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Objectives: Methods for conservation and preservation of vascular grafts are often controversially discussed. Furthermore, immunologic monitoring or immunotherapy for allogeneic graft is not considered necessary in many cases. The present study was initiated to examine the cellular vitality and functional efficiency of vein transplant during preservation., Materials and Methods: Twenty-seven human vein segments (vena saphena magna) were stored after explant in University of Wisconsin solution or histidine-tryptophan-ketoglutarate solution at 4 °C. After 3, 24, 48, 72, and 96 hours, vein functionality was tested. Ring segments were fixed by triangles in Krebs-Henseleit buffer. Contractile function was measured after addition of potassium chloride solution (80 mM) and phenylephrine (0.2, 2, or 20 μM). To investigate endothelium-dependent vasorelaxation, 1 μM acetylcholine was added., Results: Of 27 segments, 5 showed endothelium-dependent relaxation. Vasorelaxation continued for up to 48 hours after administration of acetylcholine in University of Wisconsin solution and for up to 24 hours in histidine-tryptophane-ketoglutarate solution. At 48 hours, potassium chloride solution-induced vasocontraction was 17% more effective than phenylephrine in University of Wisconsin solution. University of Wisconsin solution was significantly more effective than histidine-tryptophane-ketoglutarate solution in terms of preservation of phenylephrine (0.2, 2 μM)-induced vasocontraction. Phenylephrine (2 μM)-induced contraction was retained in University of Wisconsin solution after 24 hours by 81% and after 48 hours by 55%, with comparable results in histidine-tryptophane-ketoglutarate solution of only 62% and 34% after 24 and 48 hours., Conclusions: At 48 hours, human saphenous vein transplants had better endothelium and smooth muscle function when preserved in University of Wisconsin solution versus histidine-tryptophane-ketoglutarate solution.

Aims: Vascular oxidative stress generated by endothelial NO synthase (eNOS) was observed in experimental and clinical cardiovascular disease, but its relative importance for vascular pathologies is unclear. We investigated the impact of eNOS-dependent vascular oxidative stress on endothelial function and on neointimal hyperplasia., Results: A dimer-destabilized mutant of bovine eNOS where cysteine 101 was replaced by alanine was cloned and introduced into an eNOS-deficient mouse strain (eNOS-KO) in an endothelial-specific manner. Destabilization of mutant eNOS in cells and eNOS-KO was confirmed by the reduced dimer/monomer ratio. Purified mutant eNOS and transfected cells generated less citrulline and NO, respectively, while superoxide generation was enhanced. In eNOS-KO, introduction of mutant eNOS caused a 2.3-3.7-fold increase in superoxide and peroxynitrite formation in the aorta and myocardium. This was completely blunted by an NOS inhibitor. Nevertheless, expression of mutant eNOS in eNOS-KO completely restored maximal aortic endothelium-dependent relaxation to acetylcholine. Neointimal hyperplasia induced by carotid binding was much larger in eNOS-KO than in mutant eNOS-KO and C57BL/6, while the latter strains showed comparable hyperplasia. Likewise, vascular remodeling was blunted in eNOS-KO only., Innovation: Our results provide the first in vivo evidence that eNOS-dependent oxidative stress is unlikely to be an initial cause of impaired endothelium-dependent vasodilation and/or a pathologic factor promoting intimal hyperplasia. These findings highlight the importance of other sources of vascular oxidative stress in cardiovascular disease., Conclusion: eNOS-dependent oxidative stress is unlikely to induce functional vascular damage as long as concomitant generation of NO is preserved. This underlines the importance of current and new therapeutic strategies in improving endothelial NO generation.

The aim of the study was to evaluate the possible contribution of non-endothelial eNOS to the regulation of blood pressure (BP). To accomplish this, a double transgenic strain expressing eNOS exclusively in the vascular endothelium (eNOS-Tg/KO) has been generated by endothelial-specific targeting of bovine eNOS in eNOS-deficient mice (eNOS-KO). Expression of eNOS was evaluated in aorta, myocardium, kidney, brain stem and skeletal muscle. Organ bath studies revealed a complete normalization of aortic reactivity to acetylcholine, phenylephrine and the NO-donors in eNOS-Tg/KO. Function of eNOS in resistance arteries was demonstrated by acute i.v. infusion of acetylcholine and the NOS-inhibitor L-NAME. Acetylcholine decreased mean arterial pressure in all strains but eNOS-KO responded significantly less sensitive as compared eNOS-Tg/KO and C57BL/6. Likewise, acute i.v. L-NAME application elevated mean arterial pressure in C57BL/6 and eNOS-Tg/KO, but not in eNOS-KO. In striking contrast to these findings, mean, systolic and diastolic BP in eNOS-Tg/KO remained significantly elevated and was similar to values of eNOS-KO. Chronic oral treatment with L-NAME increased BP to the level of eNOS-KO only in C57BL/6, but had no effect on hypertension in eNOS-KO and eNOS-Tg/KO. Taken together, functional reconstitution of eNOS in the vasculature of eNOS-KO not even partially lowered BP. These data suggest that the activity of eNOS expressed in non-vascular tissue might play a role in physiologic BP regulation., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The post surgery success of coronary artery bypass grafting (CABG) is counteracted by thrombosis and de-endothelialization, intimal hyperplasia and, over the long term, atherosclerosis. There are many reasons to assume that in CABG patients vascular bioavailability of NO generated by the endothelium plays an important role for graft function. This holds true for factors such as graft type, harvesting and storage, the type of surgery, non-pharmacologic prevention of risk factors, for example, regular physical activity (if feasible), and drug therapy. Although the precise role of graft endothelial NO bioavailability for graft patency and clinical endpoints is still uncertain, current data rather speak in favor of NO indicating that the potential of vasoprotective activities of NO in the CABG patient deserves further investigation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The number of circulating stem cells with endothelial progenitor capacity (EPCs) inversely correlates with the number of cardiovascular risk factors. In this study we sought to investigate the effects of vascular H(2)O(2) on circulating EPC levels. In C57BL/6 mice 3 weeks of freely moving or forced physical activity or voluntary exercise failed to increase circulating EPCs defined as double positive for Flk-1 and CD34, CD133 or Sca-1. Likewise, neither insertion of additional genes encoding for catalase (cat(++)) or eNOS nor eNOS knock-out changed EPCs in resting mice. In striking contrast, inhibition of catalase by aminotriazole strongly reduced circulating EPCs in sedentary cat(++) and their transgen-negative littermates (cat(n)), while forced or voluntary exercise training of cat(++) mice significantly increased the number of circulating EPCs. The latter effect was completely inhibitable by aminotriazole. These data suggest that endogenous vascular H(2)O(2) likely contributes to the impairment of important stem cell-induced vascular repair mechanisms in cardiovascular disease.

The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.

Background: The efficacy of angiotensin-converting enzyme (ACE) inhibitors has been demonstrated in large clinical trials, but knowledge of the underlying mechanisms remains incomplete. Therefore, this study investigated the impact of ACE inhibitor therapy on cardiac nitric oxide (NO) synthases in patients with coronary artery disease (CAD) or heart failure., Patients and Methods: The mRNA expression was quantified by standard calibrated competitive RT-PCR, protein expression by Western blotting and NOS activity by monitoring the conversion of [3H]arginine to [3H]citrulline during enzymatic formation of NO in tissue homogenates of myocardium of patients with, or without, ACE inhibitor treatment before elective coronary artery bypass grafting or heart transplantation., Results: The mRNA expression (amol microg(-1) RNA) of endothelial NO synthase (eNOS) was higher (22.5 +/- 4.8, n = 23) in the atrial myocardium of patients taking ACE inhibitor treatment, before elective coronary artery bypass grafting, compared with patients not taking this therapy (8.9 +/- 0.7, n = 33, P < 0.0001). The ACE inhibitor therapy increased eNOS protein expression from [(9 +/- 0.7) relative units (RUs) to (12 +/- 0.9) RUs, P < 0.05, respectively] and cardiac NOS activity from 17.6 +/- 1.3 to 23.7 +/- 1.1 pmol mg protein(-1) min(-1) (P < 0.001, respectively). Inducible and neuronal NO synthase expression was not changed by the ACE inhibition. A similar up-regulation of eNOS by ACE inhibition was found in the left ventricles of patients with heart failure. The augmented endothelial NOS expression and activity was not the result of differences in clinical characteristics and concomitant therapy between the patient groups., Conclusion: Increased eNOS expression and activity might contribute to the beneficial effects of ACE inhibitor therapy in the treatment of CAD and heart failure.

Hydrogen peroxide (H2O2) has been implicated as a component of oxidative ischaemia/reperfusion stress. We investigated the role of H2O2 in cardiovascular ischaemia/reperfusion stress in hearts from mice overexpressing catalase in their endothelial cells. Hearts of transgenic (TG, n = 9) and age-matched wild-type (WT, n = 7) mice were perfused at constant flow (2.2 mlmin(-1)) and subjected to brief ischaemia and reperfusion. Intrinsic function and the effects of norepinephrine (3 nM-3 microM) were determined. Left ventricular pressure (LVDevP; balloon method), end-diastolic pressure (LVEDP), maximum rates of pressure development (+dP/dt, -dP/dt), coronary perfusion pressure (index of vascular function) and heart rate were recorded. Apart from a slightly higher recovery of LVDevP during reperfusion (+6 mmHg), neither systolic nor diastolic function was improved during ischaemia or reperfusion in TG hearts. However, hearts from TG mice exhibited a significantly better contractile response to noradrenergic stimulation (LVDevP: +20 mHg or 1.15-fold increase; +dP/dt: +1476 mmHgs(-1) or 1.2-fold increase) (P < 0.05). Norepinephrine relaxed the coronary microvasculature and increased heart rate, but no differences were detected between groups. We conclude that overexpressing catalase in endothelial cells is only weakly protective against myocardial or vascular ischaemia/reperfusion injury, but preserves the responsiveness of the heart to adrenergic stimulation.

Objective: Recent studies from our groups have indicated that endothelial nitric oxide synthase (eNOS) expression is increased in cell culture by both shear stress and by hydrogen peroxide (H(2)O(2)). In vivo, exercise training, known to increase both endothelial shear stress and oxidative stress, also increases eNOS expression. It is unclear if H(2)O(2) contributes to an increase in eNOS expression in response to exercise training., Methods: To address this question, we generated mice overexpressing human catalase (hCat) driven by the murine Tie-2 promoter to specifically target this transgene to the endothelium (cat(++))., Results: Vessels of cat(++) expressed significantly higher levels of catalase mRNA and catalase protein and activity but normal levels of eNOS. Exercise alone had no effect on catalase expression in C57BL/6. Wild-type littermates of cat(++) showed an increase in eNOS expression with 3 weeks of exercise (2.53+/-0.42-fold) comparable to C57BL/6 (2.93+/-0.45-fold). In striking contrast, 3 weeks of exercise had no effect on aortic (1.33+/-0.32-fold) and myocardial (1.1+/-0.2-fold) eNOS expression in catalase transgenic mice., Conclusions: These data suggest that endogenous H(2)O(2) plays a key role in the endothelial adaptation to exercise training by stimulating an up-regulation of eNOS.

Objectives: We sought to determine if physical inactivity affects endothelial function in young healthy individuals., Background: Recent studies have linked exercise training to increased bioavailability of vascular nitric oxide (NO) and to improved endothelial function in patients with cardiovascular disorders. The effects of physical inactivity on normal vascular endothelial function are not known., Methods: Healthy young male C57Bl/6 mice living in groups of five in large cages, where they were running, climbing, and fighting during their active cycle, were randomly assigned to stay there or to live alone in small cages where they were predominantly resting. After five and nine weeks citrate synthase activity (a measure of mitochondrial respiratory chain activity), heart weight/body weight ratio, vascular reactivity, and protein expression of endothelial nitric oxide synthase (eNOS) were assessed., Results: Singularized mice showed a reduction of citrate synthase activity (p < 0.05), of endothelium-dependent vasorelaxation (to 65 +/- 5% of control levels; p < 0.001), and of eNOS protein expression (to 53 +/- 8% of control levels; p < 0.01). In striking contrast, vascular responses to potassium chloride, phenylephrine, and the NO-donor racemic S-nitroso-N-acetylpenicillamine were unchanged. The alterations of vascular eNOS-activity were completely reversible when singularized mice underwent exercise. In mice living in groups, exercise showed only a small effect on aortic eNOS expression., Conclusions: In young healthy individuals physical inactivity induces endothelial dysfunction, which is completely reversible by a short period of moderate exercise training. We suggest that physical inactivity, the so-called sedentary lifestyle, increases cardiovascular risk in young healthy individuals by inducing endothelial dysfunction.

Objectives: We sought to investigate the effects of orally administered, long-term, eccentric isosorbide mononitrate (ISMN) on endothelial function., Background: Previous studies have shown that nitrate tolerance induced by continuous transdermal glyceryl trinitrate (GTN) is associated with increased vascular superoxide production and endothelial dysfunction. In contrast, it is unclear whether vascular superoxide increases during eccentric administration of oral nitrates, which is a widely used therapeutic dosing regimen., Methods: New Zealand White rabbits were randomly classified into three groups (n = 10, each) that received either placebo, ISMN at 2 mg/kg body weight per day (ISMN-2), or ISMN at 200 mg/kg body weight per day (ISMN-200) in an eccentric, twice-daily scheme for four months. Animals were sacrificed 3 h after application of the last ISMN dose., Results: The continuously present, lowest ISMN plasma levels (ng/ml) were 4.8 +/- 0.2 in ISMN-2 and 14.5 +/- 4 in ISMN-200 (p = 0.026). Treatment with ISMN had no effect on aortic reactivity to phenylephrine, acetylcholine, or the nitric oxide (NO) donor S-nitroso-N-acetyl-D,L-penicillamine, while the half-maximal effective concentration of ISMN (EC(50)-value in -logM) was shifted from 5.23 +/- 0.03 (placebo) to 4.69 +/- 0.04 (ISMN-200) (p < 0.0001 by analysis of variance). This moderate in vivo nitrate tolerance was not associated with increased aortic superoxide production (5 micromol/l lucigenin). The cumulative (20-min) lucigenin signals (cpm/mg) were 211 +/- 34 (ISMN-200) and 230 +/- 22 (placebo) (p = 0.415)., Conclusions: Long-term treatment with high-dose, eccentric ISMN does not increase vascular superoxide production and/or impair endothelium-dependent vasorelaxation, despite the development of moderate nitrate tolerance. Thus, it is unlikely that long-term anti-ischemic treatment with ISMN aggravates endothelial dysfunction in coronary artery disease.

Recent results suggested that long-term treatment with a low dose of the organic nitrate pentaerythritol tetranitrate (PETN, 6 mg kg(-1) per day) for 16 weeks slightly decreases aortic superoxide production in normal rabbits. We sought to determine if PETN can preserve endothelium dependent relaxation (EDR) in atherosclerotic rabbits. Three groups of 9 - 10 New Zealand White rabbits received a cholesterol chow (0.75%) for 16 weeks. One group (CHOL16) served as control and two groups were fed for another 16 weeks a cholesterol-chow without (CHOL32) or with 6 mg PETN kg(-1) per day (PETN32). Isolated aortic rings of CHOL16 showed a typical impairment of EDR with a maximal relaxation at 1 microM acetylcholine of 28+/-16%. In CHOL32-rings EDR was completely impaired. In striking contrast, EDR in PETN32 (24+/-15%) was similar to that of CHOL16 indicating a protective effect of PETN on endothelial function. Vascular superoxide production measured with the lucigenin method was not different between the groups. Aortic lesion formation in PETN32 was smaller than in CHOL32 (P<0.008). The onset of copper-induced LDL-oxidation (lag-time) after 16 weeks of cholesterol feeding (214+/-9 min) was reduced in CHOL32 (168+/-24 min, P=0.035) but not in PETN32 (220+/-21 min). This indicates prevention of increased LDL oxidation by PETN. The halfmaximal effective vasodilator concentrations of PETN (in -logM) were identical in CHOL16 (7.9+/-0.1), CHOL32 (7.6+/-0.2) and PETN32 (7.7+/-0.2). Similar results were obtained with S-nitroso-N-acetyl-D,L-penicillamine. These data suggest that PETN can reduce the progression of lesion formation, endothelial dysfunction and of LDL-oxidation in established atherosclerosis.

1. The differential responsiveness of various sections and regions in the vascular system to the vasodilator activity of organic nitrates is important for the beneficial antiischaemic effects of these drugs. In this study we examined the vasodilator activity of organic nitrates in cerebral arteries, where vasodilation causes substantial nitrate induced headache. 2. Isolated porcine basilar and coronary arteries were subjected to increasing concentrations of glyceryl trinitrate (GTN), isosorbide-5-nitrate (ISMN) and pentaerythritol tetranitrate (PETN). S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and endothelium-dependent vasodilation was investigated for comparison purpose. 3. The vasodilator potency (halfmaximal effective concentration in -logM) of GTN (4.33+/-0.1, n=8), ISMN (1.61+/-0.07, n=7) and PETN (>10 microM, n=7) in basilar arteries was more than 100 fold lower than that of GTN (6.52+/-0.06, n=12), ISMN (3.66+/-0.08, n=10) and PETN (6.3+/-0.13, n=8) observed in coronary arteries. 4. In striking contrast, the vasodilator potency of SNAP (halfmaximal effective concentration in -logM) was almost similar in basilar (7.76+/-0.05, n=7) and coronary arteries (7.59+/-0.05, n=9). Likewise, no difference in endothelium dependent relaxation was observed. 5. Denudation of the endothelium resulted in a small increase of the vasodilator potency (halfmaximal effective concentration in -logM) of GTN (4.84+/-0.09, n=7, P<0.03) in basilar arteries and similar results were obtained in the presence of the NO-synthase inhibitor N(omega)-nitro-L-arginine (4.59+/-0.05, n=9, P<0.03). 6. These results suggest that cerebral conductance blood vessels such as porcine basilar arteries seems to have a reduced expression and/or activity of certain cellular enzymatic electron transport systems such as cytochrome P450 enzymes, which are necessary to bioconvert organic nitrates to NO.

There is a growing body of evidence suggesting that numerous pathological conditions are associated with increased vascular production of reactive oxygen species. This form of vascular oxidant stress and particularly interactions between NO and oxygen-derived radicals represent a common pathological mechanism present in many so-called risk factors for atherosclerosis. Furthermore, reactive oxygen species seem to serve important cellular signalling mechanisms responsible for many of the features of vascular lesion formation. The mechanisms whereby vascular cells produce reactive oxygen species are only presently coming to light, and almost certainly will prove to be a focus for future therapies.

NO as produced by NO-synthases (NOS) contributes to the regulation of cardiovascular functions. In hypertension, there is a reduced production and/or activity of endogenous NO in the vasculature. We investigated if hypertension alters the NO-sensitivity of soluble guanylate cyclase (sGC) in blood vessels and heart muscle isolated from 15 month old spontaneously hypertensive rats (SHR15) and normal Wistar rats (WIS). Inhibition of NOS by 1 mM N omega-nitro-L-arginine decreased dP/dtmax in WIS (-27.6 +/- 3.4%) and SHR15 (-26.0 +/- 4.4%), while stimulation of NOS with 1 mM L-arginine increased dP/dtmax in WIS (9.9 +/- 0.7%) and SHR15 (8.9 +/- 2.3%). The positive inotropic response to 0.1 microM glyceryl trinitrate (GTN) was comparable in WIS (dP/dtmax: 4.5 +/- 1.7%) and SHR15 (dP/dtmax: 3.75 +/- 0.7%) as was the positive inotropic response to the NO-donor sodium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO, 1 microM) in WIS (dP/dtmax: 10.7 +/- 2.9%) and SHR15 (dP/dtmax: 5.1 +/- 1.5%, P = 0.1873). In aortas of SHR15 we found an increased superoxide production of 19.4 +/- 1.7 nM/mg/min (WIS: 6.1 +/- 0.6 nM/mg/min) in the smooth muscle and the endothelial layer. Endothelium-dependent relaxation by acetylcholine was markedly impaired in SHR15 as was the vasorelaxant activity of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), pentaerythritol tetranitrate and GTN. Maximal cGMP-production by sGC isolated from the lung and stimulated with SNAP (0.5 mM) was much lower in SHR15 (115 +/- 14 pmol/mg/min) than in WIS (348 +/- 36 pmol/mg/min). We suggest that hypertension is associated with a reduced activity of the sGC/cGMP-system in the vasculature but not in the heart muscle. Our results provide the first evidence that excess superoxide production in hypertension may trigger a desensitization of vascular sGC.

Objective: Nitric oxide (NO) has been suggested to have antiatherosclerotic effects. It has also been demonstrated that there is a greater basal release of endothelium derived relaxing factor (EDRF) in female as compared to male rabbit aorta, which also might have beneficial effects in atherosclerosis. We thus sought to determine if gender influences the severity of atherosclerosis., Methods: We studied 18 female and 18 male New Zealand White rabbits that were randomly divided in two groups of 9 animals each and fed either a standard or a cholesterol diet (0.75%) for 15 weeks., Results: In cholesterol-fed rabbits the percentage of atherosclerotic lesions in the aorta was identical in females and males and was inversely correlated with the maximal aortic relaxation to acetylcholine as assessed in organ chamber experiments (females: P < 0.0008, males: P < 0.0002). Importantly, the cholesterol diet induced a significantly (P < 0.025) more severe impairment of maximal vasorelaxation to acetylcholine in males from 78.4 +/- 1.2% to 29.4 +/- 10.2%) compared to females (from 84.4 +/- 1.2% to 60.7 +/- 8.5%). Both male gender (P < 0.0001) and the extent of impairment of endothelium-dependent relaxation (P < 0.0002) were associated with a reduced aortic sensitivity to S-nitroso-N-acetyl-D,L-penicillamine, which releases NO into the organ bath. In contrast, the aortic sensitivity to the organic nitrates pentaerythritol tetranitrate and isosorbide 5-nitrate, which release NO after enzymatic metabolization within the smooth muscle, was not reduced., Conclusions: These results suggest that the impairment of endothelium-dependent vasorelaxation induced by atherosclerosis is dependent on gender. This may be due to a greater degradation of extracellular NO in the vessel wall of males.

We wished to determine whether long-term treatment with organic nitrovasodilators has pharmacological effects on the development of atherosclerotic lesions and endothelial dysfunction in cholesterol-fed rabbits. For 15 weeks, six groups of 9 New Zealand White rabbits received a standard diet, which contained no admixture, pentaerythrityl-tetranitrate (PETN 6 mg/kg body weight/day), or isosorbide-5-mononitrate (ISMN 2 mg/kg body weight/day). In the other three groups, the same diets were further enriched with cholesterol (0,75%). Four rings of thoracic aorta were used for tension studies; these rings and the aortas from the aortic arch to bifurcation were then fixed in formol and stained with Sudan IV to determine the area of luminal atherosclerotic lesions by a computerized laser-scanning approach. The cholesterol diet increased plasma levels of cholesterol from 69.8 +/- 10.4 to 907.1 +/- 85.5 mg/dl. A similar result was obtained in the group receiving PETN/cholesterol, but the group fed ISMN/cholesterol showed a significantly higher plasma level of cholesterol (1,165 +/- 81.4 mg/dl). Plasma levels of PETN metabolites were still detectable by gas chromatography/mass spectrometry after a 24-h in vivo washout period. The cholesterol diet induced a pronounced degree of atherosclerotic lesions in the aortic arch and the thoracic and abdominal aorta: 73.3 +/- 1.9, 46.3 +/- 2.5, and 49.6 +/- 3.6%, respectively. Additional treatment with PETN resulted in a reduction of this atherosclerotic area to 58.6 +/- 2.05% (p < 0.0001), 34.7 +/- 1.98% (p < 0.01), and 39.3 +/- 3.06% (p < 0.05). In contrast, ISMN had no significant effect on this parameter. The cholesterol diet also induced an endothelial dysfunction as indicated by the diminished vasorelaxation induced by acetylcholine (ACh). Treatment with PETN completely inhibited the development of endothelial dysfunction, whereas ISMN had no effect. In the three groups receiving a cholesterol diet, an increased extent of aortic lesions significantly correlated with increased endothelial dysfunction measured in the same preparations. The long-term treatment with PETN did not affect the vasorelaxing potency of PETN in aortic rings, and similar results were obtained in the case of ISMN. We conclude that long-term treatment with doses of PETN, which do not promote the development of in vitro vascular nitrate tolerance, may protect against atherosclerosis and endothelial dysfunction. This novel, yet unknown pharmacodynamic quality of nitrovasodilators like PETN may contribute to their long-term efficacy in coronary artery disease but may also imply new therapeutic indications in the future.

1. Isolated segments of porcine vena cordis magna exhibited a reproducible contractile activity upon application of prostaglandin F2 alpha (PGF2 alpha) or KCl, that was independent of the presence of intact endothelium. Substance P (3 nM) elicited strictly endothelium-dependent relaxations amounting to 46.1 +/- 1.4% (n = 206) of contractions induced by 10 microM PGF2 alpha. 2. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a compound that spontaneously liberates nitric oxide, concentration-dependently relaxed PGF2 alpha-precontracted (50 microM) venous segments. Tolerance induction (incubation with 100 microM SNAP for 30 min) within the same segments resulted in a 3 fold attenuation of this effect, which was not further reduced after additional preincubation with glyceryl trinitrate (GTN). Removal of endothelium or the presence of N omega-nitro-L-arginine methylester (L-NAME) significantly improved the potency of SNAP before and after tolerance induction. 3. Concentration-dependent relaxations induced by GTN in non-tolerant veins were similar in the presence and absence of endothelium but much more reduced in tolerant endothelium-denuded (75 fold) compared to intact (20 fold) segments. In contrast, the presence of L-NAME significantly improved GTN-activity solely in non-tolerant veins, which, therefore, also resulted in a more pronounced attenuation of activity due to tolerance induction (100 fold). Preincubation of intact veins with SNAP also reduced GTN-activity but to a lesser extent (10 fold). 4. The more delayed but much longer, and compared to GTN somewhat weaker, acting new nitrovasodilator N-(3-nitrato-pivaloyl)-1-cysteineethylester (SPM 3672) was more potent in denuded than intact non-tolerant venous segments. Induction of tolerance by GTN resulted in a 2 fold-attenuation of potency. This effect was increased to 15 fold in denuded veins but solely due to enhanced potency of SPM 3672 caused by removal of endothelium.5. These data demonstrate that intact endothelium of porcine vena cordis magna attenuates the relaxant potency of nitrovasodilators but also probably participates in vascular bioactivation of GTN.We suggest that the reduced potency of nitrovasodilators is due to endogenous production of nitricoxide, which may affect the soluble guanylate cyclase/cyclic GMP-system or inhibit nitrate bioactivation pathways.

It is hypothesised that the well known development of tolerance to the vasodilating action of organic nitrates is contributed by intracellular depletion of free thiols occurring during repeated treatment with these drugs. Therefore, ring segments of porcine coronary arteries with and without endothelium were treated for 30 min with either vehicle or 100 microM of isosorbide-5-mononitrate, glyceryl trinitrate, S-nitroso-N-acetyl-D,L-penicillamine or N-(3-nitratopivaloyl)-1-cysteine-ethylester (SPM 3672), and the content of histochemically stained free thiols (-SH) and disulfides (S-S-) was measured densitometrically in single smooth muscle cells. In the presence of endothelium the content of -SH in smooth muscle cells of controls (n = 8) gave an extinction of 0.127 +/- 0.013 in the intima and 0.120 +/- 0.010 in the media. The corresponding values for S-S- were 0.684 +/- 0.084 and 0.535 +/- 0.120 (n = 8). Removal of endothelium reduced S-S- to 82.1 +/- 70% and increased -SH to 126.7 +/- 6.7%. Treatment with all nitrates reduced -SH in intact artery segments to a similar degree, ranging between 54.0 +/- 4.4 and 68.7 +/- 4.7% (n = 8-10). In contrast, S-S- content was less affected and reached values between 70.6 +/- 2.8 and 91.6 +/- 6.0% (n = 8-9). As evaluated by tension studies, tolerance developed for glycerol trinitrate and isosorbide-5-mononitrate but not for S-nitroso-N-acetyl-D,L-penicillamine. Induction of tolerance with glycerol trinitrate (0.1 mM) produced a significantly more pronounced attenuation in activity of isosorbide-5-mononitrate than tolerance induction with isosorbide-5-mononitrate (1 mM). In contrast, the potency of SPM 3672 was not reduced in glycerol trinitrate-tolerant arteries. We conclude that, in porcine coronary arteries, an intact endothelium modifies intracellular thiols and disulfides. In addition, nitrate tolerance is associated with, but probably not caused by, thiol depletion.

Vascular endothelium modulates the effect of various vasoconstricting mediators as well as the affinity of dihydropyridine-type calcium entry blockers. To further investigate this influence, vasoconstriction by PGF2 alpha as opposed to KCl and the affinity of nitrendipine and some related 3-ester side-chain derivatives were determined in isolated porcine basilar arteries in the presence and in the absence of intact endothelium, as well as in the presence of methylene blue. Treatment with methylene blue or mechanical endothelial damage increased the contractile work of basilar arteries stimulated by PGF2 alpha and reduced the affinity of the dihydropyridines in such precontracted vessels. Both experimental conditions resulted in nearly the same effect. In addition, the degree of intact endothelium, as determined by substance-P-induced vasodilation, significantly correlated with the corresponding efficacy of all dihydropyridines examined. In contrast, KCl-mediated contractions remained unchanged. It is suggested that the endothelium (probably due to the production and release of endothelium-derived vasorelaxing factors, such as EDRF and/or prostacyclin) may attenuate PGF2 alpha-induced transmembrane calcium influx through receptor operated calcium channels, whereas potential operated calcium channels seems to be unaffected.