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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 μM l-NAME), blockade of ET A - and ET B -receptors (10 μM bosentan) and stimulation of the endothelium with 0.001-10 μM 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 μM) 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 μM 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.

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 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. 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-κB 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 cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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 α1-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±μm) constricted by the α1-adrenoceptor agonist phenylephrine. Tamsulosin (0.1nM) decreased phenylephrine sensitivity 17-fold (n=10). Quercetin (5, 10 and 20µM) also caused a decrease (2-, 4- and 6-fold respectively) of phenylephrine sensitivity, while 10µM 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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.

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( partial differential/ partial differential) 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.

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.

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.

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.

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 beta(1D)-integrin, melusin, Akt, GSK3beta, 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). beta(1D)-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-GSK3beta to GSK3beta (+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-GSK3beta, and MLP, indicating a strong drive to compensated hypertrophy.

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 microg/g) or high (100 microg/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.

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 beta-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 beta-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 beta-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 beta-adrenergic stimulation (both P<0.01). No net Ang II release across adipose tissue and skeletal muscle could be detected in both groups of subjects. However, AGT was released from adipose tissue and muscle during beta-adrenergic stimulation in obese subjects (both P<0.05). In conclusion, locally produced Ang II in adipose tissue and skeletal muscle exerts no endocrine role in lean and obese subjects. In contrast, AGT is released from adipose tissue and muscle in obese subjects during beta-adrenergic stimulation, which may contribute to the increased plasma Ang II concentrations during beta-adrenergic stimulation in obese subjects.

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 micromol 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 micromol 100 ml forearm tissue(-1) min(-1), p = 0.81) and IMTG content (before: 45.4 +/- 18.8, after: 48.8 +/- 27.5 micromol/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.

It is assumed that the combined effects of multiple common genetic variants explain a large part of variation of high-density lipoprotein cholesterol (HDL-C) plasma levels, but little evidence exists to corroborate this assumption. It was our objective to study the contribution of multiple common genetic variants of HDL-C-related genes to variation of HDL-C plasma levels. A well-characterized cohort of 546 Caucasian men with documented coronary artery disease was genotyped for common functional variants in genes that control reverse cholesterol transport: ATP-binding cassette transporter A1, apolipoprotein A-I and apolipoprotein-E, cholesteryl ester transfer protein, hepatic lipase, lecithin : cholesterol-acyl transferase, lipoprotein lipase, and scavenger receptor class B type 1. Multivariate linear regression showed that these variants, in conjunction, explain 12.4% (95% confidence interval: 6.9-17.9%) of variation in HDL-C plasma levels. When the covariates smoking and body mass index were taken into account, the explained variation increased to 15.3% (9.4-21.2%), and when 10 two-way interactions were incorporated, this percentage rose to 25.2% (18.9-31.5%). This study supports the hypothesis that multiple, mildly penetrant, but highly prevalent genetic variants explain part of the variation of HDL-C plasma levels, albeit to a very modest extent. Multiple environmental and genetic influences on HDL-C plasma levels still have to be elucidated.

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.

Changes in arterial stiffness and structure occur during cardiovascular diseases and can be modified by angiotensin-converting enzyme (ACE) inhibitors. In the present study we investigated the role of membrane-bound ACE (t-ACE) in the regulation of arterial structure and mechanics. Large and small arteries of t-ACE-/- mice were isolated to determine the passive pressure-diameter relationship. We observed that t-ACE-/- mice exhibit a reduced arterial distensibility compared to t-ACE+/+ mice. This reduced arterial distensibility was also observed after 9 weeks of captopril treatment (80 mg/kg/ day). We hypothesized that bradykinin type 2 receptor (BK(2)) stimulation might be involved in the regulation of arterial stiffness. t-ACE-/- and t-ACE+/+ mice were treated with Hoe 140 (1 mg/kg/day) for 14 days. After Hoe 140 treatment, both the structural and mechanical changes observed in the t-ACE-/- carotid artery were abolished. Although Hoe 140 administration increased blood pressure in both groups by approximately 10 mm Hg, the pressure difference between the two groups did not change. Thus, t-ACE is involved in the regulation of arterial distensibility. The changes observed in t-ACE-/- mice are not caused by an altered fetal development. Moreover, it is likely that the regulation of arterial distensibility by ACE involves stimulation of the BK(2) receptor., (Copyright 2004 S. Karger AG, Basel)

Objective: Tissue kallikrein (TK) participates in acute flow-induced dilatation (FID) of large arteries. We investigated whether TK deficiency blunts FID and alters chronic flow-related arterial structural and functional changes in resistance-sized muscular arteries., Methods and Results: Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, 18- to 19-day pregnant, and 7-day postpartum TK-/- and TK+/+ littermate mice. In TK-/- mice, values of diameter, medial cross-sectional area (CSA), myogenic tone, and dilatation in response to acetylcholine were comparable to those values in TK+/+ mice, but FID (0 to 100 microL/min) was significantly reduced (55+/-4% versus 85+/-4% in TK+/+ mice). In both mouse strains, pregnancy resulted in significant increases in diameter and medial CSA and in the Nw-nitro-l-arginine methyl ester-sensitive component of FID. By 7 days after pregnancy, uterine arterial diameter and CSA values no longer differed from nonpregnant values, and FID was markedly reduced in TK-/- and TK+/+ mice., Conclusions: These observations (1) confirm at the level of resistance arteries the key role of TK in FID and (2) indicate that TK deficiency does not compromise arterial remodeling and changes in the contribution of NO to FID during and after pregnancy.

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.

Objective: Since renal sympathetic nerves are involved in the regulation of sodium excretion, we investigated whether treatment with urapidil, an alpha1-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.

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 microm [LCA] and 723+/-14 microm [RCA] for V-/- versus 808+/-20 microm [LCA] and 796+/-20 microm [RCA] for V+/+) and medial cross-sectional area (CSAm) were significantly smaller in V-/- (21+/-1 and 22+/-2 x 10(3) microm(2) for LCA and RCA, respectively) than in V+/+ (28+/-2 and 28+/-3 x 10(3) microm(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 microm), and CSAm was significantly increased (5 x 10(3) microm(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 microm) and CSAm was increased to a larger extent (10 x 10(3) microm(2)) than in V+/+. In contrast to V+/+, the high-flow RCA of V-/- displayed a significant increase in diameter (52 microm) and a significant increase in CSAm (5 x 10(3) microm(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.

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, P<0.01) and 11beta-HSD2 protein (4-fold, P<0.01), whereas 11beta-HSD1 protein was decreased (no longer detected, P<0.05). 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.

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.

We evaluated the relationship between the presence of adrenergic nerves and the presence of alpha-1 adrenoceptors (alpha-1 AR) in the arterial tree of the rat. The thoracic aorta and the carotid, mammary, renal and femoral arteries were isolated from 20-week-old male WKY rats, along with the superior mesenteric artery and small (first order) and resistance-sized (third order) side branches of this vessel. Norepinephrine content ([NE]) and specific binding of 300 pM [3H]prazosin were determined. To estimate the total density of alpha-1 AR ([alpha-1 AR]) as well as the density of alpha-1A AR ([alpha-1A AR]), binding experiments were performed with and without pretreatment of the preparations with the irreversible alpha-1B AR and alpha-1D AR antagonist chloroethylclonidine and in the absence and presence of the alpha-1A AR selective ligand (+)-niguldipine (30 nM). Also the presence of mRNA for alpha-1A AR was evaluated by use of reverse transcriptase-polymerase chain reaction (RT-PCR). In intact rats, arterial [NE] ranged between 0.1 and 15 ng/microgram DNA, arterial [alpha-1 AR] ranged between 12.4 and 46.8 fmol/mg protein and [alpha-1A AR] ranged between 0.05 and 27.9 fmol/mg protein. There was no significant correlation between [alpha-1 AR] and [NE]. However, with respect to the [alpha-1A AR] a significant correlation between [NE] and [alpha-1A AR] was observed. RT-PCR analysis confirmed the expression of alpha-1A AR in the densely innervated mesenteric resistance-sized arteries. Two weeks after chemical sympathectomy of the rats with 6-hydroxydopamine (i) arterial [NE] was markedly reduced, and (ii) a distinct reduction in the [alpha-1A AR] as percentage of the total [alpha-1 AR] density in mesenteric artery side branches was noted. These findings indicate that there is a positive and reversible relationship between the presence of adrenergic nerves and that of alpha-1A AR in rat arteries.

The influence of hypervolemia on hemodynamics and interdialytic blood pressure, as well as in relation to vascular compliance, was investigated in 10 hemodialysis patients who were not receiving vasoactive medication. All subjects were studied during a relative normovolemic interdialytic period (from 1 kg below dry weight postdialytic until dry weight predialytic) and a hypervolemic interdialytic period (from 1 kg above dry weight postdialytic until 3 kg above dry weight predialytic). Interdialytic blood pressure was measured with an ambulatory blood pressure monitor. Cardiac output was echographically measured and total peripheral resistance calculated postdialytic, mid-interdialytic, and predialytic. At the same time, a blood sample was drawn for analyzing vasoactive hormones, sodium, and hematocrit. In all patients, ideal dry weight was estimated by echography of the caval vein. Arterial and venous compliance were measured with an ultrasound vessel wall movement detector system and a strain-gauge plethysmograph. After fluid load, an increase in intravascular volume, an increase in caval vein diameter and cardiac output, and a decrease in peripheral resistance was observed. No significant influence of a 3-L fluid load was found on interdialytic blood pressure course (153+/-24 mm Hg/90+/-19 mm Hg in the hypervolemic period and 146+/-27 mm Hg/89+/-22 mm Hg in the normovolemic period). Sodium and osmolality were similar in the hypervolemic and normovolemic interdialytic periods. After fluid load, a decrease in arginine vasopressin and angiotensin II was observed, which probably contributed to the decreased systemic vascular resistance. Catecholamines were not influenced by fluid load, but increased during the interdialytic period, suggesting accumulation after dialysis. Three of the 10 patients had higher systolic but not diastolic blood pressures after fluid load (159+/-13 mm Hg/81+/-22 mm Hg in the hypervolemic period and 135+/-16 mm Hg/81+/-22 mm Hg in the normovolemic period). No correlation could be found between arterial or venous compliance and blood pressure changes. We concluded that a 3-L interdialytic fluid load does not result in higher blood pressure in most hemodialysis patients.

Unlabelled: 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 ED50 and peak pressor response to acute ANG II injections were comparable in all groups, confirming the presence of functionally intact AT1 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.

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.

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 (> or = 1.5). Patients with renal artery stenosis were older (P < .01) and had higher systolic pressures (P < .05) than patients with essential hypertension. Arterial levels of ANP were significantly higher in patients with unilateral or bilateral renal artery stenosis than in patients with essential hypertension (P < .05). Patients with hypertension and left ventricular hypertrophy had significantly higher arterial ANP levels than those with no hypertrophy (40 versus 26 pmol/L, P < .05), but in patients with renal artery stenosis, arterial ANP levels were similar in those with or without hypertrophy. Renal venous ANP levels were significantly higher in stenotic than in normal kidneys. Moreover, in unilateral renal artery stenosis, stenotic kidneys of patients with an elevated renin ratio (stenotic kidney/contralateral kidney > or = 1.5) had a significantly higher renal venous ANP level than stenotic kidneys of patients with normal renin ratio (30 versus 17 pmol/L, P < .05). However, the median fractional extraction of ANP was similar, around 0.50 (range, 0 to 0,83), in normal kidneys of hypertensive patients and in stenotic and contralateral kidneys of patients with renal artery stenosis. A significant inverse correlation between arterial ANP and renal venous active plasma renin concentration was found for normal kidneys (r= -.62, P < .01) of hypertensive patients without hypertrophy. However, for stenotic kidneys, no such relationship was apparent. A significant correlation between arterial ANP and the arteriovenous difference of ANP (r = +.92, P < .001) was found. This relationship was similar for normal and stenotic kidneys. In conclusion, an inverse relationship between arterial ANP and renal venous active plasma renin concentration exists in normal kidneys of essential hypertensive patients without left ventricular hypertrophy. Furthermore, data of ANP extraction through normal and stenotic kidneys suggest that saturation of ANP extraction does not occur. Increased levels of ANP in renal artery stenosis are likely caused by enhanced cardiac secretion of this peptide.

In forty six hypertensive patients in whom a high level of clinical suspicion for renovascular hypertension was present on the basis of clinical clues, a captopril test was performed with either 25 mg of captopril or placebo on 2 separate days to determine prospectively the value of the captopril test. Blood pressure (BP) and peripheral renin were used as response variables. All patients had discontinued their anti-hypertensive medication and were not salt depleted. In all patients selective renal angiography was performed irrespective of the results of the captopril test. Twenty patients proved to have uni- or bilateral renal artery stenosis (RAS) giving a prevalence of 43%. After the placebo and after captopril there were no significant changes (absolute or proportional) in BP values between patients with essential hypertension or RAS, either for all measurements or if only the fall in BP was taken into account. The receiver operator characteristic curves of both baseline and post-captopril peripheral renin levels indicate that the captopril test does not discriminate appropriately between patients with essential hypertension and RAS. Therefore, we would not advise the use of the captopril test as a screening test for RAS in hypertensive patients in whom a high level of clinical suspicion for RAS is already present.

Immunoreactive endothelin (ir-ET) levels were measured in the renal veins and aorta of 43 untreated hypertensive patients immediately before renal angiography. None of the patients used antihypertensive medication. Twenty-seven patients had renal artery stenosis, 17 of which were unilateral and 10 bilateral. Seven of the 17 patients with unilateral renal artery stenosis had an elevated renin ratio. Of the 16 patients with essential hypertension 6 had a unilateral small kidney with a normal blood supply. Although there was a trend towards higher levels of ir-ET in patients with renal artery stenosis, no significant differences in endothelin levels (venous or arterial) were found between different groups of patients or groups of kidneys. More than 75% of kidneys extracted endothelin, there being no significant differences between groups of kidneys. In conclusion, our data demonstrate that endothelin levels and renal endothelin extraction are comparable in essential hypertension and in hypertension associated with renal artery stenosis. Whereas renal uptake or endothelin is the rule, some kidneys, however, release this peptide irrespective of the presence or absence of renal artery stenosis.

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.

Objectives: To compare DNA synthesis in isolated arteries of normotensive and hypertensive rats and to evaluate whether removal of the endothelium affects this process., Design: Carotid and renal artery segments were isolated from normotensive Wistar, Wistar-Kyoto (WKY) and Sprague-Dawley rats, and from spontaneously hypertensive rats (SHR), transgenic Sprague-Dawley rats harbouring the mouse Ren-2 gene and from WKY rats rendered hypertensive by aortic coarctation., Methods: Artery segments were exposed in vitro to serum with or without previous gentle removal of the endothelium. Nuclear incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine was visualized by immunocytochemistry and the percentage of labelled medial nuclei was determined., Results: In both types of artery, obtained from 6-week-old WKY rats and from 6-week-old SHR, removal of endothelium increased the percentage of 5-bromo-2'-deoxyuridine-labelled medial nuclei (L%). Also, in the arteries of 20-week-old Wistar rats, WKY rats and WKY rats rendered hypertensive by aortic coarctation and in vessels of 11-week-old Sprague-Dawley rats and Sprague-Dawley rats harbouring the mouse Ren-2 gene, removal of endothelium increased L%. Conversely, in the arteries of 20-week-old SHR removal of the endothelium did not alter L%. Furthermore, maximally stimulated DNA synthesis was considerably smaller in de-endothelialized arteries of adult SHR than in denuded vessels from the other strains and models., Conclusion: These findings confirm that the endothelium can reduce DNA synthesis in the intact rat arterial smooth muscle. This effect is not modified by hypertension, but is selectively reduced in the arteries of adult SHR.

We evaluated long-term actions of angiotensin II (ATII) on arterial smooth muscle. In isolated sympathectomized renal artery segments that had been denuded of endothelium, isometric force was recorded during 3 days of incubation in nutrient medium with and without 1 microM ATII. The peptide induced, after an acute transient contraction and a latency of at least 12 hr, a slowly developing tonic increase in wall tension. This chronic effect was not influenced by indomethacin but could be reversed by sodium-nitroprusside. In vessels that had been chronically exposed to ATII, acute contractile responses to high potassium, serotonin and ATII were not altered. Also effects on agonist-induced tone of removal and readministration of extracellular potassium were not modified. Relaxing responses after exposure to NH4Cl were attenuated. During continuous exposure of isolated arteries to ATII, release of a stable contractile factor could not be detected, nuclear incorporation of the thymidine analog 5-bromo-2'-deoxyuridine was not stimulated, but the media cross-sectional area was significantly increased. These observations indicate that ATII induces in arterial smooth muscle, besides its well known acute contractile effect and its trophic action, a long-term tonic increase in tone. The mechanism largely remains to be established, but may involve altered cellular handling of hydrogen ions.

We evaluated whether chronic alpha 1-adrenergic stimulation, angiotensin II (AII), or increased blood pressure (BP) alters resistance arterial structure and function. Structural parameters and wall tension were recorded in mesenteric small arteries (MrA) isolated from 6-week-old normotensive Wistar Kyoto rats that had been infused for 4 days with saline (WKY), 2 mg/kg/day phenylephrine (WKY + PHE), or 0.3 mg/kg/day AII (WKY + AII) and from saline-infused spontaneously hypertensive rats (SHR). During the experimental period, systolic BP (SBP) did not change in WKY but increased in WKY + PHE, WKY + AII, and SHR. Relative cardiac mass did not differ between SHR and WKY, but was increased in WKY + PHE and WKY + AII. Stiffness and optimal lumen diameter of MrA did not differ between WKY and SHR and were not altered in WKY + PHE or WKY + AII. Maximal contractile responses and sensitivities for vasconstrictors and calcium in vessels of WKY + AII and SHR did not differ from those in WKY. In vessels of WKY + PHE, maximal responses to vasoconstrictors and sensitivities for norepinephrine (NE) and PHE were reduced. Relaxing responses to isoproterenol (ISO) and Na-nitroprusside did not differ between SHR and WKY and were not altered in WKY + PHE and WKY + AII. Those to acetylcholine (ACh) were reduced in WKY + PHE. Media cross-sectional area and media thickness were significantly larger in WKY + AII and SHR as compared with WKY but were not altered in WKY + PHE. These data indicate that in young rats AII leads to small artery hypertrophy and that neither increased BP or increased vasconstriction appear to be involved therein. Chronic alpha 1-adrenergic stimulation, on the other hand, did not modify small artery structure but resulted in nonselective reduction of arterial smooth muscle contractile reactivity.

Observations in cell culture and during arterial organoid culture indicate that the endothelium may influence arterial wall mass. The endothelium can inhibit and promote stimulation of DNA synthesis in the underlying smooth muscle depending on the experimental conditions, the type of vessel, and the species. The process seems to be altered during neointima formation and in a genetic model of essential hypertension. In these respects, endothelial influences on arterial smooth growth resemble endothelial influences on arterial smooth muscle tone. The mediators that are involved, however, may differ.

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.

We compared the distribution of DNA synthesis over the arterial tree of young normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) with marginally elevated blood pressure. Six-week-old male SHR and WKY rats were therefore infused with 5-bromo-2'-deoxyuridine (BrdUrd) for 2 days and the nuclear incorporation of the thymidine analogue in the media of various arteries was determined by immunohistochemistry. In WKY rats, 2.5% of the arterial smooth muscle nuclei in elastic, muscular and resistance arteries incorporated BrdUrd. In SHR, DNA synthesis was more marked in large arteries than in resistance arteries. It was in addition significantly larger in the aorta, superior mesenteric, renal and femoral arteries of the SHR than in those of the WKY rats. However, nuclear incorporation of BrdUrd in vivo did not differ between SHR and WKY rats in aortic endothelium, carotid arterial smooth muscle, nor in mesenteric or renal resistance arteries. Between 6 and 20 weeks of age, the number of nuclear profiles per media cross-section did not increase in large arteries of WKY rats and SHR. During this period of time, however, carotid artery and thoracic aorta weight and DNA content increased. SHR large arteries gained more DNA than those of WKY rats. These data indicate that DNA synthesis is uniformly distributed over the arterial system in young WKY rats and that DNA synthesis is elevated in the smooth muscle of large arteries of 6-week-old SHR but not in their resistance arteries.

To evaluate whether intravascular phenomena contribute to local differences in growth responses of the arterial wall, we evaluated responses to organoid culture in a broad variety of arterial preparations. Arterial segments were isolated from adult, normotensive rats, sympathectomized, denuded from endothelium, and suspended in medium supplemented with serum. As judged from the nuclear incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdUrd), this induced a transient stimulation of DNA synthesis in only a fraction of the arterial smooth muscle cells in all types of arteries. This intramedial DNA synthesis was more marked in renal arteries than in carotid arteries or aortae and was least pronounced in main pulmonary, femoral, and superior mesenteric artery and in mesenteric resistance-sized arteries. Organoid culture of isolated arteries did not increase the cross-sectional area of the media or the number of medial cells. It rather resulted in proliferation of smooth-muscle-like cells outside the media. In addition, smooth-muscle-like cells migrated out of the isolated arterial segments during culture. The rate of proliferation of these isolated cells did not differ between large arteries of different anatomical origin. However, isolated cells derived from mesenteric resistance arteries proliferated at a rate that was 4 times slower than that of large artery cells. The presence of endothelium significantly reduced medial DNA synthesis in carotid and renal artery segments, but not in mesenteric resistance-sized preparations. These data indicate that growth responses of the arterial wall differ quantitatively with the anatomical location and branching order of the vascular segment. In addition to the regional heterogeneity of endothelial effects on mitogenic responses of arterial smooth muscle, this seems to be due to regional differences in the susceptibility of arterial smooth muscle to exogenous growth factors. In this respect, we speculate that subsets of growth-resistant and growth-prone arterial smooth muscle cells could be heterogeneously distributed over the arterial tree.

We evaluated effects of inhibitors of angiotensin converting enzyme (ACE) on growth responses in isolated rat carotid arteries (CA) during continuous exposure to exogenous growth factors in vitro. In freshly isolated CA of adult Wistar-Kyoto rats (WKY) that had been denuded of endothelium, angiotensin-I (AT-I) and angiotensin-II (AT-II) induced concentration dependent contractions (1 nM to 1 microM). Captopril (100 microM) and lisinopril (10 microM) did not affect responses to AT-II, but increased the ED50 for AT-I 50-fold. To study the effects of ACE inhibition on arterial growth responses in vitro, we measured nuclear incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdUrd), during organoid culture of isolated CA segments in continuous presence of 20% calf serum. During 4 days of organoid culture 7-10% of the medial smooth muscle nuclei incorporated BrdUrd. During long-term culture (15 days) a new layer of cells developed at the adventitial border of the arterial segments. Continuous presence of captopril (10 microM to 100 microM) or lisinopril (1 microM to 10 microM) did not affect the extent of DNA synthesis in the media or in the newly formed cell layer. Unlike endothelin-1 (ET-1), exogenous AT-II did not stimulate DNA synthesis in isolated renal artery segments. These observations indicate the presence of ACE accessible to exogenous AT-I in arterial compartments other than the endothelium. Interference with this enzyme did not alter growth responses of the isolated arterial wall to serum. Since, in addition, exogenous AT-II did not stimulate intra-arterial DNA synthesis, effects of ACE-inhibitors on arterial growth responses in vivo are most likely due to an indirect effect.

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.

In a double-blind, placebo-controlled cross-over study, the plasma atrial natriuretic peptide (ANP) levels of nine young and ten elderly hypertensive patients were compared after placebo and after treatment with 120 mg verapamil given three times daily over 4 weeks. During placebo, plasma ANP levels proved to be higher in elderly patients than in young subjects. Chronic treatment with verapamil induced a rise in ANP levels in both young and elderly patients with hypertension.