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Preeclampsia (PE) is a pregnancy-related disorder characterized by hypertension and proteinuria that affects 3-10% of all pregnancies. Although its pathophysiology remains obscure, placental hypoxia-induced oxidative stress and alterations in vascular function, morphology, and endothelial barrier integrity are considered to play a key role in the development of preeclampsia. In this study, placental villous explants of noncomplicated placentae and BeWo cells were subjected to hypoxia. The effect of placental hypoxic-conditioned medium (HCM) on intraluminal-induced contraction and endothelial barrier integrity in chorionic arteries was investigated using pressure myography. The impact of BeWo cell HCM on endothelial cell viability, reactive oxygen species formation and inflammation was also determined. Alterations in arterial morphology and contractile responsiveness to the thromboxane A2 analog (U46619) after exposure to placental HCM were examined immunohistochemically and by wire myography, respectively. Intraluminal administration of placental HCM induced vasoconstriction and increased the endothelial permeability for KCl, which was concentration-dependently prevented by quercetin. Placental and BeWo cell HCMs decreased endothelial cell viability, increased the production of reactive oxygen species and enhanced the secretion of IL-6 and IL-8. The cross-sectional area of the arterial media was increased upon exposure to placental HCM, which was associated with increased vascular proliferation and contractile responsiveness to U46619, and all of these effects were prevented by the antioxidants quercetin and RRR-alpha-tocopherol. This study is the first to comprehensively demonstrate the link between factors secreted by placental cells in response to hypoxia and vascular abnormalities and paves the way for new diagnostic approaches and therapies to better protect the maternal vasculature during and after a preeclampsia-complicated pregnancy.

Contains fulltext : 238811.pdf (Publisher’s version ) (Open Access) Preeclampsia complicates 5-8% of all pregnancies worldwide, and although its pathophysiology remains obscure, placental oxidative stress and mitochondrial abnormalities are considered to play a key role. Mitochondrial abnormalities in preeclamptic placentae have been described, but the extent to which mitochondrial content and the molecular pathways controlling this (mitochondrial biogenesis and mitophagy) are affected in preeclamptic placentae is unknown. Therefore, in preeclamptic (n = 12) and control (n = 11) placentae, we comprehensively assessed multiple indices of placental antioxidant status, mitochondrial content, mitochondrial biogenesis, mitophagy, and mitochondrial fusion and fission. In addition, we also explored gene expression profiles related to inflammation and apoptosis. Preeclamptic placentae were characterized by higher levels of oxidized glutathione, a higher total antioxidant capacity, and higher mRNA levels of the mitochondrial-located antioxidant enzyme manganese-dependent superoxide dismutase 2 compared to controls. Furthermore, mitochondrial content was significantly lower in preeclamptic placentae, which was accompanied by an increased abundance of key constituents of glycolysis. Moreover, mRNA and protein levels of key molecules involved in the regulation of mitochondrial biogenesis were lower in preeclamptic placentae, while the abundance of constituents of the mitophagy, autophagy, and mitochondrial fission machinery was higher compared to controls. In addition, we found evidence for activation of apoptosis and inflammation in preeclamptic placentae. This study is the first to comprehensively demonstrate abnormalities at the level of the mitochondrion and the molecular pathways controlling mitochondrial content/function in preeclamptic placentae. These aberrations may well contribute to the pathophysiology of preeclampsia by upregulating placental inflammation, oxidative stress, and apoptosis. Graphical Abstract. 01 augustus 2021

Background: The regulation of cerebral arterial vasomotor tone involves several mechanisms. The role of sympathetic nerves and the adrenergic neurotransmitter, noradrenaline (NA), has been the subject of debate for decades. Moreover, the specific role of endothelin-1 (ET-1) in cerebral arterial vasoconstriction has not been elucidated to date. In this study, we evaluated the contribution of NA and ET-1 to cerebral artery vasoconstriction.Methods: Arterial responses of rat middle cerebral arteries, and human pial cerebral arteries to cumulative concentrations of NA and ET-1, and to Electrical Field Stimulation (EFS), were evaluated. To assess the role of NA and ET-1 when EFS was applied, experiments were performed in the presence of adrenergic, neurogenic, and endothelin-1 receptor modulators.Results: We found that vasoconstriction of cerebral arteries following EFS requires the application of exogenous NA, whereas neither EFS nor NA alone induced vasoconstriction. The observed vasoconstriction was abolished by a-adrenoreceptor antagonist, catecholamine-release inhibitor, blockade of the perivascular neurons, and by the endothelin-2 receptor antagonist (BQ123).Conclusion: Based on our results, cerebral artery vasoconstriction requires simultaneous neurogenic and adrenergic activation and is ET-1 dependent. We hypothesize that NA modulates the release of ET-1. Upon release, ET-1 binds to the ETA-receptor on smooth muscle cells inducing cerebral artery vasoconstriction.

Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 mu M L-NAME), blockade of ETA-and ETB-receptors (10 mu M bosentan) and stimulation of the endothelium with 0.001-10 mu M acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. L-NAME (1) increased arterial contractile responses to K+ (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 mu M) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of L-NAME during contractions induced by either 10 mu M PHE or 40 mM K+ were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.

Introduction An impediment for the acceptance of Traditional Chinese Medicine (TCM) in the West is that the improper use of some TCM is associated with serious side effects. This is exemplified by Ma Huang Decoction (MHD), a Ma Huang containing TCM formula used to treat asthma. The reported cardiovascular side effects often involve the inappropriate use of Ma Huang in isolation, while in TCM clinical practice Ma Huang is combined with other herbs in MHD to mitigate side effects. The aim of the study was to investigate the potential of Ma Huang as it is used in TCM, explore how it may affect the cardiovascular system and compare this to Ma Huang as it is used in the West. Methods The contractile response of isolated arteries from human placenta to Ma Huang, MHD, and MHD without Ma Huang was used in a bioassay to evaluate their cardiovascular side effects. Results The contractile response by Ma Huang in isolation is much higher than that of Ma Huang in combination with the other herbs in MHD. This confirms that the interplay of the various compounds in MHD may mitigate the side effects of Ma Huang. Previous studies showed that the other herbs in MHD also increase the therapeutic efficacy of Ma Huang. This indicates that the incorrect use of Ma Huang in isolation, and without the supervision of knowledgeable professionals, is likely to be the reasons why the cardiovascular side effects are reported in the West. Conclusion To fully value and appreciate its benefit, TCM should be used appropriately as developed over centuries in China.

The food supplement quercetin is used as self-medication for prostate disorders and is known to induce vasorelaxation. The drug tamsulosin is used in the treatment of benign prostatic hyperplasia. A major side effect of tamsulosin is orthostatic hypotension, mediated by vasorelaxation resulting from alpha1-adrenoceptor blockade. The overlapping profile prompted us to investigate the pharmacodynamic interaction of quercetin with tamsulosin. Since quercetin is extensively metabolized in the intestines and the liver, the metabolites quercetin-3-glucuronide and 4'O-methyl-quercetin were also examined. Vasorelaxation induced by the compounds was tested in rat mesenteric arteries (average diameter: 360+/-mum) constricted by the alpha1-adrenoceptor agonist phenylephrine. Tamsulosin (0.1nM) decreased phenylephrine sensitivity 17-fold (n=10). Quercetin (5, 10 and 20microM) also caused a decrease (2-, 4- and 6-fold respectively) of phenylephrine sensitivity, while 10microM of quercetin-3-glucuronide and 4'O-methyl-quercetin decreased this sensitivity (1.5- and 2-fold) only slightly (n=6). The combination of tamsulosin with quercetin or quercetin metabolites proved to be far more potent than the compounds in isolation. The combination of quercetin, quercetin-3-glucuronide or 4'O-methyl-quercetin with tamsulosin decreased the phenylephrine sensitivity approximately 200-, 35- and 150-fold (n=6). The strong pharmacodynamic interaction between the food supplement quercetin and tamsulosin underlines the potential of the impact of supplement-drug interactions that warrant more research.

Background: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems.Methods and results: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3 h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keapl. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and, upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-KB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function.Conclusion and significance: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD.General significance: The newly found mechanism behind the long-term protection of rutin against cardiovas-cular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) μm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) μm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 μm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) μm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 μm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 μm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 μm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 μm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

For decades, oxidative stress has been discussed as a key mechanism of endothelial dysfunction and cardiovascular disease. However, attempts to validate and exploit this hypothesis clinically by supplementing antioxidants have failed. Nevertheless, this does not disprove the oxidative stress hypothesis. As a certain degree of reactive oxygen species (ROS) formation appears to be physiological and beneficial. To reduce oxidative stress therapeutically, two alternative approaches are being developed. One is the repair of key signalling components that are compromised by oxidative stress. These include uncoupled endothelial nitric oxide (NO) synthase and oxidized/heme-free NO receptor soluble guanylate cyclase. A second approach is to identify and effectively inhibit the relevant source(s) of ROS in a given disease condition. A highly likely target in this context is the family of NADPH oxidases. Animal models, including NOX knockout mice and new pharmacological inhibitors of NADPH oxidases have opened up a new era of oxidative stress research and have paved the way for new cardiovascular therapies. LINKED ARTICLES This article is part of a themed issue on Vascular Endothelium in Health and Disease. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.164.issue-3

Introduction. This study investigated whether variation in the genes encoding for ACE, AGT and AGTR1 modifies the risk of myocardial infarction (MI) related to ACE inhibitors and AT II antagonists. Methods. A nested case-control study among users of antihypertensive drugs, in whom the polymorphisms ACE-G4656C, ACE-T3892C, AGT-C235T and AGTR1-A1166C were genotyped. Results. Among 613 cases and 3630 controls, the risk of MI was significantly lower among users of ACE inhibitors compared with that in users of other antihypertensives (adjusted OR, 0.78; 95% CI, 0.63—0.97). In patients using ACE inhibitors the largest risk reduction was found in patients carrying the ACE-4656-G allele (GC and GG genotypes) compared with patients carrying the CC genotype (OR, 0.68; 95% CI, 0.53—0.86 and OR, 1.26, 95% CI, 0.78—2.02, respectively). The synergy index for this interaction was statistically significant (SI, 0.58; 95% CI, 0.35—0.95). The risk of MI was reduced in those who were current users of ACE inhibitors those who had been prescribed dosages lower than the equivalent of 1 defined daily dose (DDD) and those having the AGTR1-1166AC or AA genotype compared with that in users of ACE inhibitors with the AGTR1-1166CC genotype (SI, 3.67; 95% CI,1.18—11.4). None of the polymorphisms modified the effectiveness of AT II antagonists regarding the risk of MI. Conclusion. This study shows an interaction between the use of ACE inhibitors and ACE-G4656C polymorphism, and in low doses also with AGTR1-A1166C polymorphism, in the prevention of MI.

Introduction Preeclampsia is a major health problem in human pregnancy, severely complicating 5-8% of all pregnancies. The emerging molecular mechanism is that conditions like hypoxic stress trigger the release of placental messengers into the maternal circulation, which causes preeclampsia. Our objective was to develop an in vitro model, which can be used to further elucidate the molecular mechanisms of preeclampsia and which might be used to find a remedy. Methods Human non-complicated term placentas were collected. Placental explants were subjected to severe hypoxia and the conditioned media were added to chorionic arteries that were mounted into a myograph. Contractile responses of the conditioned media were determined, as well as effects on thromboxane-A2 (U46619) induced contractility. To identify the vasoactive compounds present in the conditioned media, specific receptor antagonists were evaluated. Results Factors released by placental explants generated under severe hypoxia induced an increased vasoconstriction and vascular contractility to thromboxane-A2. It was found that agonists for the angiotensin-I and endothelin-1 receptor released by placental tissue under severe hypoxia provoke vasoconstriction. The dietary antioxidant quercetin could partially prevent the acute and sustained vascular effects in a concentration-dependent manner. Discussion Both the acute vasoconstriction, as well as the increased contractility to U46619 are in line with the clinical vascular complications observed in preeclampsia. Data obtained with quercetin supports that our model opens avenues for e.g. nutritional interventions aimed at treating or preventing preeclampsia.

Oxidative stress is a key factor driving the aging of cells and arteries. Studies suggest that white blood cell (WBC) telomere length is an index of systemic aging. We, therefore, investigated the association between WBC telomere length and oxidized-LDL, and vascular aging, expressed by the distensibility of the carotid artery. We studied a random population sample of 216 non-smokers and 89, smokers. In all subjects, age and gender- adjusted telomere length was inversely correlated with plasma oxidized-LDL (regression coefficient = -0.656 kb/mg/dL; p=0.0006). Independent of gender, age and mean blood pressure, carotid distensibility increased with telomere length (2.33+/- 1.18 10-3/kPa/kb; p=0.05) but decreased with higher plasma levels of oxidized LDL (-10.7+/- 3.91 10-3/kPa/ mg/dL; p=0.006). Adjusted for gender and age, smokers' telomere length was shorter (6.72 vs 6.91 kb; p=0.014) and plasma oxidized-LDL level higher (0.52 vs 0.46 mg/dL; p=0.03) than in non-smokers. Higher level of oxidized-LDL, is associated with shorter WBC telomeres and increased stiffness of the carotid artery. Smoking is marked by increased oxidative stress in concert with shortened WBC telomere length. ispartof: Frontiers in Bioscience vol:2 issue:3 pages:1164-1168 ispartof: location:Singapore status: published

Purpose In epidemiological studies, non-response may introduce bias and limit generalizability. In genetic pharmacoepidemiological research, collection of DNA might be a major reason for non-response. We determined reasons for non-response and compared characteristics of non-responders and responders in a pharmacogenetic case-control study. Methods Myocardial infarction (MI) cases and controls, who were antihypertensive drug users, were recruited through community pharmacies that participate in the Pharmaco-Morbidity-Record-Linkage-System (PHARMO). The PHARMO database comprises drug dispensing histories of about 2 000 000 subjects from a representative sample of Dutch community pharmacies linked to the national registry of hospital discharges. Independent samples t-test and ANOVA-statistics were used to analyse the differences in continuous variables between responders and non-responders. χ2 statistics and logistic regression were used to compare categorical variables. Results We approached 1871 cases and 14 102 controls of whom 794 MI cases (42.4%) and 4997 controls (35.4%) responded. We could not approach 2194 patients of whom 63.1% had died and 31.2% moved to another pharmacy. Main reasons for non-response were health problems or hospital stays (16.2%, OR 1.47; 95%CI: 1.00–2.16). Other reasons were old age or dementia (16.9%, OR 1.82; 95%CI: 1.24–2.65). Only a small percentage (1.1%, OR 1.43; 95%CI: 0.41–5.03) mentioned DNA sampling as a reason. About 30% of the non-responders did not give a reason. Women were statistically significantly (p

This study was designed to examine the contribution of six polymorphisms to the occurrence of cardiovascular disease (CVD) in a Dutch primary care population with a high prevalence of cardiovascular risk factors. In this cross-sectional case-control study, 232 patients with CVD and 571 event-free controls were studied. Patients were genotyped for the AGTR1 (A1166C), AGT (M235T), ACE (4656rpt), NOS3 (E298D), GNB3 (C825T) and ADD1 (G460W) polymorphisms. Univariate and multivariate odds ratios (ORs) were calculated to assess the relationship between genotypes and CVD. Receiver operating characteristic (ROC) analysis was used to quantify the contribution of the polymorphisms to the prediction of CVD. No differences in either genotype or allele frequencies were found between CVD cases and controls. Multivariate analyses, corrected for multiple testing according to Bonferroni, showed significant protective associations for the T-allele of AGT (OR=0.55 (0.34-0.84)) and for the T-allele of ADD1 (OR=0.52 (0.31-0.82)). ROC analysis showed only a very small improvement of CVD risk prediction by adding the six polymorphisms to a model with traditional risk factors. Our data suggest that a major attribution of the six polymorphisms to the cardiovascular risk prediction in a primary care population such as HIPPOCRATES is unlikely.

Buccal cells are an important source of DNA in epidemiological studies, but little is known about factors that influence amount and purity of DNA. We assessed these factors in a self-administered buccal cell collection procedure, obtained with three cotton swabs. In 2,451 patients DNA yield and in 1,033 patients DNA purity was assessed. Total DNA yield ranged from 0.08 to 1078.0 microg (median 54.3 microg; mean 82.2 microg +/- SD 92.6). The median UV 260:280 ratio, was 1.95. Samples from men yielded significantly more DNA (median 58.7 microg) than those from women (median 44.2 microg). Diuretic drug users had significantly lower purity (median 1.92) compared to other antihypertensive drug users (1.95). One technician obtained significantly lower DNA yields. Older age was associated with lower DNA purity. In conclusion, DNA yield from buccal swabs was higher in men and DNA purity was associated with age and the use of diuretics.

The renin-angiotensin system has been implicated in obesity-related hypertension and insulin resistance. We examined whether locally produced components of the renin-angiotensin system in adipose tissue and skeletal muscle play an endocrine role in vivo in humans. Furthermore, the effects of β-adrenergic stimulation on plasma concentrations and tissue release of renin-angiotensin system components were investigated. Systemic renin-angiotensin system components and arteriovenous differences of angiotensin II (Ang II) and angiotensinogen (AGT) across abdominal subcutaneous adipose tissue and skeletal muscle were assessed in combination with measurements of tissue blood flow before and during systemic β-adrenergic stimulation in 13 lean and 10 obese subjects. Basal plasma Ang II and AGT concentrations were not significantly different between lean and obese subjects. Ang II concentrations were increased in obese compared with lean subjects during β-adrenergic stimulation (12.6±1.5 versus 8.1±1.0 pmol/L; P =0.04), whereas AGT concentrations remained unchanged. Plasma renin activity increased to a similar extent in lean and obese subjects during β-adrenergic stimulation (both P P

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease.The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model.Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor β1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries.Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form.

AIMS: In engineered cells, endothelin ETA and ETB receptors can heterodimerize. We tested whether this can also be observed in native tissue.MAIN METHODS: Rat mesenteric resistance arteries (rMRA) were maintained in organ culture for 24h to upregulate ETB-mediated contractions in addition to their normal ETA-mediated responses. They were then exposed to 100 nM linear ET-1 (ETB-agonist) labeled with Oregon Green 488 (OG488/L.-ET-1) and/or to 16nM intact ET-1 (ETA/ETB-agonist) labeled with the rhodamine dye TAMRA (TAMRA/ET-1). Two photon laser scanning microscopy (TPLSM) was used for the visualization of their binding in the tissue. Fluorescence Lifetime Imaging Microscopy (FLIM) was employed for measurements of the OG488/L.-ET-1 lifetime in the absence and presence of TAMRA/ET-1.KEY FINDINGS: After incubation with the labeled ligands, medial smooth muscle cells (SMCs) were efficiently stained and became visible under TPLSM. TAMRA/ET-1 bound to all SMCs whereas OG488/L.-ET-1 stained only groups of SMCs. Interaction of the two receptor subtypes in SMC was investigated in double staining experiments. Fluorescence lifetime of OG488/L.-ET-1 was reduced in the presence of TAMRA/ET-1, which indicates the occurrence of Fluorescence Resonant Energy Transfer (FRET) and suggests close proximity of the two receptor subtypes within the arterial wall.SIGNIFICANCE: The methodology that is introduced by these new observations may be useful to assess ET-receptor heterodimerization in biopsies from relevant experimental animal models and human patients.

Renal 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) are subject to modulation by various endogenous factors. 11beta-HSDs convert glucocorticoids into inactive 11-ketones and thereby determine tissue levels of active glucocorticoids and thus the extent of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation. As such, modulation of the activity of renal 11beta-HSDs may contribute to the cascade of regulatory events involved in renal electrolyte water handling. We investigated whether renal 11beta-HSDs are modulated by elevated circulating angiotensin II. In rats infused for 2 wk with angiotensin II (250 ng/[kg x min] subcutaneously), plasma angiotensin II, aldosterone, and corticosterone were raised 5.1-, 10.7-, and 2.3-fold, respectively, compared with control rats. Angiotensin II infusion raised corticosterone 11beta-oxidation 1.46- and 1.35-fold in renal cortical proximal and distal tubules (enriched by Percoll centrifugation), respectively, but had no effect on 11beta-HSD1 and 11beta-HSD2 mRNA levels (semiquantitative reverse transcriptase polymerase chain reaction), except for distal tubular 11beta-HSD1 mRNA, which was decreased to 50%. In vitro treatment of freshly isolated tubules with angiotensin II for 45 min prior to assessment of 11beta-HSD activity showed no direct acute effects of angiotensin II on tubular corticosterone 11beta-oxidation. The enhanced renal tubular corticosterone 11beta-oxidation in vivo may partly protect renal GR and MR from elevated plasma corticosterone on angiotensin II infusion.

Glucocorticoid access to renal corticosteroid receptors is regulated by 11beta-hydroxysteroid dehydrogenases (11beta-HSDs), converting 11beta-hydroxyglucocorticoids into inactive 11-ketones. This mechanism plays a key role in maintaining normal salt-water homeostasis and blood pressure. To study whether renal cortical proximal and distal tubular 11beta-HSDs are modulated, upon shifting the electrolyte status (and may thereby contribute to adjusting the salt-water homeostasis), rats were treated for 14 days with diets with low (0.058 w/w%), normal (0.58%, which is the KCl content of standard European laboratory rat food) or high (5.8%) potassium chloride content. In proximal tubules, dietary KCl had no effect regarding corticosterone 11beta-oxidation in intact cells as well as 11beta-HSD1 and 11beta-HSD2 protein (Western blotting) and mRNA levels (semi-quantitative RT-PCR). In distal tubules, the low KCl diet also had no effect. However, distal tubules of rats fed the high KCl diet showed increased corticosterone 11beta-oxidation rates (1.6-fold, P0.01) and 11beta-HSD2 protein (4-fold, P0.01), whereas 11beta-HSD1 protein was decreased (no longer detected, P0.05). Distal tubular 11beta-HSD mRNA levels were not changed upon dietary treatment. Our results suggest that upon dietary KCl loading distal tubular mineralocorticoid receptor selectivity for aldosterone is increased because of enhanced corticosterone 11beta-oxidation. This may contribute to the fine-tuning of salt-water homeostasis by the kidney.

OBJECTIVE: The objective was to test the hypothesis that women with a recent history of preeclampsia have abnormalities in renal hemodynamics and volume status. STUDY DESIGN: We studied a group of 26 primiparous women with history of preeclampsia and a group of 12 parous women with a history of uneventful pregnancies (control group). At least 4 months post partum we compared the following variables between these groups: effective renal plasma flow, glomerular filtration rate, plasma volume, plasma concentration of active renin, plasma concentration of angiotensin II, plasma concentration of aldosterone, and plasma concentration of atrial natriuretic peptide. RESULTS: Both plasma volume and plasma concentration of atrial natriuretic peptide were lower in the formerly preeclamptic group. Compared with the control subjects, the formerly preeclamptic group also had a lower effective renal plasma flow, a higher filtration fraction, and a higher renal vascular resistance. Intergroup differences in plasma concentration of active renin, plasma concentration of angiotensin II and plasma concentration of aldosterone were small and inconsistent. CONCLUSIONS: Women with history of preeclampsia are relatively hypovolemic and tend to have lower effective renal plasma flow and higher renal vascular resistance and filtration fraction than do control subjects. These findings support the hypothesis that otherwise healthy women with a history of preeclampsia show abnormalities in their volume status and renal hemodynamics, irrespective of their blood pressure. (Am J Obstet Gynecol 1998;179:690-6.)

Background: Neurohormonal activation plays an important role in the progression of heart failure. In this study, we investigated the progression of neurohormonal activation in conjunction with the hemodynamic status of the rat after myocardial infarction (MI). Methods and Results: Male Wistar rats were subjected to sham or MI surgery. At 1, 3, 5, and 13 weeks after surgery, cardiac output (CO), mean arterial pressure (MAP), and total peripheral resistance (TPR), were measured. In separate groups of rats, blood was sampled at 1, 5, and 13 weeks after surgery and analyzed for various neurohormones. At 1 week after surgery, CO and TPR were not altered in MI rats, but plasma neurohormonal levels were elevated. At 3 and 5 weeks after surgery, reduced CO, increased TPR, and normal MAP were measured in MI rats compared to sham rats, but only endothelin levels were elevated. At 13 weeks after surgery, MAP was reduced in MI rats and CO and TPR were comparable between groups. Neurohormonal activation was again apparent in MI rats. Conclusions: Myocardial infarction in the rat induces early neurohormonal activation, which normalizes hemodynamic parameters. A compensatory phase follows. At 13 weeks after MI, plasma concentrations of neurohormones are again elevated, perhaps as a sign of transition to decompensation.

1. We evaluated responses of peripheral resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling. 2. Plasma samples and third order mesenteric artery side-branches were obtained from Wistar rats after induction of left ventricular infarction (M1) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [1H]-prazosin. 3. At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted. 4. At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10(6) mol l-1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24 +/- 0.04 vs 5.98 +/- 0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l-1 calcium (2.26 +/- 0.28 vs 3.53 +/- 0.34 N m-1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81 +/- 0.22 vs 3.74 +/- 0.16) were reduced. Responses to the agonist in calcium-free solution and the calcium sensitivity in the presence of 125 mmol l-1 potassium or of phorbol myristate acetate (PMA, 10(-6) mol l-1) were not altered. 5. At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04 +/- 1.40 vs 2.29 +/- 0.21 fmol microgram-1 DNA in controls). 6. In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation. This seems to involve impaired agonist-stimulated calcium influx.

Abstract The renin-angiotensin-aldosterone system plays a major role in renovascular hypertension, but the relationship between renin release and the renal fractional extraction of atrial natriuretic peptide (ANP) in this condition is not well defined. We measured ANP levels in the renal veins and aortas of 49 untreated hypertensive patients studied under standardized conditions immediately before renal angiography. Twenty-one patients had renal artery stenosis, 13 of which were unilateral and 8 bilateral. Five of the 13 patients with unilateral renal artery stenosis had an elevated renin ratio (≥1.5). Patients with renal artery stenosis were older ( P P P P P r =−.62, P r =+.92, P

BACKGROUND: Endothelin-1 (ET1) is a potent vasoconstrictor peptide with pro-mitogenic and pro-inflammatory properties and is therefore of interest in the development of endothelial dysfunction, endothelium-dependent flow-related remodeling, and hypertension-related remodeling. ET1 can be formed through cleavage of big ET1 by endothelin-converting enzyme (ECE) and neutral endopeptidase (NEP).METHOD: We investigated whether the dual NEP/ECE inhibitor SOL1 improves resistance artery function and structure in 12 weeks old spontaneously hypertensive rats (SHRs) and whether arterial structural responses to decreased (-90%) or increased (+100%) blood flow are impaired in young SHRs. To this end two groups of SHRs received chronic 4-week treatment at two different time points (4-8 and 8-12 weeks) prior to the experiment. We compared in-vitro effects of cyclo-oxygenase inhibition (1 μmol/l indomethacine), nitric oxide synthase inhibition (100 μmol/l N(ω)-L-nitro arginine methyl ester), and stimulation of the endothelium by 0.001-10 μmol/l acetylcholine (ACh) in isolated third-order mesenteric arteries of SHRs and aged-matched Wistar-Kyoto (WKY) rats.RESULTS: SOL1 had no effect on blood pressure in SHRs or WKY rats. ACh caused biphasic effects in mesenteric arteries of SHRs. The contractile component (endothelium-derived contractile factor) was absent in WKY and abolished by acute indomethacin administration or chronic SOL1 treatment. Endothelium-derived nitric oxide-type responses did not differ in both strains and were not influenced by SOL1 treatment. Endothelium-derived hyperpolarizing factor-type responses were severely impaired in SHRs as compared to WKY rats and were normalized by chronic SOL1 treatment. In first-order mesenteric arteries, outward flow-induced remodeling was impaired in SHRs. Chronic SOL1 treatment did not restore this response.CONCLUSION: Thus chronic SOL1 treatment during the development of hypertension in SHRs has no effect on blood pressure but improves several aspects of endothelium-dependent vasomotor responses but not arterial remodeling.

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

We evaluated the effects of mediators that can be produced by smooth muscle and endothelial cells on growth responses in isolated arteries. Segments of carotid and renal arteries, denuded of endothelium, were isolated from adult rats and studied during tissue culture in the presence of indomethacin. Three days of culture in the presence of serum stimulated DNA synthesis in the media. During long-term culture new layers of cells developed at the borders of the arterial segments. Medial DNA synthesis depended less on serum than extramedial cell proliferation. During moderate stimulation, basic fibroblast growth factor and endothelin-1 enhanced and interleukin-1 and transforming growth factor-beta reduced medial DNA synthesis, whereas insulin-like growth factor-1, platelet-derived growth factor AA, platelet-derived growth factor BB, and angiotensin II were without effect. Of these factors, only endothelin-1 stimulated extramedial cell proliferation. In addition, serum-stimulated but not basic fibroblast growth factor-stimulated medial DNA synthesis was less marked in arteries that had not been denuded of endothelium than in ee-endothelialized arteries. Differences between preparations with and without endothelium persisted in the absence of L-arginine and in the presence of an inhibitor of nitric oxide synthase. These observations confirmed that DNA synthesis in the arterial media and extramedial cell proliferation are influenced by different factors. They further indicated that endothelial modulation of medial DNA synthesis does not seem to involved endothelium-derived prostaglandins, nitric oxide, or interleukin-1 and that it can be blunted by basic fibroblast growth factor.

Background— Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. Methods and Results— Here, we report that VEGF ∂/∂ mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. Conclusions— These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.

Introduction. We assessed the influence of genetic polymorphisms in the renin-angiotensin system on the risk of diabetes associated with the use of angiotensin II receptor blockers and angiotensin-converting enzyme (ACE) inhibitors. Materials and methods. We performed a matched case-control study among antihypertensive drug users. Pharmacy records and questionnaires were used to ascertain incident diabetes (cases), antihypertensive drug use, and risk factors. Controls did not (yet) have diabetes. We genotyped ACE (G4656C, which is in complete linkage disequilibrium with the ACE insertion/deletion polymorphism), angiotensinogen (M235T), and angiotensin II type 1 receptor (A1166C). Results. Among 495 cases of incident diabetes and 2,624 controls, homozygous 1166C carriers of angiotensin II type 1 receptor who used angiotensin II receptor blockers had an increased risk of diabetes compared to 1166A carriers (interaction odds ratio 5.3 (95% confidence interval: 1.8-16.1)). Homozygous ACE GG subjects who used ACE inhibitors ≥ 1 defined daily dose/day had a higher risk of diabetes compared to subjects with the ACE C allele (interaction odds ratio 2.3 (95% confidence interval: 1.2-4.5)). Conclusions. Angiotensin II receptor blockers increase the occurrence of diabetes in homozygous 1166C carriers of angiotensin II type 1 receptor, but not in 1166A carriers. ACE inhibitors at doses ≥ 1 defined daily dose/day increase the risk of diabetes among homozygous ACE GG carriers, but not in 4656C carriers. angiotensin- converting enzyme inhibitors, angiotensin II receptor blockers, diabetes mellitus, hypertension, pharmacogenetics

BACKGROUND Variation in the renin-angiotensin system (RAS) and salt-sensitivity genes may influence the effect of thiazides on the risk of diabetes. We assessed whether polymorphisms in RAS and salt-sensitivity genes influenced the risk of diabetes associated with thiazides. METHODS Nested case-control study was conducted among antihypertensive drug users. Pharmacy records and questionnaires were used to assess new onset diabetes (cases), to ascertain antihypertensive use and risk factors for diabetes. Cases were matched to controls (up to five) who were not (yet) diagnosed with diabetes mellitus. We genotyped angiotensin-converting enzyme (ACE) (G4656C), angiotensinogen (AGT) (M235T), angiotensin II type 1 receptor, (AGTR1) (A1166C), adducin 1 (alpha) (ADD1) (G460T), guanine nucleotide binding protein (G protein), beta-polypeptide 3 (GNB3) (C825T). RESULTS Among 497 incident cases of type 2 diabetes and 2,633 controls, AGTR1 CC genotype carriers had no increased risk of diabetes due to thiazides (odds ratio (OR) 0.63 (95% confidence interval (CI): 0.28-1.40)) compared to AGTR1 1166A allele carriers (OR 1.79 (95% CI: 1.43-2.23)) receiving thiazides (synergy index (SI) for interaction 0.32 (95% CI: 0.15-0.68)). Although homozygous ACE GG subjects and ACE C allele carriers both had an increased risk of diabetes associated with thiazide use, this risk was more increased for ACE GG subjects (SI 1.70 (95% CI: 1.08-2.66)), particularly at doses > or =1 daily defined dose (DDD) (=25 mg hydrochlorothiazide)/day (SI 2.0 (95% CI: 1.20-3.32)). Among GNB3 T allele carriers, the risk of diabetes due to thiazide use was less increased than among homozygous GNB3 CC subjects (SI 0.62 (95% CI: 0.41-0.93)). CONCLUSION The risk of diabetes due to thiazide use was not increased among AGTR1 1166 CC homozygous subjects and less increased among GNB3 T allele carriers. The ACE 4656 GG genotype enhanced the risk of diabetes due to thiazides.

We evaluated changes in DNA synthesis, structure, and mechanical activity in isolated arteries during exposure to growth factors. Renal arteries were isolated from rats, sympathectomized, denuded of endothelium, and maintained in tissue culture. Up to 4 days of culture did not affect maximal contractile responses to depolarization. From the results of nuclear incorporation of the thymidine analogue 5-bromo-2-deoxyuridine (BrdUrd), culture stimulated DNA synthesis. In the media, incorporation of BrdUrd was maximal after 3 days but fell precipitously thereafter. Culture of arterial segments did not, however, increase the cross-sectional area of the media, the ploidy of the arterial nuclei, or the number of medial cells. In contrast, new layers of cells, part of which displayed smooth musclelike properties, developed at the border of the segments. The outermost edge of this newly formed layer continued to incorporate BrdUrd for at least 2 wk. These data demonstrate that stimulation of DNA synthesis by continuous exposure of the arterial wall to exogenous growth factors is 1) transient in the media; 2) does not, at least initially, compromise contractile reactivity; 3) does not alter gross medial structure; but 4) leads to proliferation of smooth musclelike cells outside the media. These findings suggest that the number of smooth muscle cells in the arterial media is maintained constant in the presence of even strong mitogenic stimuli.

Introduction The GNB3 C825T polymorphism has been shown to affect lipid parameters, atherosclerosis progression, and incidence of myocardial infarction (MI). Therefore, we assessed whether the effectiveness of statins in reducing the risk of MI was modified by the GNB3 C825T polymorphism. Methods In a population-based registry of pharmacy records linked to hospital discharge records (PHARMO), we used a nested case-control design. We selected patients hospitalized for MI as cases if they used antihypertensive drugs and had a diagnosis of hypercholesterolemia before their first MI. Controls met the same eligibility criteria, but were not hospitalized for MI. Logistic regression analysis was used to calculate odds ratios (OR) and synergy index with corresponding 95% confidence intervals (CI), and to adjust for potential confounding factors. Results We included 459 cases and 1805 controls. The risk of MI was significantly lower among participants exposed to statins compared with participants not exposed to statins (adjusted OR: 0.37, 95% CI: 0.29-0.47). The GNB3T allele was associated with a reduced risk of MI (adjusted OR: 0.74, 95% Cl: 0.60-0.92). Among homozygous wild-type (CC) individuals (n = 1119), exposure to statins was associated with a lower risk of MI (OR: 0.48, 95% Cl: 0.34-0.67). However, T allele carriers (CT and TT) who used statins had an even stronger reduced risk of M I (OR: 0.27, 95% Cl: 0.19-0.39). Overall, the interaction between exposure to statins and the GNB3 C825T polymorphism was significantly increased on the multiplicative scale (synergy index: 1.67, 95% Cl: 1.06-2.65). Conclusion Our findings show that T allele carriers of the GNB3 C825T polymorphism have less risk of MI and are more likely to benefit from statin therapy in a hypercholesterolemic population of antihypertensive drug users. Pharmacogenetics and Genomics 18:631-636 (c) 2008 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Background: Genetic dissection of complex diseases requires innovative approaches for identification of disease-predisposing genes. A well-known example of a human complex disease with a strong genetic component is Type 2 Diabetes Mellitus (T2DM). Methods: We genotyped normal-glucose-tolerant subjects (NGT; n = 54), subjects with an impaired glucose metabolism (IGM; n = 111) and T2DM (n = 142) subjects, in an assay (designed by Roche Molecular Systems) for detection of 68 polymorphisms in 36 cardiovascular risk genes. Using the single-locus logistic regression and the so-called haplotype entropy, we explored the possibility that (1) common pathways underlie development of T2DM and cardiovascular disease -which would imply enrichment of cardiovascular risk polymorphisms in "pre-diabetic" (IGM) and diabetic (T2DM) populations- and (2) that gene-gene interactions are relevant for the effects of risk polymorphisms. Results: In single-locus analyses, we showed suggestive association with disturbed glucose metabolism (i.e. subjects who were either IGM or had T2DM), or with T2DM only. Moreover, in the haplotype entropy analysis, we identified a total of 14 pairs of polymorphisms (with a false discovery rate of 0.125) that may confer risk of disturbed glucose metabolism, or T2DM only, as members of interacting networks of genes. We substantiated gene-gene interactions by showing that these interacting networks can indeed identify potential "disease-predisposing allele-combinations". Conclusion: Gene-gene interactions of cardiovascular risk polymorphisms can be detected in prediabetes and T2DM, supporting the hypothesis that common pathways may underlie development of T2DM and cardiovascular disease. Thus, a specific set of risk polymorphisms, when simultaneously present, increases the risk of disease and hence is indeed relevant in the transfer of risk.

It is poorly understood how mechanical stimuli influence in vivo myocardial remodeling during chronic hemodynamic overload. Combined quantitation of ventricular mechanics and expression of key proteins involved in mechanotransduction can improve fundamental understanding. Adult anesthetized dogs ( n = 20) were studied at sinus rhythm (SR) and 0, 3, 10, and 35 days of complete atrioventricular block (AVB). Serial left ventricular (LV) myofiber mechanics were measured. Repeated LV biopsies were analyzed for mRNA and/or protein expression of β1D-integrin, melusin, Akt, GSK3β, muscle LIM protein (MLP), four-and-a-half LIM protein 2 (fhl2), desmin, and calpain. Upon AVB, increased ejection strain (0.29 ± 0.01 vs. 0.13 ± 0.02, SR) and end-diastolic stress (4.8 ± 1.1 vs. 2.7 ± 0.4 kPa) dominated mechanical changes. Brain natriuretic peptide plasma levels were correspondingly high (33 ± 4 vs. 19 ± 1 pg/ml, SR). β1D-Integrin protein expression increased chronically after AVB. Melusin was temporarily overexpressed (+33 ± 9%, 3 days AVB vs. SR), followed by elevated ratios of phosphorylated (P)-Akt to Akt and P-GSK3β to GSK3β (+26 ± 6% and +30 ± 8% at 10 days AVB vs. SR). These changes corresponded to peak hypertrophic growth at 3 to 10 days. MLP increased gradually to maxima at chronic AVB (+36 ± 7%). In contrast, fhl2 (−22 ± 3%, 3 days) and desmin (−30 ± 9%, 10 days AVB) transiently declined but recovered at chronic AVB. Calpain protein expression remained unaltered. In conclusion, volume overload after AVB causes a transient compromise of cytoskeletal integrity based, at least partly, on transcriptional downregulation. Subsequent cytoskeletal reorganization coincides with the upregulation of melusin, P-Akt, P-GSK3β, and MLP, indicating a strong drive to compensated hypertrophy.

Background There is ample evidence that genetic factors contribute to cardiovascular disease risk. The present study aimed to assess the relation between polymorphisms of the angiotensin II type 1 receptor (AGTR1 A 1166 C) and endothelial nitric oxide synthase (NOS3 G 894 T) and the risk of stroke. Methods We performed a case-cohort study on all first fatal and nonfatal stroke events ( n = 74) and a 10% random sample ( n = 1523) of a population-based cohort of women aged 49 to 70 years ( n = 15,236; median follow-up 4.3 years). Univariate and multivariate unweigthed Cox proportional hazards regression models were used to assess the relation between the polymorphisms, their interactions with coexisting risk factors, and the risk of stroke. Results The relation between the AGTR1 CC genotype and stroke risk (unadjusted hazards ratio [HR] 1.62; 95% confidence interval [CI], 0.81–3.28) was modified by increasing age (>56 years: adjusted HR 2.77; 95% CI, 1.17–6.56) and systolic blood pressure (BP) (>130 mm Hg: adjusted HR 2.58; 95% CI, 1.12–5.93). The NOS3 G 894 T polymorphism, however, was not associated with stroke risk. Conclusions In the presence of other coexisting risk factors the AGTR1 A 1166 C but not the NOS3 G 894 T polymorphism increased the risk of stroke. The CC genotype may help identify those individuals who are at greatest risk and who may need (early) treatment or careful follow-up.

Arterial calcification (AC) is generally regarded as an independent risk factor for cardiovascular morbidity and mortality. Matrix Gla protein (MGP) is a potent inhibitor of AC, and its activity depends on vitamin K (VK). In rats, inactivation of MGP by treatment with the vitamin K antagonist warfarin leads to rapid calcification of the arteries. Here, we investigated whether preformed AC can be regressed by a VK-rich diet. Rats received a calcification-inducing diet containing both VK and warfarin (W&K). During a second 6-week period, animals were randomly assigned to receive either W&K (3.0 mg/g and 1.5 mg/g, subsequently), a diet containing a normal (5 μg/g) or high (100 μg/g) amount of VK (either K1 or K2). Increased aortic calcium concentration was observed in the group that continued to receive W&K and also in the group changed to the normal dose of VK and AC progressed. Both the VK-rich diets decreased the arterial calcium content by some 50%. In addition, arterial distensibility was restored by the VK-rich diet. Using MGP antibodies, local VK deficiency was demonstrated at sites of calcification. This is the first study in rats demonstrating that AC and the resulting decreased arterial distensibility are reversible by high-VK intake.

AIMS/HYPOTHESIS: This study was designed to investigate the effect of short-term ACE inhibitor treatment on insulin sensitivity and to examine possible underlying metabolic and haemodynamic effects in obese insulin-resistant subjects. METHODS: A randomised, double-blind placebo-controlled trial was performed in 18 obese insulin-resistant men (age, 53 +/- 2 years; BMI, 32.6 +/- 0.8 kg/m(2); homeostasis model assessment of insulin resistance, 5.6 +/- 0.5; systolic blood pressure [SBP], 140.8 +/- 3.2; diastolic blood pressure [DBP], 88.8 +/- 1.6 mmHg), who were free of any medication. The aim was to examine the effects of 2 weeks of ACE inhibitor treatment (ramipril, 5 mg/day) on insulin sensitivity, forearm blood flow, substrate fluxes across the forearm, whole-body substrate oxidation and intramuscular triacylglycerol (IMTG) content. RESULTS: Ramipril treatment decreased ACE activity compared with placebo (-22.0 +/- 1.7 vs 0.2 +/- 1.1 U/l, respectively, p < 0.001), resulting in a significantly reduced blood pressure (SBP, -10.8 +/- 2.1 vs -2.7 +/- 2.0 mmHg, respectively, p = 0.01; DBP, -10.1 +/- 1.3 vs -4.2 +/- 2.1 mmHg, respectively, p = 0.03). Ramipril treatment had no effect on whole-body insulin-mediated glucose disposal (before: 17.9 +/- 2.0, after: 19.1 +/- 2.4 mumol kg body weight(-1) min(-1), p = 0.44), insulin-mediated glucose uptake across the forearm (before: 1.82 +/- 0.39, after: 1.92 +/- 0.29 mumol 100 ml forearm tissue(-1) min(-1), p = 0.81) and IMTG content (before: 45.4 +/- 18.8, after: 48.8 +/- 27.5 mumol/mg dry muscle, p = 0.92). Furthermore, the increase in carbohydrate oxidation (p < 0.001) and forearm blood flow (p < 0.01), and the decrease in fat oxidation (p < 0.001) during insulin stimulation were not significantly different between treatments. CONCLUSIONS/INTERPRETATION: Short-term ramipril treatment adequately reduced ACE activity and blood pressure, but had no significant effects on insulin sensitivity, forearm blood flow, substrate fluxes across the forearm, whole-body substrate oxidation and IMTG content in obese insulin-resistant subjects.

In resistance-sized arteries, a chronic increase in blood flow leads to increases in arterial structural luminal diameter and arterial wall mass. In this review, we summarize recent evidence that outward remodeling of resistance arteries 1) can help maintain and restore tissue perfusion, 2) is not intimately related to flow-induced vasodilatation, 3) involves transient dedifferentiation and turnover of arterial smooth muscle cells, and 4) is preceded by increased expression of matricellular proteins, which have been shown to promote disassembly of focal adhesion sites. Studies of experimental and physiological resistance artery remodeling involving differential gene expression analyses and the use of knockout and transgenic mouse models can help unravel the mechanisms of outward remodeling.

Objective— To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE −/− and t-ACE +/+ mice. Results— In CA of t-ACE −/− mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE −/− than t-ACE +/+ mice. In UA of t-ACE −/− mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains. Conclusions— t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.

Objective Since renal sympathetic nerves are involved in the regulation of sodium excretion, we investigated whether treatment with urapidil, an α 1 -adrenoceptor blocking agent which also lowers sympathetic activity, alters sodium excretory capacity in patients with essential hypertension. Design A double-blind, randomized, parallel-group study. Methods Studies were carried out in 26 patients who were randomized to treatment with either placebo or urapidil for 8 weeks. Before and after treatment blood pressure, renal haemodynamics and various neurohormones were measured, as well as the response of these variables to a hypertonic saline infusion. Results Urapidil had no effect on renal haemodynamics or neurohormones at rest However, as compared to placebo the saline-induced rises in renal plasma flow and glomerular filtration rate lasted longer during treatment with urapidil. Responses of renin, angiotensin II and catecholamines were not modified by urapidil. On the other hand, aldosterone was less suppressed while atrial natriuretic peptide was less stimulated following the saline load when patients had been treated with urapidil. Cumulative sodium excretion during a 3 h period from the moment of saline infusion was similar whether patients had been treated with placebo or with urapidil. Conclusions Our data show that urapidil interferes with renal haemodynamics after sodium loading but that any tendency to promote sodium output may be offset by changes in aldosterone and atrial natriuretic peptide. We conclude that urapidil, under the circumstances tested, does not affect the sodium excretory capacity of the kidney.

Abstract —The endothelial cytoskeleton plays a key role in arterial responses to acute changes in shear stress. We evaluated whether the intermediate filament protein vimentin is involved in the structural responses of arteries to chronic changes in blood flow (BF). In wild-type mice (V+/+) and in vimentin-deficient mice (V−/−), the left common carotid artery (LCA) was ligated near its bifurcation, and 4 weeks later, the structures of the occluded and of the contralateral arteries were evaluated and compared with the structures of arteries from sham-operated mice. Body weight and mean carotid artery BF did not differ between the strains, but LCA and right carotid artery (RCA) diameter (737±14 μm [LCA] and 723±14 μm [RCA] for V−/− versus 808±20 μm [LCA] and 796±20 μm [RCA] for V+/+) and medial cross-sectional area (CSAm) were significantly smaller in V−/− (21±1 and 22±2×10 3 μm 2 for LCA and RCA, respectively) than in V+/+ (28±2 and 28±3×10 3 μm 2 for LCA and RCA, respectively). In V+/+, LCA ligation eliminated BF in the occluded vessel (before ligation, 0.35±0.02 mL/min) and increased BF from 0.34±0.02 to 0.68±0.04 mL/min in the RCA. In V−/−, the BF change in the occluded LCA was comparable (from 0.38±0.05 mL/min to zero-flow rates), but the BF increase in the RCA was less pronounced (from 0.33±0.02 to 0.50±0.05 mL/min). In the occluded LCA of V+/+, arterial diameter was markedly reduced (−162 μm), and CSAm was significantly increased (5×10 3 μm 2 ), whereas in the high-flow RCA of V+/+, carotid artery diameter and CSAm were not significantly modified. In the occluded LCA of V−/−, arterial diameter was reduced to a lesser extent (−77 μm) and CSAm was increased to a larger extent (10×10 3 μm 2 ) than in V+/+. In contrast to V+/+, the high-flow RCA of V−/− displayed a significant increase in diameter (52 μm) and a significant increase in CSAm (5×10 3 μm 2 ). These observations provide the first direct evidence for a role of the cytoskeleton in flow-induced arterial remodeling. Furthermore, they dissociate (1) between acute and chronic arterial responses to altered BF, (2) between alterations of lumen diameter and wall mass during arterial remodeling, and (3) between developmental and imposed flow-induced arterial remodeling.

Abstract The present experiments were designed to test the hypothesis that the activation of the renin-angiotensin system during compensated heart failure may have adverse effects on cardiac function and change the peripheral vascular structure. ANG II (250 ng/kg/min) or saline (0.9% NaCl) were infused in myocardial-infarcted and sham-operated rats. After 2 weeks, cardiac function and peripheral vascular changes were investigated. Results: ANG II infusion reduced baseline cardiac index in sham rats but did not further reduce this index in ANG II-infused MI rats. Total peripheral resistance was similarly increased in ANG II-infused infarcted and sham rats, and also plasma ANG II concentrations were comparable. ANG II elevated systolic blood pressure by approximately 70 mm Hg in sham rats and increased the medial cross-sectional area of the superior mesenteric artery by 33%. However, ANG II infusions in MI rats resulted in only a minor increase in blood pressure, whereas the cross-sectional area of the superior mesenteric artery did not change. ANG II infusion had no effect on vessel dimensions of the resistance arteries of the pulmonary and mesenteric vascular bed of either group. Calculated ED 50 and peak pressor response to acute ANG II injections were comparable in all groups, confirming the presence of functionally intact AT 1 receptors. The increases in plasma atrial natriuretic peptide (ANP) and nitric oxide (NO) synthase activity (estimated by aortic cyclic GMP concentrations) were higher in ANG II-infused MI rats than in ANG II-infused sham rats. Conclusion: ANG II infusion in rats with and without MI has comparable negative effects on cardiac function but has different effects on blood pressure and vascular structure. The concomitant increases in plasma ANP and NO synthase activity in ANG II-infused MI rats suggest that the growth stimulatory and hypertensive actions of ANG II in sham rats may be counter-regulated by activation of inhibitory neurohumoral systems such as ANP or NO in MI rats.

The endothelium can modulate the supply of growth factors to the underlying smooth muscle. In vitro experiments suggest that it may also influence the responsiveness of arterial smooth muscle to mitogens. In these experiments, we measured DNA synthesis in segments of carotid and renal arteries that were isolated from Wistar-Kyoto (WKY) rats and exposed to serum. Nuclear incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdUrd), was visualized by immunocytochemistry and the percentage of labeled nuclei (BrdUrd L%) was determined in the tunica media. In both types of artery isolated from 6- and 20-week-old WKY rats, mechanical removal of endothelium increased the BrdUrd L% in the tunica media. In carotid arteries of 20-week-old WKY rats, gentle denudation increased the incorporation of [3H]thymidine but not [14C]leucine. In denuded renal arteries of adult WKY rats, exogenous prostaglandin E2, iloprost, and transforming growth factor-beta (TGF-beta) reduced media labeling, which was not affected by Na nitroprusside. In renal arteries with endothelium, methylene blue and indomethacin did not affect medial DNA synthesis. These findings demonstrate that in arteries of young and adult rats, the endothelium reduces stimulated DNA synthesis. It is unlikely that basal production of nitric oxide or prostaglandins is involved herein. Endothelial inhibition of DNA synthesis but not protein synthesis in arteries indicates that the endothelium may influence the extent of arterial smooth muscle hypertrophy and hyperplasia.

The volumes of distribution of the beta-adrenoceptor blocking agents propranolol and atenolol, and the calcium antagonist verapamil, during exercise have been investigated. Changes in the plasma concentrations of atenolol and propranolol during exhaustive exercise at 70% of maximal aerobic power were compared after 1 week of oral treatment (propranolol 80 mg b.d. and atenolol 100 mg once daily) in 12 healthy volunteers. In a second study the effect of 10 min exercise at 50% of maximal aerobic power on steady state plasma concentrations of propranolol, atenolol and verapamil was compared in 7 healthy subjects. The drugs were administered by a continuous intravenous infusion. The plasma concentration of atenolol was not changed by exercise in either study, but the plasma concentrations of propranolol and verapamil were significantly increased in both studies. However, after correction for changes in plasma volume during exercise, the plasma propranolol concentration was not significantly elevated in the second study. From the results it is concluded that exercise led to a reduction in the volume of distribution of propranolol during prolonged exercise (25 min) at 70% Wmax, which was not clearly demonstrable during 10 min exercise at 50% Wmax. The volume of distribution of verapamil was reduced during 10 min exercise at 50% Wmax. No change in the volume of distribution of atenolol during exercise could be shown. The changes in the volumes of distribution of propranolol and verapamil during exercise may contribute to preventing an increase in the half-life of these drugs in patients performing prolonged physical exercise.