Ulrich Förstermann

Selective attenuation of endothelium-mediated vasodilation in atherosclerotic human coronary arteries.

This study was undertaken to determine whether atherosclerosis impairs relaxations mediated by endothelium-derived relaxing factor (EDRF) in human coronary arteries. Epicardial coronary arteries were obtained from the hearts of cardiac transplantation patients with or without histologically documented coronary atherosclerosis (atherosclerotic arteries were from patients aged 42–55 years, nonatherosclerotic arteries were from patients aged 14–24 years). Transverse strip preparations were mounted in organ baths for isometric tension recording. Tension was induced with prostaglandins F2a. Indomethacin (10−9 M) was present to prevent possible interference from endogenously formed prostaglandins. The EDRF-mediated relaxations in response to substance P (10−10 to 10−5 M), bradykinin (10−9 to 10−7 M), and Ca2+-Monophore A23187 (10−9 to 10−7 M) were significantly attenuated in atherosclerotic arteries. In deendothelialized tissues these compounds had no effect. In contrast, endothelium-independent relaxations induced by isoprenaline (10−7 to 10−5 M) were not affected by atherosclerosis. Atherosclerotic arteries showed also normal relaxations with high concentrations of glyceryl trinitrate (10−3 to 10−7 M), but reduced relaxations with a lower concentration of the compound (10−9 M). Acetylcholine (10−7 to 10−6 M) only produced endothelium-dependent relaxations in 8 of 60 arterial preparations (with or without atherosclerosis). In. most of the arteries, it was a direct vasoconstrictor (which may have masked EDRF release in many cases). Omission of indomethacin from the bath solution increased the incidence of moderate acetylcholine-induced relaxations (9 of 16 preparations). It is concluded that atherosclerosis attenuates EDRF-mediated vasodilation in epicardial human coronary arteries and that this impairment could promote coronary vasospasm and myocardial ischemia.

Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets☆

The present study was designed to investigate whether endothelium-derived relaxing factor (EDRF) produced by cultured human endothelial cells (HEC) inhibits the aggregation of human platelets. Microcarrier beads covered with HEC from umbilical veins (approximately 2 X 10(6)) or empty beads (as controls) were co-incubated in an aggregometer with washed human platelets (approximately 5 X 10(7)). Indomethacin was present throughout and no prostacyclin production (measured as 6-keto-PGF1 alpha by radioimmunoassay) could be detected. The presence of HEC markedly inhibited thrombin-induced platelet aggregation and this inhibition was further enhanced by bradykinin, a stimulator of EDRF production. The anti-platelet aggregatory effect was blocked by treating the HEC with the inhibitor of EDRF production gossypol, by treating the platelets with the inhibitor of soluble guanylate cyclase methylene blue, or by adding the EDRF scavenger oxyhemoglobin to the aggregation mixture. The antiaggregatory material was labile, since the supernatant of indomethacin-treated cultured HEC did not inhibit aggregation. In the absence of indomethacin, the prostacyclin-mediated antiaggregatory effect of HEC was not inhibited by gossypol, methylene blue or hemoglobin. These data strongly suggest that the EDRF formed by HEC is an inhibitor of platelet aggregation and may constitute an important defense mechanism against vasospasm and platelet aggregation.

Ciclosporin A inhibits endothelium-dependent vasodilatation and vascular prostacyclin production

Aortic rings dissected from rats treated with ciclosporin A (30 mg/kg per day for five days) showed reduced relaxation induced by the endothelium-dependent vasodilator acetylcholine, but unchanged responses induced by glyceryl trinitrate. After eight weeks of ciclosporin A treatment, the relaxation induced by both acetylcholine and glyceryl trinitrate was inhibited. In addition, phenylephrine-induced contractions were slightly enhanced and vascular prostacyclin production was reduced by more than 80%. These effects may participate in the hypertensive and thrombo-embolic complications associated with the clinical use of ciclosporin A.

Human endothelial cells inhibit platelet aggregation by separately stimulating platelet cyclic AMP and cyclic GMP

Thrombin-induced platelet aggregation was monitored in a mixture of washed human platelets and cultured human endothelial cells on microcarrier beads. Endothelial cells completely inhibited platelet aggregation and enhanced the content of both cyclic AMP and cyclic GMP in the platelets. Inhibition of endothelial prostacyclin synthesis with indomethacin abolished the cyclic AMP increase in the platelets, whereas the cyclic GMP increase was unimpaired. A significant component of the endothelial anti-aggregatory effect persisted in the presence of indomethacin. This anti-aggregatory component and the elevation of cyclic GMP were blocked by the inhibitors of endothelium-derived relaxing factor (EDRF), gossypol, haemoglobin and methylene blue. These inhibitors did not affect the endothelium-induced increases in cyclic AMP or the cyclic AMP-mediated anti-aggregatory effects. Exogenous prostacyclin stimulated only platelet cyclic AMP, whereas sodium nitroprusside selectively enhanced cyclic GMP. These data suggest that the endothelium can inhibit platelet aggregation by two completely separate mechanisms, one mediated by prostacyclin and cyclic AMP, and the other by EDRF and cyclic GMP.

Mechanisms of action of lipoxygenase and cytochrome P-450-mono-oxygenase inhibitors in blocking endothelium-dependent vasodilatation

1 Acetylcholine, ionophore A23187 and melittin induced endothelium‐dependent relaxations of preconstricted strips of rabbit aorta. These relaxations are likely to be mediated by endothelium‐derived relaxing factor (EDRF). 2 Relaxations in response to acetylcholine (1 μm) were inhibited by the following lipoxygenase inhibitors, with the approximate IC50 values indicated in parentheses: gossypol (1.5 μm), nordi‐hydroguairetic acid (NDGA, 5 μm), AA 861 (20 μm), phenidone (30 μm), quercetin (40 μm), BW 755C (300 μm), and piriprost (500 μm); with cirsiliol 50% inhibition was not achieved. Acetylcholine‐induced relaxations were also blocked by the cytochrome P‐450‐mono‐oxygenase inhibitors proadifen (SKF 525A, 4 μm), metyrapone (300 μm), and cimetidine (300 μm); 7,8 benzoflavone had no effect up to 100 μm. 3 The more potent inhibitors were also tested against relaxations induced by A23187 (0.1 μm) and melittin (1 μm) and produced partial inhibition of these relaxations. 4 The mechanism of action of the more potent inhibitors was investigated in a bioassay system. EDRF was produced in columns filled with cultured human endothelial cells. The factor was bioassayed with endothelium denuded segments of rabbit femoral artery. When added to effluent of the column, NDGA, AA861, proadifen and metyrapone inhibited the EDRF‐induced vasodilatation, whereas gossypol had no effect. Gossypol, however, blocked EDRF production when infused through the column. 5 The more potent inhibitors were also tested to determine their effect on purified soluble guanylate cyclase. While gossypol, NDGA and proadifen had no appreciable effects, basal and nitroprusside (50 μm)‐stimulated guanylate cyclase activity was inhibited by AA861 and metyrapone. 6 These data suggest that many of the above compounds inhibit EDRF by mechanisms other than lipoxygenase‐ or cytochrome P‐450‐mono‐oxygenase inhibition.

Endothelium-dependent relaxation of human epicardial coronary arteries: frequent lack of effect of acetylcholine

Strip preparations of human epicardial coronary arteries (free of atherosclerosis) relaxed in an endothelium-dependent fashion to substance P and Ca2+-ionophore A23187. Acetylcholine generally caused contraction in the same strips. Glyceryl trinitrate and isoproterenol induced relaxation irrespective of the presence or absence of endothelium. Nordihydroguaiaretic acid abolished the relaxation produced by substance P and A23187. Mepacrine only blocked substance P relaxation. Haemoglobin and methylene blue inhibited substance P and A23187 relaxations but also reduced the response to glyceryl trinitrate. These inhibitor experiments indicate that human coronary arteries are relaxed by the endothelium-derived relaxing factor.

Selective inhibition by gossypol of endothelium-dependent relaxations augments relaxations to glyceryl trinitrate in rabbit coeliac artery.

1 Acetylcholine, substance P, prostaglandin E, and the nitrovasodilator glyceryl trinitrate induced concentration‐dependent relaxations of endothelium‐intact strips of rabbit coeliac artery precontracted with noradrenaline. 2 Endothelium‐denuded strip preparations contracted to acetylcholine and showed no response to substance P. The relaxant response to prostaglandin E1 was unimpaired after removal of endothelium, whereas the response to glyceryl trinitrate was increased. 3 A 20 min exposure of endothelium‐intact strips to gossypol, an irreversible inhibitor of the production and/or release of endothelium‐derived relaxing factor, abolished vasodilatation in response to the endothelium‐dependent agents acetylcholine and substance P, did not change relaxations to prostaglandin E1, but significantly enhanced relaxations in response to glyceryl trinitrate. 4 In view of the assumed common mechanism of action of endothelium‐derived relaxing factor and nitrovasodilators, these results suggest an interference of the two active principles at the level of the vascular smooth muscle cell.

The inhibition of α1-adrenoceptor-mediated contractions of rabbit pulmonary artery by Ca2+-withdrawal, pertussis toxin and N-ethylmaleimide is dependent on agonist intrinsic efficacy

SummaryContractions were induced in rings of rabbit pulmonary artery with the preferential al-adrenoceptor agonists, phenylephrine, methoxamine and St 587 [2-(2-chloro-trifluoromethyl-phenylimino)imidazolidine and the preferential α2-adrenoceptor agonists, clonidine and B-HT 920 [6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d) azepine]. Phenylephrine and methoxamine acted as full agonists whereas St 587, clonidine and B-HT 920 were partial agonists (intrinsic activities 0.62, 0.38 and 0.42, respectively). Experiments with α1- and α2-adrenoceptor antagonists indicated that the receptors involved are of the al type only. Removal of extracellular Ca2+ inhibited maximal contractions to phenylephrine and methoxamine by 30% and 49%, respectively. The remaining contraction components of the full agonists were abolished by the “intracellular Ca2+ antagonist” TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate]. Contractions to St 587, clonidine and B-HT 920 were virtually abolished in Ca2+-free medium. Pretreatment of the donor rabbits with pertussis toxin (2.5 μg/kg i. v., 5–6 days before sacrifice) attenuated the efficacies of the full agonists, phenylephrine and methoxamine by only 24% and 17%, respectively, whereas maximal contractions to the partial agonists, St 587, clonidine and B-HT 920, were inhibited by 46%, 61% and 75%, respectively. Also the sulfhydryl reagent, N-ethylmaleimide (10 μM), reduced contractile efficacies of phenylephrine and methoxamine to a lesser degree than those of St 587, clonidine and B-HT 920. When agonists were used at equieffective concentrations (i.e. EC30–40 for phenylephrine and methoxamine, EC70−80 for St 587 and EC99 for clonidine and B-HT 920) the degree of inhibition by removal of extracellular Ca2+ pertussis toxin and N-ethylmaleimide was similar for all agonists. These data suggest that a unitary α1-receptor may stimulate contractions via two different mechanisms. At a low degree of receptor stimulation, contractions are mediated by a pertussis toxin- and N-ethylmaleimide-sensitive influx of external Ca2+. At a higher degree of receptor stimulation, an additional mechanism is activated which is insensitive to the two G protein inhibitors and mediated by Ca2+ mobilization from intracellular sites.

Gossypol attenuates selectively the blood pressure lowering effect of endothelium-dependent vasodilators in the rabbit in vivo.

The in vivo blood pressure lowering effect of three vasodilators whose in vitro effects are endothelium-dependent (acetylcholine, substance P and adenosine triphosphate) was reduced by 40-60% after systemic treatment of the animals with gossypol. In vitro, gossypol is an inhibitor of the production and/or release of endothelium-derived relaxing factor. Systemically administered gossypol had no effect on blood pressure decreases produced by the endothelium-independent agents prostaglandin E1 (alprostadil) and glyceryl trinitrate. These results suggest that endothelium-mediated vasodilator mechanisms could participate in blood pressure regulation.

Platelets, Endothelium-Dependent Responses and Atherosclerosis

Basal release of endothelium-derived relaxing factor (EDRF) and prostacyclin from intact vascular endothelium may inhibit continuously platelet aggregation. If local platelet aggregation occurs, platelet-derived adenine nucleotides stimulate the release of EDRF. Stimulated EDRF release may override the direct vasoconstrictor effects of other platelet products such as thromboxane and serotonin resulting in local vasodilatation. In addition, stimulation of EDRF release by adenine nucleotides may inhibit further platelet adhesion and aggregation by a feedback mechanism. Thus, intact vascular endothelium may play an important role in the defense against platelet deposition and vasospasm. In atherosclerosis, basal and stimulated release of EDRF is markedly reduced. Endothelial dysfunction will impair this protective mechanism and will favour vasoconstriction and further platelet disposition. Occurrence of occlusive thrombus formation in patients with coronary artery disease may be pathophysiologically related to this impairment of endothelial defense.