Hypercholesterolemia causes generalized impairment of endothelium-dependent relaxation to aggregating platelets in porcine arteries

The role of the endothelium in response to aggregating platelets was examined in porcine coronary and peripheral (carotid, femoral and renal) arteries from normal and hypercholesterolemic pigs. Male Yorkshire pigs were fed either a normal diet or a 2% high cholesterol diet for 10 weeks. Endothelium-dependent responses were examined in vitro. In all arteries from control animals, aggregating platelets caused endothelium-dependent relaxations, which were augmented by ketanserin (a 5-HT2-serotonergic blocker), attenuated by apyrase (an adenosine diphosphatase and triphosphatase) or methiothepin (a combined 5-HT1 and 5-HT2-serotonergic blocker) and were almost abolished by a combination of apyrase and methiothepin. The platelet-induced relaxations were most pronounced in the coronary arteries. Adenosine diphosphate caused endothelium-dependent relaxations, which were significantly attenuated by apyrase. Serotonin also caused endothelium-dependent relaxations, which were significantly attenuated by methiothepin but augmented by ketanserin. The endothelium-dependent relaxations to adenosine diphosphate were most pronounced in coronary arteries and those to serotonin in coronary and renal arteries. In cholesterol-fed animals, the endothelium-dependent relaxations to aggregating platelets, adenosine diphosphate and serotonin were impaired in all four arteries. These experiments indicate that 1) the endothelium exerts inhibitory effects against aggregating platelets in porcine coronary and peripheral arteries; 2) platelet-induced endothelium-dependent relaxations are achieved by purinergic and 5-HT1-serotonergic receptors on the endothelium; and 3) hypercholesterolemia reduces the endothelium-dependent relaxations to aggregating platelets in a generalized manner because it impairs the relaxations to adenosine diphosphate and serotonin released from the platelets.     

Impaired endothelium-dependent relaxation to aggregating platelets and related vasoactive substances in porcine coronary arteries in hypercholesterolemia and atherosclerosis

Vasoconstrictor responses are augmented in porcine coronary arteries in hypercholesterolemia and atherosclerosis, leading to an occurrence of coronary vasospasm in the latter condition. The role of the endothelium in the vascular hyperreactivity in hypercholesterolemic and atherosclerotic coronary arteries was examined, particularly in response to aggregating and related vasoactive substances. Male Yorkshire pigs underwent balloon endothelial denudation of the left anterior descending coronary artery (LAD) and 2% high-cholesterol feeding for 10 weeks. Electron microscopic examination demonstrated a full lining of endothelial cells in the LAD and the left circumflex coronary artery (LCX). Endothelium-dependent responses were examined in vitro. In cholesterol-fed animals, endothelium-dependent relaxations to aggregating platelets, serotonin, ADP, bradykinin, thrombin, and the calcium ionophore A23187 were depressed in LAD (atherosclerosis), while the relaxations to aggregating platelets, serotonin and ADP were depressed in LCX (hypercholesterolemia). Serotonin-induced contractions were endothelium-dependently augmented in atherosclerotic LAD; the endothelium-dependent component of the contractions was inhibited by blockers of cyclooxygenase. Bioassay studies demonstrated a depressed release of endothelium-derived relaxing factor(s) from the atherosclerotic LAD in response to serotonin. These experiments indicate that the endothelium-dependent relaxations to aggregating platelets and related vasoactive substances are severely impaired in atherosclerosis and moderately impaired in hypercholesterolemia. Since coronary atherosclerosis was induced by a combination of balloon endothelial injury (and regeneration) and high-cholesterol feeding in this study, the combined effects of those factors must account for the severely impaired responses in atherosclerosis. The depressed release of the endothelium-derived relaxing factor(s) and the concomitant release of vasoconstrictor product(s) of cyclooxygenase appear to be responsible for the impaired relaxations.     

Long-term impairment of endothelium-dependent relaxations to aggregating platelets after reperfusion injury in canine coronary arteries

Experiments were designed and performed to determine whether endothelial function remained chronically impaired after coronary artery reperfusion. Canine left anterior descending coronary arteries were exposed to ischemia (60 minutes) followed by reperfusion (12 weeks). Rings (3-4 mm wide) of the reperfused artery and of normal left circumflex (control) coronary artery segments were suspended in organ chambers containing physiological saline solution (37 degrees C, gassed with 95% O2-5% CO2) for isometric force measurement. Endothelium-independent contractions to KCl or prostaglandin F2 alpha and endothelium-independent relaxations to nitric oxide or isoproterenol were comparable in control and chronically reperfused arteries. However, chronically reperfused coronary arteries exhibited impaired endothelium-dependent relaxations to aggregating platelets. In addition, the reperfused coronary arteries exhibited impaired endothelium-dependent relaxations to the platelet-derived compounds adenosine diphosphate, serotonin, and thrombin. However, the endothelium-dependent relaxations to acetylcholine were comparable between control and reperfused arteries. Thus, after 12 weeks of reperfusion, previously occluded coronary arteries exhibited a selective impairment of endothelium-dependent relaxation evoked by aggregating platelets. In vivo, this phenomenon could favor platelet adhesion, aggregation, and platelet-induced contraction of coronary smooth muscle and thus facilitate ischemic events such as vasospasm and coronary thrombosis.     

Acute impairment of endothelium-dependent relaxations to aggregating platelets following reperfusion injury in canine coronary arteries

Experiments were designed to determine whether endothelial injury contributes to augmented coronary vascular tone seen during myocardial reperfusion. Canine left anterior descending coronary arteries were exposed to ischemia followed by reperfusion (60 minutes each). Rings (3-4 mm) of the reperfused artery and of normal left circumflex (control) coronary artery segments were prepared. Rings were suspended for isometric force measurement in organ chambers containing modified Krebs' Ringer bicarbonate solution (37 degrees C, 95% O2-5% CO2). Endothelium-independent contractions to KCl and prostaglandin F2 alpha were unaltered after reperfusion. Endothelium-dependent relaxations to nitric oxide, sodium nitroprusside, and isoproterenol were comparable in control and reperfused arteries. However, reperfused coronary arteries contracted with prostaglandin F2 alpha lost the ability to express endothelium-dependent relaxations to aggregating platelets. Reperfused arterial rings also exhibited impaired endothelium-dependent relaxations to acetylcholine, the calcium ionophore A23187, and the platelet-derived compounds ADP and serotonin. Quiescent (noncontracted) reperfused arterial rings exhibited larger contractions than controls when exposed to aggregating platelets. In such quiescent rings, the endothelium-dependent increase in tension to hemoglobin was unaltered after reperfusion. Thus, coronary reperfusion impairs the normal endothelium-dependent relaxations to aggregating platelets and vasoactive drugs. This impairment of platelet-mediated coronary relaxation could help explain the increased vascular tone and tendency toward vasospasm commonly observed after reperfusion of the coronary arteries.     

Porcine coronary arteries with regenerated endothelium have a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin

To test the ability of regenerated endothelium to evoke endothelium-dependent relaxations, male Yorkshire pigs underwent balloon endothelial denudation of the proximal left anterior descending coronary artery. Endothelium-dependent responses were examined in vitro, in rings of coronary segments taken from the denuded area or from the proximal left circumflex coronary artery. The experiments were performed 8 days or 4 weeks after the denudation. Endothelial regrowth was confirmed by histologic examination 8 days after the denudation and by demonstrating the presence of endothelium-dependent relaxations to bradykinin; at that time aggregating platelets evoked normal endothelium-dependent responses. However, 4 weeks after the denudation, the relaxations to aggregating platelets were markedly depressed although continuous endothelial lining was present, and the endothelium-dependent responses to bradykinin, adenosine diphosphate, the Ca2+-ionophore A23187, platelet activating factor, and thrombin were unaltered. Four weeks after denudation, endothelium-dependent relaxations to serotonin were depressed. Higher concentration of serotonin induced endothelium-dependent contractions in quiescent rings with regenerated endothelium, suggesting that regenerated endothelial cells may produce endothelium-derived constricting factor(s) and release less endothelium-derived relaxing factor(s) when exposed to the monoamine. The endothelium-dependent relaxation to serotonin was not reduced by the S2-serotonergic antagonist ketanserin but prevented by the combined S1- and S2-serotonergic blocker methiothepin. The platelet-induced relaxation was due to released serotonin and adenine nucleotides in control left circumflex coronary arteries, but in left anterior descending coronary artery with regenerated endothelium, it was due solely to the latter. The platelet-induced contractions were due to activation of receptors on the smooth muscle cells. Four weeks after denudation, regenerated endothelial cells were morphologically different from native cells; they were elongated and cuboidal, and the number of the cells had increased twofold. At this state, eccentric myointimal thickening was present in the previously denuded portion. These experiments indicate that the protective role of endothelial cells against the vasoconstriction induced by aggregating platelets is depressed in the chronic regenerated state. A lack of responsiveness to serotonin appears to be the cause for the endothelial dysfunction.     

Dietary cod-liver oil improves endothelium-dependent responses in hypercholesterolemic and atherosclerotic porcine coronary arteries

This study examined the effects of dietary supplementation with cod-liver oil on impaired endothelium-dependent relaxations in hypercholesterolemia and in atherosclerosis in porcine coronary arteries. Sixteen male Yorkshire pigs underwent balloon endothelium removal of the left coronary arteries and were fed a 2% high-cholesterol diet for 10 weeks, with or without dietary supplementation of cod-liver oil (30 ml/day) (oil-fed and cholesterol-fed groups, respectively). This model allowed the simultaneous examination of the effects of dietary cod-liver oil on vascular reactivity in hypercholesterolemia alone (right coronary artery) and in atherosclerosis (left coronary artery). After 10 weeks of feeding, the dietary treatment with cod-liver oil caused an increase in plasma levels of eicosapentaenoic acid and a decrease in the plasma levels of arachidonic acid, whereas the treatment had no significant effect on the increases in plasma lipid levels induced by the high-cholesterol feeding. Morphometric analysis showed significant inhibition of coronary atherosclerosis by the treatment. Endothelium-dependent responses were examined in vitro in ring preparations and in bioassay experiments. Endothelium-dependent relaxations to bradykinin, serotonin, and adenosine 5'-diphosphate were larger in both right and left coronary arteries from oil-fed than from cholesterol-fed animals. Aggregating platelets from cholesterol-fed and oil-fed pigs induced comparable, larger endothelium-dependent relaxations in rings from oil-fed than from cholesterol-fed pigs. The contractions induced by serotonin or aggregating platelets were significantly inhibited in rings with endothelium from oil-fed pigs, whereas they were comparable in rings without endothelium in both groups. Relaxations to sodium nitroprusside and contractions to potassium chloride or serotonin were comparable in rings without endothelium in both groups. The bioassay experiments revealed that the release of endothelium-derived relaxing factor in response to bradykinin and the relaxations of vascular smooth muscle to the endothelial factor were greater after the fish-oil diet. These results indicate that dietary supplementation of cod-liver oil delays the impairment of endothelium-dependent relaxations in hypercholesterolemia and in atherosclerosis, partly because of an improved release of endothelium-derived relaxing factor and partly because of an improved relaxation of coronary smooth muscle to the factor.     

Angiographic demonstration of hyperconstriction induced by serotonin and aggregating platelets in porcine coronary arteries with regenerated endothelium

In isolated porcine coronary arteries, the endothelium inhibits the contractions induced by serotonin and aggregating platelets. This effect is reduced by regeneration after balloon denudation. The present study was designed to examine whether those in vitro observations could be reproduced in vivo. Sixteen male Yorkshire pigs underwent balloon removal of the endothelium from either the left anterior descending (n = 9) or left circumflex (n = 7) coronary artery. An angiographic study was performed before and 4 weeks after endothelial denudation; in the latter condition the presence of a full endothelial lining was confirmed histologically. Before endothelial denudation, intracoronary injection of serotonin (10 micrograms/kg) caused only mild constriction in both arteries. However, 4 weeks after the denudation, intracoronary serotonin repeatedly caused marked hyperconstriction in the previously denuded artery compared with the control coronary artery. Intracoronary injection of aggregating platelets stimulated by collagen caused constriction in the previously denuded artery but not in the control artery. The hyperconstriction induced by serotonin and aggregating platelets was inhibited by intravenous injection of ketanserin (a 5-HT2-serotonergic blocker). These data indicate that 1) the normal endothelium blunts the coronary vasoconstriction induced by serotonin and aggregating platelets in vivo, and 2) the inhibitory effect of the endothelium is reduced after regeneration, allowing coronary hyperconstriction or vasospasm, or both.     

Effects of dietary supplementation with cod-liver oil on endothelium-dependent responses in porcine coronary arteries

To study the effect of dietary supplementation with fish oil on endothelium-dependent responses, Yorkshire pigs were maintained on a normal diet or on a low (0.6 ml/kg/day) or a high (1.0 ml/kg/day) dose of cod-liver oil for 4 weeks. Endothelium-dependent responses were examined in vitro in rings of proximal left anterior descending coronary arteries taken from control and treated animals studied in parallel. Endothelium-dependent relaxations in response to bradykinin, serotonin, adenosine diphosphate, and thrombin were facilitated in arteries from treated but not in those from control animals, whereas the relaxations in response to A23187 were unaltered. The facilitated relaxations were not altered by indomethacin but significantly inhibited by methylene blue. Aggregating platelets from control and treated pigs induced comparable, facilitated endothelium-dependent relaxations in rings taken from treated pigs. The platelet-induced contractions were significantly reduced in rings with endothelium taken from treated pigs, and they were comparable in rings without endothelium in both groups. Aggregating platelets from control and treated pigs released comparable amounts of serotonin and thromboxane A2. Endothelium-dependent relaxations induced by arachidonic acid and eicosapentaenoic acid were unaltered, whereas transient endothelium-dependent contractions induced by arachidonic acid were significantly reduced by the treatment with cod-liver oil. Relaxations to sodium nitroprusside or isoproterenol,and contractions to potassium chloride or serotonin were not different in rings without endothelium from control or treated pigs. These results indicate that dietary supplementation with cod-liver oil facilitates endothelium-dependent relaxations and inhibits endothelium-dependent contractions in porcine coronary arteries.     

Endothelium-dependent relaxation to aggregating platelets in isolated basilar arteries of control and hypercholesterolemic pigs

The role of the endothelium was examined in the response to aggregating platelets in cerebral arteries from normal and hypercholesterolemic animals. Male Yorkshire pigs were fed either a normal diet or a 2% high-cholesterol diet for 10 weeks. Endothelium-dependent responses were examined in vitro. In rings of basilar arteries from control animals aggregating platelets caused endothelium-dependent relaxations, which were significantly inhibited by apyrase, an adenosine diphosphatase and triphosphatase, but were augmented by methiothepin, a combined S1- and S2-serotonergic blocker. In quiescent rings platelets induced contractions that were inhibited by the presence of the endothelium; these contractions were significantly inhibited by methiothepin, but not by ketanserin (an S2-serotonergic blocker) or dazoxiben (a thromboxane-synthetase blocker) in the presence or absence of SQ29548 (a thromboxane-receptor blocker). Adenosine diphosphate but not serotonin caused endothelium-dependent relaxations. In cholesterol-fed animals the endothelium-dependent relaxations in response to aggregating platelets and adenosine diphosphate were impaired. These experiments indicate that 1) the endothelium inhibits the vasoconstrictor effect of aggregating platelets in porcine cerebral arteries; 2) platelet-induced relaxations are achieved mainly by a purinergic mechanism, while platelet-induced contractions are mediated by activation of S1-serotonergic receptors with little contribution of thromboxanes; and 3) hypercholesterolemia impairs the endothelium-dependent relaxations in response to aggregating platelets due to the impaired responses to adenosine diphosphate.     

Myocardial inotropic responses to aggregating platelets and modulation by the endocardium

Ventricular mural thrombi complicate many cardiac diseases. The endocardial endothelium can modulate the mechanical performance of subjacent myocardium and mediate responses to certain physiopharmacologic agents. We studied the effects of aggregating platelets on the contractile performance of isolated cardiac muscle. The role of the endocardium was investigated by selectively damaging it by very brief (1 second) exposure to 1% Triton X-100 in some muscle preparations before experiments. Cat papillary muscles (n = 54) were attached to an electromagnetic length-tension transducer in organ baths containing Krebs-Ringer solution (1.25 mM Ca2+, 35 degrees C), and stimulated electrically at 0.2 Hz. Homologous washed platelets (final concentration 3 x 10(11)/l) aggregated spontaneously on addition to baths. Mechanical performance increased significantly more in muscles with damaged endocardium than in intact muscles (p less than 0.05); total peak isometric twitch tension increased by 31.8 +/- 7.8% (with damaged endocardium) and 11.8 +/- 2.6% (with intact endocardium), and peak isotonic twitch shortening increased by 36.7 +/- 7.8% (with damaged endocardium) and 9.6 +/- 2.0% (with intact endocardium). Increases in maximum velocity of unloaded shortening were similar in both muscle groups. Time to half isometric twitch tension decline decreased in intact muscles (3.6 +/- 1.0%) but increased in Triton-treated muscles (2.5 +/- 1.3%, p = 0.003 for difference between groups). The inotropic response to platelets in muscles with intact endocardium was unaltered by pretreatment of muscles with indomethacin (10 microM) or by stimulation of platelet aggregation with thrombin (0.1 unit/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonged cold storage abolishes endothelium-dependent relaxing responses to A23187 and substance P in porcine coronary arteries.

In the presence of potassium (K+), A23187 and substance P elicited endothelium-dependent relaxations of porcine coronary arteries. Isoproterenol or hypoxia elicited endothelium-independent relaxations. Rubbing the artery potentiated the contractile response to a low K+ concentration (15.4 mM). After intact arteries had been stored at 5 degrees C for 3 days, K(+)-induced maximal tension was not affected, but contractile responses to 15 mM K+ were potentiated with a decrease in ED50, suggesting that cold storage produces a supersensitivity to K+. Endothelium-dependent relaxations were abolished after 3 days of cold storage, while endothelium-independent relaxations were not inhibited. Cold storage of arteries with l-arginine (1 mM) for 3 days did not alter the relaxation responses to substance P and A23187, indicating that l-arginine does not prevent the loss of endothelium-dependent relaxation. Cold storage for 5 days inhibited the maximal tension to K+ and abolished the supersensitivity. Scanning electron micrographs showed that endothelial cells can be damaged by prolonged cold storage. The changes in tension response of the artery were correlated with the time course of endothelial cell loss resulting from cold storage.     

Hypercapnia-induced contraction in isolated pulmonary arteries is endothelium-dependent

It has been demonstrated previously that isohydric hypercapnia (IH) does not affect agonist-induced tension development in pulmonary arteries. The aim of the present study was to examine the effects of IH on depolarisation-induced, steady state tension in the isolated rat pulmonary artery. Rings were submaximally contracted with high KCl under control conditions (5% CO(2)-95% air). IH was achieved by switching to a modified PSS (isosmotic substitution of NaHCO(3) for NaCl), equilibrated with 10% CO(2) in air. On switching to IH, a significant increase in mean (+/-SEM) tension (25.3+/-6.3% Tmax) was observed in endothelium intact rings (n=6). Endothelial removal significantly reduced this response. Non-specific inhibition of nitric oxide synthase (NOS) isoenzymes (L-NAME, 10(-3) M) abolished the IH-induced increase in tension while inhibition of neuronal NOS (TRIM, 10(-5) M) was without effect. The relaxant response to the nitric oxide donor sodium nitroprusside was similar in IH and control conditions. These results suggest that IH caused an endothelium-dependent increase in depolarisation-induced tension by reducing NO production.     

Endothelium-dependent relaxation in response to aggregating platelets in porcine femoral veins and its modulation by diet

The present study examined the protective role of the venous endothelium against aggregating platelets and its modulation by diet. Yorkshire pigs were fed a regular chow (control pigs), 2% high-cholesterol diet (for 10 weeks, cholesterol-fed pigs), and regular chow plus cod-liver oil (30 ml/day for 4 weeks, oil-fed pigs). Endothelium-dependent responses were examined in vitro in rings of femoral veins in the presence of the inhibitor of cyclooxygenase indomethacin. In control pigs, aggregating platelets, serotonin, and ADP caused endothelium-dependent relaxations. The platelet-induced relaxations were attenuated by methiothepin (a combined 5-HT1 and 5-HT2 serotonergic blocker) or apyrase (an ADPase and ATPase) and were abolished by the combination of the two agents. In quiescent rings, platelets caused contractions, which were reduced in the presence of endothelium; the contractions were prevented by ketanserin (a 5-HT2 serotonergic blocker) or methiothepin but not by R 68 070 (a thromboxane A2 receptor blocker) or dazoxiben (a thromboxane-synthetase blocker). In cholesterol-fed pigs, the platelet-induced relaxations were not altered, whereas in oil-fed pigs, the endothelium-dependent relaxations to platelets, serotonin, and ADP were augmented. Platelet-induced contractions were significantly reduced in rings with endothelium from oil-fed pigs, whereas the contractions were comparable in rings without endothelium among the three groups. Endothelium-dependent relaxations in response to the calcium ionophore A23187, direct relaxations in response to sodium nitroprusside, and direct contractions in response to potassium chloride were comparable among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of human coronary arteries to aggregating platelets: importance of endothelium-derived relaxing factor and prostanoids

Epicardial human coronary arteries (HCA) were obtained during heart transplantation. Strip preparations were mounted for isometric tension recording. Tension was induced with prostaglandin F2 alpha (0.3-3 microM). Aggregating human platelets (10(7)/ml and 10(8)/ml) caused marked relaxation if the endothelium of the HCA was intact, but produced modest additional constriction if the endothelium was removed. The endothelium-dependent relaxations to platelets were slightly enhanced in the presence of methysergide (1 microM) or after treatment of the platelets with the thromboxane synthase inhibitor dazmegrel (1 mM, 30 minutes). Relaxations to platelets were markedly inhibited or abolished in the presence of apyrase (1 unit/ml), after selective pretreatment of HCA with gossypol or methylene blue (both 30 microM, 30 minutes), or after addition of hemoglobin (20 microM) to the organ bath. Pretreatment of HCA (not the platelets) with aspirin (30 microM, 30 minutes) had no significant effect on platelet-induced relaxations. Adenosine 5'-diphosphate (0.1-100 microM) induced marked relaxations in endothelium-intact and much smaller relaxations in endothelium-denuded HCA. A low concentration (10 nM) of serotonin (5-HT) produced modest endothelium-dependent relaxations, higher concentrations (0.1-1 microM) led to increases in tension (also in the presence of endothelium). The thromboxane A2 mimetic U44069 (1-100 nM) was the most potent constrictor of HCA irrespective of the presence or absence of endothelium. After inhibition of thromboxane synthase, platelets produced large amounts of prostaglandins E2 and F2 alpha. Both prostaglandins constricted HCA, which can explain the limited effects of dazmegrel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of endothelium-dependent hyperpolarizations in porcine coronary arteries with regenerated endothelium

The present study was designed to test the ability of regenerated endothelium to evoke endothelium-dependent hyperpolarizations. Hyperpolarizations induced by serotonin and bradykinin were compared in isolated porcine coronary arteries with native or regenerated endothelium, 4 weeks after balloon endothelial denudation. The experiments were performed in the presence of inhibitors of nitric oxide synthase (Nomega-nitro-L-arginine) and cyclooxygenase (indomethacin). The transmembrane potential was measured using conventional glass microelectrodes. Smooth muscle cells from coronary arteries with regenerated endothelium were depolarized in comparison with control coronary arteries from the same hearts. Spontaneous membrane potential oscillations of small amplitude or spikes were observed in some of these arteries but never in arteries with native endothelium. In coronary arteries from control pigs, both serotonin and bradykinin induced concentration-dependent hyperpolarizations. In the presence of ketanserin, 10 micromol/L serotonin induced a transient hyperpolarization in control coronary arteries. Four weeks after balloon denudation, the response to serotonin was normal in arteries with native endothelium, but the hyperpolarization was significantly lower in coronary arteries with regenerated endothelium. In control arteries, the endothelium-dependent hyperpolarization obtained with bradykinin (30 nmol/L) was reproducible. Four weeks after balloon denudation, comparable hyperpolarizations were obtained in coronary arteries with native endothelium. By contrast, in arteries with regenerated endothelium, the hyperpolarization to bradykinin became voltage-dependent. In the most depolarized cells, the hyperpolarization to bradykinin was augmented. The changes in resting membrane potential and the alteration in endothelium-dependent hyperpolarizations observed in the coronary arteries with regenerated endothelium may contribute to the reduced response to serotonin and the unchanged relaxation to bradykinin described previously.     

Heterogeneity of endothelium-dependent responses to acetylcholine in canine femoral arteries and veins. Separation of the role played by endothelial and smooth muscle cells

The purpose of this study was to determine whether heterogeneity in endothelium-dependent responses to acetylcholine between canine blood vessels of different anatomical origin reflects variations in endothelial function or in responsiveness of vascular smooth muscle cells. Experiments were conducted in a bioassay system, where segments of femoral artery or vein with endothelium were perfused intraluminally and the perfusate used to superfuse rings of femoral arteries or veins without endothelium. Indomethacin was present in all experiments to prevent the synthesis of prostanoids. The blood vessels were contracted by phenylephrine. Measurement of wall tension in both the perfused segment and bioassay ring allowed simultaneous detection of endothelium-derived relaxing factor(s) released abluminally (segment) and intraluminally (ring). Intraluminal infusion of acetylcholine (ACh) induced relaxations in the perfused artery but not in vein segments. During arterial superfusion ACh induced relaxation in femoral arterial rings but contraction in venous rings. After treatment with atropine the arterial perfusate evoked relaxations in venous rings. Infusion of ACh through the femoral vein evoked only moderate relaxations in arterial rings. These data demonstrate that depressed endothelium-dependent relaxation to ACh in femoral veins compared to femoral arteries is due to a masking effect of the direct stimulating action of ACh and decreased release of the same mediator or the release of a different relaxing factor from venous endothelium.     

HIV protease inhibitor ritonavir decreases endothelium-dependent vasorelaxation and increases superoxide in porcine arteries

OBJECTIVE: Although HIV Protease inhibitors significantly reduce the viral load, they are associated with increased risk of cardiovascular disease. The aim of this study was to investigate the effects of HIV protease inhibitor ritonavir on vascular endothelial cell function. METHODS: Porcine carotid arteries were perfusion-cultured for 24 h as controls or with 15 microM of ritonavir. Vessels were precontracted with norepinephrine followed by endothelium-dependent vasorelaxation with acetylcholine. Rings of vessels were cultured as controls or with ritonavir for 24 h and basal and NADPH-stimulated superoxide levels were determined using lucigenin-enhanced chemiluminescence. Superoxide levels in situ were also examined using dihydroethidium (DHE) staining, and nitrotyrosine levels were examined using a nitrotyrosine antibody. RESULTS: Endothelium-dependent vasorelaxation was significantly reduced in ritonavir-treated vessels compared to controls. There were significant increases in basal and NADPH-stimulated superoxide production in vessel rings treated with ritonavir compared to control vessels. Dihydroethidium staining and nitrotyrosine staining were also elevated in endothelial cells of ritonavir-treated vessels, indicating increased superoxide production and increased oxidative stress, respectively, in ritonavir-treated vessels compared to controls. CONCLUSIONS: These data demonstrate that HIV protease inhibitor ritonavir causes a significant reduction in endothelium-dependent vasorelaxation in cultured porcine carotid arteries. Increased oxidative stress may be a possible mechanism of HIV protease inhibitor ritonavir-induced endothelial dysfunction.     

Heterogeneity of endothelium-dependent vasorelaxation in conductance and resistance arteries from Lyon normotensive and hypertensive rats

OBJECTIVES: The nature of endothelial factors in response to acetylcholine (ACh) was investigated in conductance and resistance arteries from Lyon normotensive (LN) and Lyon hypertensive (LH) rats. Differences in endothelial function between the two strains were evaluated. METHODS AND DESIGN: Relaxations to ACh were studied in the aorta and small mesenteric arteries (SMA). The relative contribution of nitric oxide (NO), prostanoids and endothelial-derived hyperpolarizing factor (EDHF) was assessed using appropriate inhibitors. Western blot of endothelial NO synthase was achieved. The membrane potential of smooth muscle cells was assessed using microelectrodes. RESULTS: In LN rats, endothelium-dependent relaxation to ACh involved exclusively NO in the aorta, whereas both NO and EDHF were implicated in SMA. In the latter, relaxation was almost entirely prevented by blockade of either the NO or EDHF pathway, although ACh was still able to produce hyperpolarization in the presence of NO synthase and cyclooxygenase inhibitors. In LH rats, relaxation to ACh was unchanged in SMA but moderately depressed in the aorta, despite unchanged endothelial NO synthase protein expression and sensitivity to NO. In addition, indomethacin, but not a selective cyclooxygenase-2 inhibitor, significantly reduced ACh relaxations in the aorta from LH rats but not from LN rats. CONCLUSIONS: These results document differential endothelial function in a conductance and in resistance arteries from LN rats and LH rats. They show that simultaneous participation of NO and EDHF is required to promote relaxation in SMA from both strains, whereas NO alone accounts for relaxation in aorta from LN rats. In LH rats, aortic relaxation induced by ACh is slightly decreased despite the involvement of vasodilator products from cyclooxygenase-1.     

Acetylcholine and adenosine diphosphate cause endothelium-dependent relaxation of isolated human pulmonary arteries

Endothelium-dependent vasorelaxation mediated by endothelium-derived relaxing factors (EDRF) has been extensively studied in animals but only limited studies in man are available. Demonstration of EDRF-mediated dilatation of human vessels is fundamental for understanding the mechanisms of vascular diseases in man. We have investigated endothelium-dependent relaxation of isolated human pulmonary arteries. Vascular segments, taken from uninvolved regions of resected lung from eight patients undergoing lobectomy for lung carcinoma, were cut into rings. In rings precontracted with phenylephrine, both acetylcholine (ACh) and adenosine diphosphate (ADP) induced dose-dependent relaxation in the presence of endothelium but not when the endothelium had been carefully removed. The rings without endothelium relaxed completely with sodium nitroprusside, a vasodilator agent acting directly on vascular smooth muscle. Pre-incubation with indomethacin, a cyclo-oxygenase inhibitor which blocks production of prostacyclin, did not alter the vasorelaxant responses to ACh and ADP, suggesting that one (or several) non-prostanoid EDRF(s) are responsible for the endothelium-dependent relaxation of isolated human pulmonary arteries.