, sex and BMI. Stepwise multiple regression analysis revealed that HDL-cholesterol, sex, BMI and HOMA-IR were independently correlated with the serum adiponectin level (R2 = 0.377). These findings suggest that the serum adiponectin level is negatively correlated with HOMA-IR and positively correlated with HDL-cholesterol, independent of age, sex and BMI, in the Japanese population.
Long-term angiotensin-converting enzyme inhibition with ramipril reduces thrombin generation in human hypertension
Authors: EKHOLM Mikael , WALLÉN N. Håkan , JOHNSSON Hans , ELIASSON Keith , KAHAN Thomas
Published: August 2015

Antihypertensive treatment reduces the risk of thromboembolic events in hypertension. The aim of this study was to examine the influence of angiotensin-converting enzyme inhibition on blood coagulation in subjects with mild-to-moderate essential hypertension. Fibrinogen, thrombin–antithrombin complex (TAT) and Factor VII were determined in plasma at rest and after a mental stress test following placebo for 6 weeks, or ramipril for 6 weeks or 6 months. Ramipril reduced resting TAT, and tended to reduce fibrinogen; Factor VII remained unchanged. Mental stress increased fibrinogen, but did not alter TAT or Factor VII activity. The reduced thrombin generation in patients taking ramipril may explain in part why angiotensin-converting enzyme inhibitors reduce thromboembolic complications in patients with cardiovascular disease.
Genetic contribution to renal function and electrolyte balance: a twin study
Authors: HUNTER David J. , DE LANGE Marlies , SNIEDER Harold , MAC GREGOR Alex J. , SWAMINATHAN R. , THAKKER Rajesh V. , SPECTOR Tim D.
Published: August 2015

A classical twin study was performed to assess the relative contributions of genetic and environmental factors to serum levels of calcium, phosphate and magnesium, urinary levels of calcium, sodium and potassium, and creatinine clearance. The subjects were 1747 adult female twin pairs: 539 monozygotic and 1208 dizygotic. The intraclass correlations were calculated, and maximum-likelihood model fitting was used to estimate genetic and environmental variance components. The intraclass correlations for all of the variables assessed were higher in monozygotic twin pairs. The heritabilities (with 95% confidence intervals) obtained from model fitting were: serum calcium, 33% (21–45%); serum phosphate, 58% (53–62%), serum magnesium, 27% (15–39%); 24h urinary potassium, 40% (27–51%); 24h urinary calcium, 52% (41–61%); 24h urinary sodium, 43% (30–54%); fractional excretion of sodium, 52% (44–59%); serum creatinine, 37% (25–49); calculated creatinine clearance, 63% (54–72%). This study provides evidence for the importance of genetic factors in determining urinary and blood levels of the major electrolytes involved in blood pressure regulation. Identifying heritability is the first step on the way to finding specific genes, which may improve our insight into the pathophysiology of the metabolism of these electrolytes, and thereby improve our understanding of the aetiology of complex diseases such as renal failure and hypertension.
Altered cutaneous microvascular responses to reactive hyperaemia in coronary artery disease: a comparative study with conduit vessel responses
Authors: SHAMIM-UZZAMAN Qurratul Aine , PFENNINGER Dana , KEHRER Christine , CHAKRABARTI Anjan , KACIROTTI Nico , RUBENFIRE Melvyn , BROOK Robert , RAJAGOPALAN Sanjay
Published: August 2015

Cutaneous microvascular responses to physiological stimuli are currently being investigated as indices of vascular function and to monitor responses to therapy. We attempted to systematically assess various microvascular cutaneous flow indices in response to reactive hyperaemia in control subjects and in patients with coronary artery disease (CAD), and to correlate these with brachial artery flow-mediated dilation (FMD). Groups of 24 healthy controls and 24 subjects with CAD underwent sequential brachial FMD determination in the dominant arm, and laser Doppler imaging to assess skin blood flow in the contralateral arm in response to reactive hyperaemia induced by cuff inflation and release. Laser Doppler values were expressed as: (a) AUC5min (area under the curve over 5min of release), (b) time to peak response, (c) % reactive hyperaemia, and (d) peak perfusion ratio. As expected, FMD was attenuated in CAD patients compared with controls (1.85±4.29% compared with 4.30±4.00%; P = 0.05). Percentage reactive hyperaemia (CAD, 294±290%; controls, 501±344%; P = 0.04) and the time to peak response as measured by laser Doppler imaging (CAD, 16.84±9.61s; controls, 9.13±4.43s; P = 0.001) were significantly different between the CAD and control groups, while AUC5min and the peak perfusion ratio did not show significant differences. Receiver operator curves for sensitivity/specificity analysis suggested that the time to peak response derived by laser Doppler imaging was superior to FMD for the diagnosis of CAD, with an overall specificity of 91.3% (positive predictive value of 89.4%) and a sensitivity of 73.7% (negative predictive value of 77.6%). In conclusion, laser Doppler-derived indices of microvascular flow do not correlate with conduit vessel responses. However, a time to peak response of >10s as measured by laser Doppler imaging offers superior specificity for the diagnosis of CAD compared with brachial FMD.
The Trp64Arg β3-adrenergic receptor amino acid variant confers increased sensitivity to the pressor effects of noradrenaline in Sardinian subjects
Authors: MELIS Maria G. , SECCHI Giannina , BRIZZI Patrizia , SEVERINO Cristiana , MAIOLI Mario , TONOLO Giancarlo
Published: August 2015

The β3-adrenergic receptor (β3AR) plays a critical role in lipid metabolism, and thus alterations in its function may be involved in the metabolic syndrome. Indeed, we have found previously that the Trp64Arg amino acid variant of the β3AR is associated with hypertension and higher serum triacylglycerol levels in the Sardinian population. The aim of the present study was to evaluate the effect of the Trp64Arg β3AR variant on the regulation of triacylglycerol levels and blood pressure during the exogenous infusion of noradrenaline. We studied groups of non-diabetic normotensive subjects: eight with the wild-type Trp64Trp β3AR and eight with the Trp64Arg variant. The subjects each received, on two different days (randomized, double-blind fashion), a 4h infusion of either noradrenaline (0.147nmol·min-1·kg-1) or Emagel (subjects had fasted for at least 12h). The only available subject with a homozygous mutant Arg64Arg β3AR was also studied. Blood pressure was measured every 10min using a sphygmomanometer, and blood samples were taken every 30min from the contralateral vein for biochemical determinations. After a 4h noradrenaline infusion, significant increases in diastolic blood pressure (from 83±2 to 91±3mmHg; P<0.01) and serum triacylglycerol levels (from 1.69±0.4 to 1.79±0.6mmol/l; P<0.05) were observed compared with basal values in subjects with the Trp64Arg β3AR variant, whereas subjects with the Trp64Trp β3AR did not show any significant change over the infusion period. Glycaemia had increased significantly only at the end of the first 1h of infusion in subjects with the Trp64Arg variant (from 5.0±0.1 to 5.8±0.3mmol/l; P<0.05), with no significantly different behaviour compared with those subjects with the Trp64Trp β3AR during the remaining infusion period. The effects of noradrenaline infusion were more marked in the subject with the Arg64Arg variant. In conclusion, our data indicate that the Trp64Arg amino acid variant of the β3AR confers increased sensitivity to the pressure effect of noradrenaline. Moreover, this variant also influences blood triacylglycerol levels and, to a degree, glucose metabolism.
Cystic fibrosis transmembrane conductance regulator (CFTR) activity in nasal epithelial cells from cystic fibrosis patients with severe genotypes
Authors: ANDERSSON C. , DRAGOMIR A. , HJELTE L. , ROOMANS G.M.
Published: August 2015

Cystic fibrosis is a heterogenic disease, in which the phenotype can also vary for patients with the same genotype. In the present study the function of the cystic fibrosis transmembrane conductance regulator (CFTR) in nasal epithelial cells from 19 adult patients with cystic fibrosis was investigated. All patients had severe mutations, whereby no or little functional CFTR is expected in the plasma membrane. Of the patients, 15 were homozygous for ΔF508-CFTR (i.e. CTFR lacking residue Phe-508). The others were ΔF508-heterozygous with 3659delC, 394delTT or 2183AA→G. Nasal epithelial cells, obtained by nasal brushings, were loaded with the fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide to measure Cl- efflux. In most of the cystic fibrosis patients, forskolin plus isobutylmethylxanthine was unable to elicit any response. Unexpectedly, cells from three cystic fibrosis patients (two ΔF508/ΔF508 patients and one ΔF508/3659delC patient) responded to stimulation in a wild-type manner. It was investigated whether this residual chloride transport function was associated with a milder phenotype. Clinical parameters studied were lung function, number of antibiotic courses, Shwachman score, Bhalla score, age at chronic colonization with Pseudomonas aeruginosa and the pattern of essential fatty acids in serum phospholipids. Unknown factors may affect the presence of functional CFTR in patients with severe CFTR mutations. However, we could not find a correlation between the response to cAMP and any of the phenotype parameters. It appears that functional cAMP transport in the nasal epithelium is no guarantee of a mild phenotype and, conversely, that a patient lacking cAMP-dependent chloride transport can develop a mild phenotype.
Serum levels of androgens and migraine in postmenopausal women
Authors: MATTSSON Peter
Published: August 2015

Female hormones are thought to be of importance in the aetiology of migraine, which is more common in women than in men. Little attention has been paid to androgens. This study investigates the associations between migraine and serum levels of androgens in postmenopausal women not taking oestrogens. A case-control study was carried out among women participating in a mammography screening program. A neurologist clinically assessed the participants. Headache criteria proposed by the International Headache Society were used. Each of the 15 women with migraine was matched to three controls by time since menopause and by body mass index. Serum levels of androstenedione and total testosterone were measured by radioimmunoassays. Free testosterone was calculated from total testosterone, immunoassayed sex hormone-binding globulin and albumin. The mean±S.D. serum level of androstenedione was 2.7±1.1nmol/l and 3.4±1.9nmol/l in cases and controls respectively. The mean serum level of testosterone was 0.8±0.4nmol/l and 1.0±0.5nmol/l in cases and controls respectively. The mean serum level of free testosterone was 12.5±8.5pmol/l and 14.0±7.9pmol/l in cases and controls respectively. Conditional logistic regression analysis was used to test for differences in serum levels of androstenedione, total testosterone and free testosterone between cases and controls. No statistically significant differences were found. To conclude, there was no evidence in this study that differences within normal levels of androgens play an important role in the aetiology of migraine in postmenopausal women not taking oestrogens.
Altered Na+/H+ exchanger isoform 1 activity in immortalized lymphoblasts from women with pre-eclampsia: evidence for an intermediate phenotype
Authors: LEE V.M. , QUINN P.A. , JENNINGS S.C. , HALLIGAN A.W.F. , NG L.L.
Published: August 2015

Previous studies have demonstrated a raised Na+ content in leucocytes isolated from women with pre-eclampsia. Increased Na+/H+ exchanger activity is one membrane transport abnormality that may contribute to this phenomenon and may be implicated in the abnormal volume homoeostasis and hypertension associated with the disease. Increased Na+/H+ exchanger activity has been documented in nucleated white blood cells from both pre-eclamptic and post-partum pre-eclamptic women, and may suggest the importance of genetic influences on exchanger activity. In the present study, we used lymphoblasts from women with pre-eclampsia and from age- and gestation-matched normotensive pregnant controls to determine Na+/H+ exchanger activity and intracellular resting pH using fluorimetry and the pH-sensitive dye BCECF-AM [bis(carboxyethyl)carboxyfluorescein acetoxymethyl ester]. Determination of Na+/H+ exchanger protein abundance was performed by Western blotting. Intracellular pH was not significantly different in cells from pre-eclamptic women compared with those from normotensive controls. Na+/H+ exchanger activity was measured when the intracellular pH was clamped at 6.0, and was found to be significantly higher in cells from pre-eclamptic women (20.77±0.92mmol·min-1·l-1) compared with those from normotensive controls (15.22±0.92mmol·min-1·l-1; P = 0.001). Na+/H+ exchanger protein abundance was established to be similar in the two subject groups, suggesting that the turnover number for the Na+/H+ exchanger is increased in the women with pre-eclampsia. These changes in Na+/H+ exchanger activity indicate the importance of genetic factors in determining this particular phenotype, since in this cell culture model of pre-eclampsia it is likely that environmental or hormonal influences present in vivo would have declined. Overactivity of the Na+/H+ exchanger may contribute to the raised intracellular Na+ concentration reported previously in white blood cells from women with pre-eclampsia.
Impact of periodic breathing on measurement of oxygen uptake and respiratory exchange ratio during cardiopulmonary exercise testing
Authors: FRANCIS Darrel P. , DAVIES L. Ceri , WILLSON Keith , WENSEL Roland , PONIKOWSKI Piotr , COATS Andrew J.S. , PIEPOLI Massimo
Published: August 2015

Metabolic exercise testing is valuable in patients with chronic heart failure (CHF), but periodic breathing may confound the measurements. We aimed to examine the effects of periodic breathing on the measurement of oxygen uptake (VO2) and respiratory exchange ratio (RER). First, we measured the effects of different averaging procedures on peak VO2 and RER values in 122 patients with CHF undergoing cardiopulmonary exercise testing. Secondly, we studied the effects of periodic breathing on VO2 and RER in healthy volunteers performing computer-guided periodic breathing. Thirdly, we used a Fourier analysis to study the effects of periodic breathing on gas exchange measurements. The first part of the study showed that 1min moving window gave a mean peak VO2 of 13.8mlμmin-1μkg-1 for the CHF patients. A 15s window gave significantly higher values. The difference averaged 1.0mlμmin-1μkg-1 (P<0.0001), but varied widely: 41% of subjects showed a difference greater than 1.0mlμmin-1μkg-1. RER values were also higher by an average of 0.09 (P<0.0001); in 20% of subjects the difference was greater than 0.10. In the second part of the study, we found artefactual elevations of peak VO2 (without averaging) of 2.9mlμmin-1μkg-1 (P<0.01) and of peak RER of 0.13 (P<0.001), which were still significant when 30s averaging was applied [Δ(peak VO2) = 1.8mlμmin-1μkg-1, P<0.01; ΔRER = 0.08, P<0.001]. The third, theoretical, part of the study showed that values of carbon dioxide output and VO2 oscillate with different phases and amplitudes, resulting in oscillations in their ratio, RER. Averaging over 15s or 30s can be expected to give only 10% or 36% attenuation respectively. Thus periodic breathing causes variable artefactual elevations of measured peak VO2 and RER, which can be attenuated by using longer averaging periods. Clinical reports and research publications describing peak VO2 in CHF should be accompanied by details of the averaging technique used.
Involvement of gap junctions in bradykinin-induced relaxation of bovine pulmonary supernumerary arteries before and after inhibition of nitric oxide/guanylate cyclase
Authors: TRACEY A. , MACDONALD A. , SHAW A.M.
Published: August 2015

This study evaluated the possible contribution of gap junctions to the nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated responses elicited by bradykinin in bovine pulmonary supernumerary arteries. In artery rings with an intact endothelium and treated with the cyclo-oxygenase inhibitor indomethacin (10μM), bradykinin (100pM–1μM) produced a concentration-dependent relaxation [-logEC50 (pEC50), 9.6±0.2; maximum relaxation (Rmax), 89.7±14.8%; n = 6]. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 100μM) and the NO scavenger hydroxocobalamin (200μM) each produced a rightward shift in the bradykinin concentration–response curve [pEC50: L-NAME, 8.9±0.1 (n = 6; P<0.01); hydroxocobalamin, 8.3±0.2, (n = 6; P<0.001)]. However, the soluble guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 10μM) did not significantly alter the response to bradykinin (pEC50 9.4±0.2; n = 9). The gap junction inhibitor carbenoxolone (100μM) did not affect the relaxation produced by bradykinin (pEC50, 9.7±0.1; Rmax, 100±3.2%; n = 6), but it significantly depressed Rmax when L-NAME, hydroxocobalamin or ODQ was present. Further, carbenoxolone produced a rightward shift in the bradykinin concentration–response curve in the presence of ODQ (8.4±0.1; n = 6, P<0.01). The data suggest that, in bovine pulmonary supernumerary arteries, gap junctions may, in part, facilitate the EDHF-mediated response, but not the NO-mediated response, to bradykinin. However, the additional involvement of an unidentified endothelial relaxing factor cannot be excluded.
Retinoid receptors in the developing human lung
Authors: KIMURA Yuichiro , SUZUKI Takashi , KANEKO Chika , DARNEL Andrew D. , MORIYA Takuya , SUZUKI Satoshi , HANDA Masashi , EBINA Masahito , NUKIWA Toshihiro , SASANO Hironobu
Published: August 2015

Nuclear receptors and their ligands are known to play very important roles in lung development. Among these receptors, retinoid receptors, members of the steroid/thyroid hormone receptor superfamily, are classified into retinoic acid receptor (RAR) isoforms α, β, and γ and retinoid X receptor (RXR) isoforms α, β, and γ. In addition, isoforms I and II of the orphan receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) have been shown to negatively regulate the activation of retinoid receptors. Both of these receptors have been shown to regulate lung development in the mouse. In the present study we utilized immunohistochemistry and real-time quantitative PCR to examine the expression of RAR-α, -β and -γ, RXR-α, -β and -γ and COUP-TFII in the human fetal lung at 13–16 gestational weeks, a very critical stage of human pulmonary development, in order to study possible roles in pulmonary morphogenesis by comparing these findings with those of the adult lung. RXR-γ immunoreactivity was detected at both proximal (epithelia and mesenchyme of the trachea and bronchi associated with cartilage) and distal (epithelia and mesenchyme of smaller distal bronchi) sites in the fetal lung, but was markedly weaker in the adult lung. RAR-β immunoreactivity was detected in distal mesenchymal cells of the fetal lung, but was not discernible in distal mesenchymal cells in the adult lung (bronchioles, alveolar ducts and alveolus). Relatively intense RAR-γ immunoreactivity was detected in the chondrocytes of bronchial cells. COUP-TFII immunoreactivity was detected with a similar pattern to that of RAR-β. Real-time quantitative PCR analyses revealed that mRNA levels of RXR-γ at proximal and distal sites (ratio of fetal lung/adult lung: 3.4±0.05-fold and 3.1±0.03-fold respectively; P<0.01), RAR-β at distal sites (2.4±0.01-fold; P<0.05) and RAR-γ at proximal sites (2.2±0.11-fold; P<0.05) were significantly higher in the fetus than in the adult.
Effects of obesity on endothelium-dependent reactivity during acute nitric oxide synthase inhibition: modulatory role of endothelin
Authors: TRAUPE Tobias , D'USCIO Livius V. , MUENTER Klaus , MORAWIETZ Henning , VETTER Wilhelm , BARTON Matthias
Published: August 2015

This study investigated vascular reactivity in response to acetylcholine, in the presence of acute inhibition of nitric oxide synthase, in the carotid artery and aorta of obese C57Bl6/J mice fed on a high-fat diet for 30 weeks, and of control mice. A subgroup of obese animals was also treated with the ETA receptor antagonist darusentan (50mg·kg-1·day-1). In vascular rings from control animals, acetylcholine caused endothelium-dependent contractions in the carotid artery, but not in the aorta. In vascular rings from obese mice, contractility to acetylcholine was also evident in the aorta, and that in the carotid artery was increased compared with control mice. ETA receptor blockade by darusentan treatment of the obese mice prevented enhanced vasoconstriction to acetylcholine, resulting in mild vasodilatation. Thus obesity increases endothelium-dependent vasoconstriction in the absence of endothelial nitric oxide. This effect can be completely prevented by chronic ETA receptor blockade, suggesting that endothelin modulates increased endothelium-dependent vasoconstriction in obesity.
Peroxisome proliferator-activated receptor-γ activators inhibit endothelin-1-related cardiac hypertrophy in rats
Authors: SAKAI Satoshi , MIYAUCHI Takashi , IRUKAYAMA-TOMOBE Yoko , OGATA Takehiro , GOTO Katsutoshi , YAMAGUCHI Iwao
Published: August 2015

Endothelin-1 (ET-1) causes cardiac hypertrophy, and ET receptor antagonists inhibit the development of cardiac hypertrophy in vitro and in vivo. Peroxisome proliferator-activated receptor γ (PPARγ), a member of the family of nuclear receptors, suppresses activator protein-1 (AP-1). We investigated the effects of the thiazolidinediones troglitazone and pioglitazone, activators of PPARγ, on cardiac hypertrophy due to pressure overload provoked by abdominal aortic banding (AB) in rats. Rats were divided into four groups: sham operation with vehicle treatment (n = 5); AB surgery with vehicle treatment (n = 6); AB surgery with troglitazone treatment (100mg·kg-1·day-1; n = 5); and AB surgery with pioglitazone treatment (10mg·kg-1·day-1; n = 8). Treatments were started 7 days before AB surgery, and left ventricular (LV) hypertrophy was assessed 24h after surgery. The ratio of LV weight/body weight (BW) was significantly increased in AB rats compared with sham-operated rats; treatment of AB rats with troglitazone or pioglitazone significantly inhibited the increase in LV weight/BW. Expression of ET-1 mRNA was markedly enhanced in the left ventricles of AB rats; treatment with troglitazone or pioglitazone lowered expression significantly. Suppression of cardiac hypertrophy by pioglitazone treatment was accompanied by a decrease in expression of the gene encoding brain natriuretic factor, a molecular marker for cardiac hypertrophy, in AB rats. Because the ET-1 gene has AP-1 response elements in its 5´-flanking region, the thiazolidinediones troglitazone and pioglitazone may inhibit cardiac hypertrophy partly through suppression of AP-1-induced ET-1 gene up-regulation.
Forearm vasoconstriction to endothelin-1 is impaired, but constriction to sarafotoxin 6c and vasodilatation to BQ-123 unaltered, in patients with essential hypertension
Authors: FERRO Charles J. , HAYNES William G. , HAND Malcolm F. , WEBB David J.
Published: August 2015

The importance of endothelin-1 (ET-1) in the pathophysiology of essential hypertension is unclear. We therefore compared the effects of endothelin ETA receptor blockade and the stimulation of ETA and ETB receptors, and their interaction with the sympathetic nervous system, in the forearm resistance vessels of patients with essential hypertension and healthy control subjects. A total of 27 untreated patients with essential hypertension (blood pressure >160/100mmHg) and 25 normotensive (blood pressure <140/90mmHg) age- and sex-matched control subjects participated in these studies. A total of 10 patients and 10 controls took part in each phase. Locally active doses of study drugs were infused into the non-dominant brachial artery, while forearm blood flow was measured by venous occlusion plethysmography. A 60min infusion of BQ-123 (an ETA receptor antagonist; 100nmol/min) significantly increased forearm blood flow by 40±8% in hypertensive patients and by 35±5% in controls, with no difference between groups (P = 0.49). Forearm vasoconstriction to ET-1 (an ETA and ETB receptor agonist; 5 pmol/min) for 90min was significantly blunted in hypertensive patients (21±4%) compared with control subjects (37±3%; P = 0.0001). Forearm vasoconstriction to sarafotoxin S6c (an ETB receptor agonist; 10 pmol/min) for 90min was similar in hypertensive patients (44±5%) and control subjects (48±4%; P = 0.95). Sympathetically mediated vasoconstriction produced by lower-body negative pressure was not different in hypertensive patients compared with controls, and was not affected by infusion of ET-1 or sarafotoxin S6c. There were no differences in the observed increase in forearm blood flow with a control vasodilator (sodium nitroprusside) or the observed decrease in forearm blood flow with a control vasoconstrictor (noradrenaline) between hypertensive patients and control subjects. BQ-123 produced a significant increase in forearm blood flow in hypertensive patients, consistent with the anti-hypertensive actions of this agent. In conclusion, forearm vasoconstriction to ET-1, but not to sarafotoxin S6c, was reduced in patients with essential hypertension, consistent with possible down-regulation of the ETA receptor in this condition.
Comparison of red wine extract and polyphenol constituents on endothelin-1 synthesis by cultured endothelial cells
Authors: KHAN Noorafza Q. , LEES Delphine M. , DOUTHWAITE Julie A. , CARRIER Martin J. , CORDER Roger
Published: August 2015

Regular consumption of red wine reduces mortality from coronary heart disease. This observation has been attributed to the anti-thrombotic effects of ethanol and to the antioxidant properties of polyphenolic compounds present in red wine. Here we show that an extract of red wine polyphenols causes a concentration-dependent inhibition of endothelin-1 synthesis in cultured bovine aortic endothelial cells. This action was associated with modifications in phosphotyrosine staining, indicating that the active components of red wine cause specific modifications of tyrosine kinase signalling. Thus inhibition of endothelin-1 synthesis by red wine may reduce the development of atherosclerosis, and hence decrease coronary heart disease.
Characterization of PgPepO, a bacterial homologue of endothelin-converting enzyme-1
Authors: CARSON Julie A. , ANSAI Toshihiro , AWANO Shuji , YU Weixian , TAKEHARA Tadamichi , TURNER Anthony J.
Published: August 2015

PgPepO is a homologue of endothelin-converting enzyme-1 (ECE-1), with which it shares 31% identity. PgPepO was isolated from the periodontal pathogen Porphyromonas gingivalis. Recent studies have suggested a link between periodontal and cardiovascular disease, and several groups have suggested that bacterial and viral infections may contribute to the latter. P. gingivalis possesses the ability to invade, and multiply within, aortic endothelial cells and has been localized to atherosclerotic plaques. PgPepO was expressed and purified to homogeneity and we have begun detailed functional analysis, in terms of substrate preference and inhibitor specificity, in order to provide active-site comparisons with other members of the neprilysin (NEP)/ECE family. PgPepO possesses similar substrate specificity to ECE-1 and has been shown to cleave big endothelin-1 (big ET-1), big ET-2 and big ET-3, converting the substrates into their respective mature endothelin peptides. Substance P, angiotensin I, angiotensin II and neurotensin are all cleaved at multiple sites by PgPepO and the kinetics of these reactions have been compared. The potent vasoconstrictor urotensin II is not hydrolysed by PgPepO. Cleavage of bradykinin by PgPepO occurs at the Pro7-Phe8 bond and is inhibited by the NEP and ECE-1 inhibitor phosphoramidon in a pH-dependent fashion (IC50 = 10µM at pH 7.0) but not by thiorphan, an NEP-specific inhibitor. PgPepO activity is completely inhibited by EDTA. Characterization of this enzyme is important in elucidating possible links between periodontal pathogens and cardiovascular disorders such as atherosclerosis, and provides an opportunity to gain structural information on a bacterial protein with striking similarity to human ECE-1.
Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a
Authors: KULATHILA Raviraj , CLARK Kirk , SAVAGE Paula , BOWEN Benjamin R. , EMOTO Noriaki , YANAGISAWA Masashi , JENG Arco Y.
Published: August 2015

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High FiveTM insect cells in suspension culture. Both the monomeric (85kDa) and dimeric (170kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11mg of ECE-1a monomer and 6mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the Km and kcat values for the monomer were 2.2µM and 1.6min-1 respectively, while those of the dimer were 1.4µM and 4.9min-1 respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.
CGS 34226, a thiol-based dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11
Authors: JENG Y. Arco , SAVAGE Paula , BEIL Michael E. , BRUSEO Charles W. , HOYER Denton , FINK Cynthia A. , TRAPANI Angelo J.
Published: August 2015

Endothelins (ETs) are potent vasoconstrictors and have been implicated in the pathogenesis of various cardiovascular and renal diseases. In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic agent, which is mainly degraded by neutral endopeptidase 24.11 (NEP) in vivo. Thus, compounds that can suppress the biosynthesis of ETs by inhibiting endothelin converting enzymes (ECEs), which catalyse the final step of post-translational processing of the vasoconstrictors, while simultaneously potentiating the levels of ANP by inhibiting NEP may have novel therapeutic utility. Through targeted screening of our compound library and subsequent optimization, CGS 34226 was identified as a potent, dual inhibitor of ECE-1 and NEP, inhibiting the enzymes with respective IC50 values of 11 and 4.6nM. In vivo, CGS 34226 suppressed the big endothelin-1 (big ET-1)-induced pressor response dose-dependently. At 15 and 90min after an intravenous dose of 30mg/kg in anaesthetized rats, this compound inhibited the big ET-1-induced effect by 79% and 65% respectively. In addition, CGS 34226 increased plasma ANP immunoreactivity by 120% up to 4h after an intravenous dose of 10mg/kg in conscious rats infused with ANP at a rate of 450ng/kg per min, intravenously. These results show that CGS 34226 is a potent dual inhibitor of ECE-1 and NEP in vitro and in vivo and that the compound may represent a novel agent for the treatment of cardiovascular and renal dysfunction.
Pharmacology of endothelin receptor antagonists ABT-627, ABT-546, A-182086 and A-192621: ex vivo and in vivo studies
Authors: WESSALE Jerry L. , ADLER Andrew L. , NOVOSAD Eugene I. , CALZADILLA Samuel V. , DAYTON Brian D. , MARSH Kennan C. , WINN Martin , JAE Hwan-Soo , GELDERN Thomas W.von , OPGENORTH Terry J. , WU-WONG J. Ruth
Published: August 2015

Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ETA and ETB receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627 (atrasentan), ABT-546, A-182086 and A-192621, which exhibit differences in selectivity for ETA and ETB receptors. In this report, we compare the efficacy, potency and pharmacokinetic properties of these four antagonists, including potency in inhibiting ET-1- or Sarafotoxin 6c-induced vessel constriction in isolated arteries and efficacy in antagonizing ET-1-, big ET-1- or Sarafotoxin 6c-induced pressor responses in rats.
Effects of exercise training on expression of endothelin-1 mRNA in the aorta of aged rats
Authors: MAEDA Seiji , MIYAUCHI Takashi , IEMITSU Motoyuki , TANABE Takumi , YOKOTA Tomoko , GOTO Katsutoshi , YAMAGUCHI Iwao , MATSUDA Mitsuo
Published: August 2015

Aging impairs endothelial function and the vascular tone regulation, although the precise mechanism remains unclear. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide produced by vascular endothelial cells. Because ET-1 has a potent vasoconstrictor effect on vessels, it may be involved in the regulation of vascular tonus. We hypothesized that aging causes a decrease in ET-1 expression in aorta, and that exercise training improves the aging-induced decrease in ET-1 expression in aorta. This study was performed to examine whether gene expression of ET-1 in the aorta of rats is altered by aging and subsequent exercise training. We studied expression of ET-1 mRNA in the aortas of sedentary young rats (Sedentary young group, 4 months old), sedentary aged rats (Sedentary aged group, 23 months old), and swim trained aged rats (Training aged group, 23 months old; swimming training for 8 weeks, 5days/week, 90min/day). The expression of ET-1 mRNA in the aorta was analysed by real-time quantitative PCR. Body weight and resting heart rate were significantly lower in the Training aged group compared with the Sedentary aged group. These results suggest that the Training aged rats exhibited physiological effects from exercise training. The expression of ET-1 mRNA in the aorta was markedly lower in Sedentary aged group compared with the Sedentary young group, whereas it was significantly higher in Training aged group compared with the Sedentary aged group. These results show that the expression of ET-1 mRNA in the aorta is decreased by aging, and that the expression is increased by exercise training. Therefore, the present study provides a possibility that exercise training improves endothelial function through up-regulation of the aging-induced decrease in ET-1 expression in the aorta.
Modulation of ETB receptor-induced arginine-vasopressin secretion by N-methyl-D-aspartate (NMDA) and γ-aminobutyric acid (GABA)-dependent mechanisms in hypothalamo-neurohypophysial explants
Authors: ROSSI Noreen F. , CHEN Haiping
Published: August 2015

The endothelins (ETs) stimulate the secretion of arginine-vasopressin (AVP) in vivo and in vitro. The activation of hypothalamic ETB receptors increases AVP release, but the neurotransmitters mediating these responses are not known. In the compartmentalized hypothalamo–neurohypophysial explant model, the overall basal release of AVP was 53±17pg·h-1·PP-1 (where PP is posterior pituitary). ETB receptor activation in hypothalamic sites by 1nM IRL1620 dose-dependently increased AVP secretion, with a maximal response of 340±70% of basal·h-1·PP-1, whereas 1nM ET-1, the ETA receptor-selective agonist, inhibited AVP release to 44±8%·h-1·PP-1. Addition of MK801 along with IRL1620 inhibited AVP release to a value no different from basal (122±41%·h-1·PP-1). In contrast, 10µM DNQX [6,7-dinitroquinozaline-2,3-(1H,4H)-dione] did not block ETB receptor-induced AVP release (326±73%·h-1·PP-1), and nor did non-selective α-adrenergic receptor antagonism. The GABAA (where GABA is γ-aminobutyric acid) receptor agonist muscimol (10µM) inhibited AVP release in response to IRL1620 (127±30%·h-1·explant-1). These data suggest that AVP release induced by activation of hypothalamic ETB receptors is mediated by a hypothalamic N-methyl-D-aspartate (NMDA) receptor-mediated mechanism. In turn, the local release of GABA associated with NMDA activation may exert an inhibitory influence and dampen the AVP secretory response.
Enhancement of glycolysis in cardiomyocytes elevates endothelin-1 expression through the transcriptional factor hypoxia-inducible factor-1 α
Authors: KAKINUMA Yoshihiko , MIYAUCHI Takashi , SUZUKI Takahiko , YUKI Koichi , MURAKOSHI Nobuyuki , GOTO Katsutoshi , YAMAGUCHI Iwao
Published: August 2015

We investigated whether the type of energy metabolism directly affects cardiac gene expression. During development, the heart switches from glycolysis to fatty acid β-oxidation in vivo, as demonstrated by the developmental switching of the major isoform of myosin heavy chain (MHC) from β to α. However, the β-MHC isoform predominates in monocrotaline-induced pulmonary hypertension, a model of right ventricular hypertrophy in vivo. Cultured cardiomyocytes showed a predominance of β-MHC expression over that of α-MHC, the same pattern as in the hypertrophied heart, suggesting that the in vitro condition itself causes the energy metabolism of cardiomyocytes to be switched to glycolysis. Electrical stimulation of cultured cardiomyocytes decreased the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxia-inducible factor-1α (HIF-1α), but not that of peroxisome-proliferator-activated receptor-γ co-activator, suggesting that electrical stimulation suppresses the glycolytic system. Furthermore, a higher oxygen content (50%) decreased drastically the expression of GAPDH, HIF-1α and endothelin-1 (ET-1), and increased [3H]palmitate uptake. These findings indicate that the intrinsic energy metabolic system in cultured cardiomyocytes in vitro is predominantly glycolysis, and that the gene expression of cardiac ET-1 parallels the state of the glycolytic system. An antisense oligonucleotide against HIF-1α greatly decreased the gene expression of ET-1 and GAPDH, suggesting that cardiac ET-1 gene expression is regulated by cardiac energy metabolism through HIF-1α. In conclusion, it is suggested that the pattern of gene expression of ET-1 reflects the level of the glycolytic system in cardiomyocytes, and that enhanced glycolysis regulates the cardiac gene expression of ET-1 via HIF-1α.