Regulation of vascular tone and endothelial function and its alterations in cardiovascular disease

2 Regulation of vascular tone and endothelial function and its alterations in cardiovascular disease EDWARD WIGHT MD Consultant

GEORG NOLL

MD

Assistant Professor

THOMAS F. LUSCHER* MD Professor and Head of Cardiology Department of Obstetrics and Gynaecology, University Hospital Ziirich, Frauenklinikstrasse 10, 8091 Ziirich, and Cardiology, University Hospital Ziirich, Rgimistrasse 100, 8091 Ziirich, Switzerland

The endothelium, located between the circulating blood and the vascular smooth muscle cells, is exposed to physical, metabolic, hormonal and pharmaceutical influences, to which it reacts by secreting factors modulating the activity of the underlying vascular smooth muscle cells in a predominantly paracrine fashion. Under physiological conditions, endothelial mediators promote, as an overall effect, vasodilatation, prevent the adhesion of platelets and monocytes and, in addition, inhibit the proliferation and migration of vascular smooth muscle cells. Complex interactions between the numerous endothelial mediators so far described allow the fine tuning of vascular reactivity and the adaptations of the vasculature to changing demands. Endothelial dysfunction, on the other hand, is characterized by enhanced vasoconstrictor responses and by increased risks of thrombus formation and atherosclerosis. Ageing and chronic diseases such as hyperlipidemia, atherosclerosis and hypertension are typically associated with restrictions of endothelial function; in addition, some acute disorders seem to be mediated by the same pathomechanism. Certain drugs exert their vascular effects on the endothelial level by directly or indirectly supplying nitric oxide (nitrates and oestrogens) or by inhibiting the action of other endothelial mediators (calcium-channel blockers, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers and endothelin antagonists). In conclusion, the endothelium holds a * Correspondence: Thomas E Liischer, Professor and Head of Cardiology, University Hospital, CH 8091 Ziirich, Switzerland. Bailli~re's Clinical Anaesthesiology-Vol. 11, No, 4, December 1997 ISBN 0-7020-2360M 0950-3501/97/040531 + 30 $12.00/00

5 31 Copyright 9 1997, by Bailli~re Tindall All rights of reproduction in any form reserved

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central regulatory role in vascular physiology and disease, and seems to be the target of relevant therapeutic interventions.

Key words: endothelium; endothelial dysfunction; nitric oxide; prostacyclin; endothelin1; hypertension; vascular structure. Hypertension is a disorder of the circulation associated with an increase in pressure on the arterial side of the circulation, most commonly as the result of an increase in peripheral vascular resistance. The crucial anatomical structures determining peripheral vascular resistance are arteries with a diameter of 200 ~tm or less, referred to as resistance arteries. The contractile state of resistance arteries is controlled by neuronal effects (in particular from the sympathetic nervous system) and circulating vasoactive hormones such as noradrenaline, adrenaline, the renin-angiotensin system, vasopressin and bradykinin, as well as by local mechanisms within the vessel wall. The importance of these local, endothelium-derived, regulatory mediators has only recently been recognized. The endothelium is in a strategical anatomical position within the blood vessel wall, located between the circulating blood and vascular smooth muscle cells. It can respond to mechanical and hormonal signals from the blood. Of particular importance is the fact that the endothelium is a source of mediators, which can, in a predominantly paracrine fashion, modulate the contractile state and proliferative responses of vascular smooth muscle cells, platelet function, coagulation and monocyte adhesion. Additionally sexual steroids exert part of their influence on the endothelial cell. Under physiological conditions, the endothelium plays a protective role as it prevents adhesion of circulating blood cells, keeps the vasculature in a vasodilated state and inhibits vascular smooth muscle proliferation and migration. In disease states, on the other hand, endothelial dysfunction contributes to enhanced vasoconstrictor responses and the adhesion of platelets and monocytes, as well as the proliferation and migration of vascular smooth muscle cells, events all known to occur in atherosclerosis and especially coronary artery disease. The clinical manifestations of different illnesses, such as the haemolytic uraemic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP, Moschcowitz) and also preeclampsia, are believed to be mediated by an endothelial dysfunction, probably secondary to very different primary causes (Roberts et al, 1989). The endothelium is also the target of pharmacological influences from many different drugs in prophylactic and therapeutic settings. E N D O T H E L I A L C O N T R O L OF VASCULAR F U N C T I O N AND S T R U C T U R E

Endothelium-derived relaxing factors The endothelium can undergo relaxation when stimulated by neurotransmitters, hormones and substances derived from platelets and the

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coagulation system (Figure 1) (Furchgott and Zawadzki, 1980; Ltischer and Vanhoutte, 1990). In addition, shear forces exerted by the circulating blood induce endothelium-dependent vasodilation (Joannides et al, 1995a), an important adaptive response of the vasculature during exercise. The endothelial mediator of these responses, originally named endotheliumderived relaxing factor (EDRF), is a diffusible substance with a half-life of a few seconds, which has been identified as the free radical nitric oxide (NO) (Palmer et al, 1988). Nitric oxide is formed from L-arginine by the oxidation of its guanidine-nitrogen terminal. The catalysing enzyme NO synthase (NOS) is constitutively expressed and exists in several isoforms in endothelial cells, platelets, macrophages, vascular smooth muscle cells and the brain (Bredt et al, 1990). In endothelial cells, NOS gene expression although constitutively activated, is upregulated by oestrogens and during pregnancy (Van Buren et al, 1992; Weiner et al, 1994). The activity of the enzyme is inhibited by asymmetrical dimethyl-arginine (ADMA), an amino acid, which accumulates in patients with renal failure (Vallance et al, 1992). An inducible form of the enzyme exists in vascular smooth muscle, endothelium and macrophages (Wright et al, 1992). The enzyme is calcium-independent and produces large amounts of NO; it is induced by cytokines such as endotoxin, interleukin-l[3 and tumour necrosis factor (TNF), and is hence activated in inflammatory processes and endotoxic shock. NO-mediated, endothelium-dependent relaxation can be pharmacologically inhibited by analogues of L-arginine such as L-NC-monomethyl arginine (L-NMMA) or L-nitroarginine methyl ester (L-NAME), which compete with the natural precursor L-arginine at the catalytic site of the NOS (Figure 1) (Rees et al, 1990; Yang et al, 1991b). In isolated arteries, such inhibitors cause endothelium-dependent contraction (Tschudi et al, 1990). In perfused hearts, the inhibition of NO formation markedly decreases coronary flow (Chu et al, 1991; Amrani et al, 1992). Local infusion of L-NMMA in the human forearm circulation induces an increase in peripheral vascular resistance (Vallance et al, 1989; Joannides et al, 1995b). In pregnant animals, medication with L-NAME produces a preeclampsia-like syndrome (MolMr et al, 1994). When infused intravenously, L-NMMA induces longlasting increases in blood pressure (Rees et al, 1989). This demonstrates that the vasculature is in a constant state of vasodilation due to the continuous basal release of NO by the endothelium. The intracellular mechanism by which NO causes relaxation in vascular smooth muscle cells involves the formation of cyclic Y, 5'-guanosine monophosphate (cGMP) via the enzyme soluble guanylyl cyclase (Figure 1)(Rapoport and Murad, 1983). Nitric oxide is released abluminally as well as luminally, where it interacts with circulating blood cells and proteins (Figure 1). Certain plasma proteins, such as albumin, become nitrosylated and may act as circulating reservoirs of NO. In platelets, an increase of intracellular cGMP is associated with reduced adhesion and aggregation. Platelets themselves possess a L-arginine/NO pathway, which regulates their aggregability (Radomski and Moncada, 1991). Platelets release substances such as

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adenosine diphosphate and triphosphate, as well as serotonin, which activate the release of NO and prostacyclin from the endothelium (Figure 1) (Cohen et al, 1983; Yang et al, 1991a). At sites where platelets are stimulated, coagulation is also activated, leading to the formation of thrombin. Thrombin, the major enzyme of the coagulation cascade, is responsible for the formation of fibrin from fibrinogen and, in addition, stimulates the release of NO and prostacyclin by the endothelium (Ltischer et al, 1988). Hence, at sites where platelets and the coagulation cascade are activated, intact endothelial cells immediately release NO and in turn cause vasodilation and platelet inhibition, thereby preventing vasoconstriction and thrombus formation. On the other hand, in the absence of functional endothelial cells, aggregating platelets cause profound vasoconstriction, which is mediated through the activation of vascular smooth muscle cells by platelet-derived thromboxane A 2 (TXA2) and serotonin (Yang et al, 1991a). Prostacyclin (PGI2) is the major product of vascular cyclo-oxygenase (Moncada and Vane, 1979). In addition to NO, prostacyclin is released by endothelial cells in response to shear stress, hypoxia and several substances (see above), which also release NO. Prostacyclin increases cyclic 3', 5'-adenosine monophosphate (cAMP) in smooth muscle and platelets (Moncada and Vane, 1979). However, in most blood vessels the contribution of prostacyclin to endothelium-dependent relaxation is negligible (Richard et al, 1990; Yang et al, 1991b), and its platelet inhibitory effects are probably more important, especially as NO and prostacyclin synergistically inhibit platelet aggregation, suggesting that the activity of both mediators is required to exert full anti-platelet activity (Radomski et al, 1987a). In the coronary circulation, and even more prominently in intramyocardial vessels, not all endothelium-dependent relaxation is prevented by inhibitors of the L-arginine pathway (L-arginine analogues, haemoglobin and methylene blue) (Richard et al, 1990; Nakashima et al, 1993). In particular, the vasodilation in response to bradykinin is only slightly reduced by L-NMMA and not at all influenced by indomethacin. These types of relaxation therefore seem independent of NO and prostacyclin. As under these conditions, vascular smooth muscle cells become hyperpolarized, an endothelium-derived hyperpolarizing factor (EDHF) of unknown chemical structure has been proposed (Figure 1) (Vanhoutte, 1987). EDHF appears to activate ATP-sensitive K+ channels and/or the Na+/K+-ATPase in smooth muscle cells (Feletou and Vanhoutte, 1988). Indirect evidence suggests that EDHF may be a product of the lipoxygenase or the cytochrome P450 pathway, but other substances may also be candidates (Cohen and Vanhoutte, 1995).

Endothelium-derivcd contracting factors Endothelium-derived contracting factors include vasoconstrictor prostanoids such as thromboxane A2 and prostaglandin H2, as well as the 21 amino acid peptide endothelin and components of the renin-angiotensin system.

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In canine and human veins, arachnidonic acid, acetylcholine, histamine and serotonin can evoke endothelium-dependent contraction (Moncada and Vane, 1979; Miller and Vanhoutte, 1985). In the presence of indomethacin, however, the contractile responses are blocked and endothelium-dependent relaxation is unmasked (Yang et al, 1991b). Products of cyclo-oxygenase, such as TXA 2 and endoperoxides (prostaglandin H2: PGH2), mediate these endothelium-dependent contractions after stimulation with acetylcholine and histamine respectively (Yang et al, 1991b). TXA 2 and PGH: both activate the thromboxane receptor on vascular smooth muscle cells and platelets and hence counteract the protective effects of NO and prostacyclin on the two cell types (Moncada and Vane, 1979). Furthermore, the endothelial cyclo-oxygenase pathway is a source of superoxide anions, which cause contraction either by accelerating the breakdown of NO or by directly affecting the vascular smooth muscle cell (Vanhoutte and Katusic, 1988; Katusic and Vanhoutte, 1989). Among the three endothelin isoforms--endothelin-1, endothelin-2 and endothelin-3--endothelial cells produce exclusively endothelin-1 (Yanagisawa et al, 1988). Translation of mRNA generates pre-proendothelin, which is converted to big-endothelin; its conversion to the mature peptide endothelin-1 by the endothelin-converting enzymes (ECE-1 and ECE-2) is necessary for the development of full vascular activity (Ohnaka et al, 1993; Xu et al, 1994; Emoto and Yanagisawa, 1995). The expression of mRNA and the release of the peptide is stimulated by thrombin, transforming growth factor [3,, interleukin-1, epinephrine, angiotensin II, arginine vasopressin, calcium ionophore and phorbol ester (Figure 1) (Yanagisawa et al, 1988; Boulanger and Lfischer, 1990), as well as through other stimuli (hypoxia, ischaemia, shear stress, cyclosporin A and oxidized low-density lipoproteins (Boulanger et al, 1992; Grieff et al, 1993; Goerre et al, 1995). Most of the endothelin produced by endothelial cells is released abluminally towards vascular smooth muscle cells rather than luminally (Yoshimoto et al, 1991). Hence circulating levels of this vasoactive peptide only poorly reflect the local vascular production and are very low under physiological conditions. Furthermore, this fact stresses the concept that endothelin is an autocrine and paracrine vascular regulatory mechanism rather than a circulating hormone. Plasma endothelin-1 is cleared up to 90% by the lungs during first passage (de Nucci et al, 1988). In addition, very little of this peptide is normally produced, owing to absence of stimuli and particularly to the presence of potent inhibitory mechanisms involving cGMP (liberated via NO, atrial natriuretic peptide and prostacyclin) (Boulanger and Lfischer, 1990; Saijonmaa et al, 1990), cAMP (Yokokawa et al, 1991) and an inhibitory factor produced by vascular smooth muscle cells (Stewart et al, 1990). After inhibition of the endothelial L-arginine pathway, the thrombin- or angiotensin-induced endothelin production is augmented (Boulanger and Lfischer, 1990). Endothelin can, by itself, release NO and prostacyclin from endothelial cells, which in turn reduce endothelin production, acting as a negative feedback mechanism (Warner et al, 1989; Dohi and Lfischer, 1991a).

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Endothelin-1 causes vasodilation at very low concentrations but marked and sustained contractions at higher concentrations, which makes it the most potent endogenous vasoconstrictor yet identified (Kiowski et al, 1990; Haynes et al, 1996), eventually leading in the heart to ischaemia, arrhythmias and death. Intramyocardial vessels are more sensitive to the vasoconstrictor effects of endothelin-I than are epicardial coronary arteries, suggesting that the peptide is particularly important in the regulation of flow (Kting et al, 1995). In general, veins are more sensitive to endothelin1 than are arteries (Ltischer et al, 1990b; Yang et al, 1990a). All three isoforms of endothelin bind to two types of endothelin receptor: the ETa and ETB receptors (Arai et al, 1990; Sakurai et al, 1990). Both are Gcprotein coupled with seven transmembrane domains and are linked to phospholipase C and protein C. ETa receptors are expressed on vascular smooth muscle cells, have a 10 times higher binding affinity to endothelin1 compared with endothelin-3 and mediate mainly the vasoconstrictor (Simonson and Dunn, 1990) and proliferative (Simonson and Herman, 1993) actions of endothelin, although ETB receptors in some vascular beds also contribute to this effect. Endothelial cells predominantly express the ET B receptor, which binds endothelin-1 and endothelin-3 with similar affinity. In endothelial cells, the ET~ receptor is linked to the formation of NO and prostacyclin, which explains the transient vasodilatory effects of endothelin when infused in intact organs or organisms (Warner et al, 1989; Fukuda et al, 1990; Kiowski et al, 1990; Le Monnier de Gouville et al, 1990; Sorensen et al, 1994). Endothelin-1 probably has a role in the maintenance of basal vasomotor tone as the local administration of a selective ETa receptor antagonist leads to vasodilation and an increase in local blood flow (Haynes and Webb, 1994; Wenzel et al, 1994). Endothelin potentiates the vasoconstrictory effect of catecholamines, which in turn potentiate the action of the former (Tabuchi et al, 1989a). Furthermore, threshold concentrations of endothelin-1, which by themselves do not cause contraction, potentiate the contractile effects of serotonin and norepinephrine (Yang et al, 1990b). Vasoconstriction by endothelin is also increased in atherosclerotic vessels, in which the opposing effect of NO is diminished or lost (Lopez et al, 1990). Plasma endothelin concentrations are increased after myocardial infarction and correlate with prognosis in these patients (Battistini et al, 1993; Omland et al, 1994), suggesting a pathogenic role for endothelin by augmenting myocardial damage after acute ischaemia. Increased levels of endothelin have also been described in patients with congestive heart failure (Wei et al, 1994), ischaemic cerebral infarction (Ziv et al, 1992) and subarachnoid haemorrhage (Suzuki et al, 1992) and in women with pre-eclampsia (Kamoi et al, 1990), while conflicting results concerning endothelin levels are found in essential hypertension (Ltischer et al, 1993; Vanhoutte, 1993). Finally, the endothelium is involved in the renin-angiotensin system. ACE, which transforms angiotensin I into angiotensin II, is expressed in endothelial cells, and ACE activity has been documented in the vessel wall (Figure 2). There is evidence that, besides the traditional view of the renin-angiotensin system being a classical endocrine system, there also

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exist local renin-angiotensin systems in the endothelium and other tissues (Danser et al, 1992). ACE is identical to kinase II, which breaks down bradykinin. Whether or not other components of the renin-angiotensin system are produced in endothelial cells is controversial. Angiotensin II can activate endothelial angiotensin receptors; these receptors stimulate the production of endothelin (Yanagisawa and Masaki, 1989) and possibly also of other mediators such as plasminogen activator inhibitor (Vaughan et al, 1995).

Interactions between endothelium-derived relaxing and contracting factors The endothelium is a source of several relaxing and contracting factors. With increasing complexity of the endothelial organ, interactions of these factors at the level of the endothelium itself or at the level of the vascular smooth muscle cell become more and more important. Endothelin stimulates endothelial NO production at the level of the endothelium via the ETB receptor subtype, thereby limiting its own effects (Warner et al, 1989; Dohi and Liischer, 1991a). Nitric oxide inhibits endothelin production by activating the soluble guanylyl cyclase, which results in formation of cGMP in endothelial cells (Boulanger and L~ischer, 1990). Decreasing endothelial NO production by the blockade of NO synthase with L-arginine analogues such as L-NMMA or L-NAME unmasks a tonic pressor influence of endothelin (Richard et al, 1996) and augments the thrombin-induced, but not the basal, production of

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endothelin from the intact porcine aorta (Boulanger and LiJscher, 1990). Superoxide dismutase, which inhibits the breakdown of NO by superoxide anions and in turn prolongs the half-life of NO (Gryglewski et al, 1986), markedly inhibits endothelin synthesis in the above-mentioned experimental model. Finally, inhibitors of the soluble guanylyl cyclase, such as methylene blue, increase, but activators of this enzyme, such as nitroglycerin or the active metabolite of molsidomine (3-morpholino-sydnonimine, SIN-l), prevent, thrombin- and angiotensin H-induced endothelin production (Boulanger and LiJscher, 1990; Kohno et al, 1991). This provides an alternative mechanism of action of nitrovasodilators, i.e. the inhibition of vascular endothelin production. Certain agonists of endothelin production, such as oxidized LDL (Boulanger et al, 1992) are, however, insensitive to cGMP-dependent inhibition, indicating that NO interferes with some, but not all, pathways of endothelin production (Boulanger and Liischer, 1991). The character and degree of interaction between NO and endothelin may also change in different vascular beds, as NO is a potent inhibitor of the effects of endothelin in vascular smooth muscle cells of the isolated conduit and larger resistance arteries (Ltischer et al, 1990b), whereas in the human forearm circulation endothelin dominates the effects of NO (Kiowski et al, 1990). Intraluminal infusions of endothelin-1 evoke endothelium-dependent relaxation in isolated perfused rat mesenteric arteries. Since this response is prevented by indomethacin but not by L-NMMA, prostacyclin or PGE: is the most likely mediator (Dohi and Liischer, 1991 a). In endothelial cells in culture, inhibition of prostacyclin synthesis by indomethacin augments endothelin formation, suggesting that prostacyclin, via the activation of cAMP does, like NO, exert a negative feedback inhibition on endothelin production (Yokokawa et al, 1991). The interaction of the vascular renin-angiotensin system with endothelium-derived vasoactive factors is not yet fully understood (Liischer and Vanhoutte, 1990). It is of interest, however, that angiotensin II induces endothelin gene expression in a variety of experimental settings (Dohi et al, 1992b). In the spontaneously hypertensive rat (SHR), the induction of endothelin production by angiotensin II augments the contractile response to norepinephrine in an endothelium-dependent manner. Since this effect can be prevented by phosphoramidon (an inhibitor of the endothelin-converting enzyme (Sawamura et al, 1991)) or by antibodies against endothelin, angiotensin II is believed to stimulate the local production of endothelin-1, thereby increasing vascular reactivity to catecholamines. Angiotensin II also increases endothelin production in vascular smooth muscle cells in culture, but it is a much weaker stimulator than is platelet-derived growth factor, transforming growth factor ~1 or arginine vasopressin (Hahn et al, 1990). The interaction of NO and prostacyclin showed conflicting results in different experimental settings. In porcine coronary arteries, prostacyclin stimulates the release of NO and augments the relaxing activity of NO released in the presence of prostacyclin (Shimokawa et al, 1988), while in cultured bovine aortic endothelial cells, NO inhibits the production of prostacyclin (Doni et al, 1988). In human washed platelets stimulated with collagen, subthreshold concentrations of either NO or prostacyclin, which

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by themselves exert no anti-aggregatory effects, can profoundly potentiate each other's inhibitory action (Radomski et al, 1987b). This may be of physiological importance and indicates that the luminal release of both NO and prostacyclin is required to ensure the full anti-thrombotic function of the endothelium. Certain stimuli, for example, mechanical forces or agonists activating the L-arginine/NO pathway, such as acetylcholine--the prototype agonist for endothelium-dependent relaxation---cause the co-release of NO and cyclo-oygenase-derived contracting factors from the endothelium. These endothelium-dependent contracting factors (EDCFs), such as TXA2, PGH2 and superoxide radicals, all reduce the effects of NO (Katusic and Vanhoutte, 1989; Liischer and Vanhoutte, 1990; Ltischer et al, 1992). The final vascular response depends on the relative amounts and potency of the factors released. In general, ageing is associated with a decreased formation of relaxing and an increased synthesis of contracting factors after stimulation with acetylcholine, thereby leading to depressed endothelium-dependent relaxation (Koga et al, 1989; Ltischer and Vanhoutte, 1990; Taddei et al, 1995). Endothelial influence on vascular structure

Removal of the endothelium, for example, mechanically by a balloon catheter, invariably leads to the immediate deposition of platelets and white blood cells and, after days to weeks, to intimal hyperplasia at the site of injury (Baumgartner and Studer, 1963; Ross, 1986). This suggests that the endothelium also regulates vascular structure and that its presence assures the quiescence of vascular smooth muscle cells (Figure 3). Endothelial dysfunction, on the other hand, seems to be an important factor in atherosclerosis, re-stenosis, coronary bypass graft disease (Ltischer et al, 1988) and hypertensive vascular disease. Vascular structure is mainly determined by vascular smooth muscle cells and, in disease states, by white blood cells invading the intima. Endothelial cells may exert either direct or indirect effects on vascular structure. Nitric oxide and prostacyclin inhibit the adhesion of platelets to the vessel wall (Radomski et al, 1987b). If, at sites of endothelial dysfunction or denudation, platelets adhere to the blood vessel wall, they cause contraction (through the release of TXA2 and serotonin) (Yang et al, 1991a) and stimulate the proliferation and migration of vascular smooth muscle cells (via the release of platelet-defived growth factor: PDGF) (Ross, 1993). In addition, NO inhibits the adhesion of monocytes, which are an important component of the atherosclerotic plaque and also capable of releasing growth factors and cytokines. Furthermore, endothelial cells are a source of growth promoters and inhibitors. It is thought that, under physiological conditions, growth inhibitors prevail and that this may explain why the blood vessel wall is normally quiescent and does not exhibit proliferative responses (Figure 3). Heparin, heparin sulphates, transforming growth factor [5~, and most probably also NO and prostacyclin, are potent inhibitors of vascular smooth muscle migration and proliferation (Hannan et al, 1988; Garg and Hassid,

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1989; Battegay et al, 1990; DiCorletto and Fox, 1990). Nitric oxide and prostacyclin can, besides directly influencing the growth of the vascular smooth muscle cell (Garg and Hassid, 1989; Scott-Burden and Vanhoutte, 1993), exert indirect inhibitory effects on proliferative responses in the blood vessel wall; indeed, the inhibition of platelet function by these two mediators prevents platelet adhesion and aggregation (Busse et al, 1987; Radomski et al, 1987a) and, in turn, the local release of PDGE On the other hand, at least under certain conditions, endothelial cells may also stimulate the proliferation of vascular smooth muscle cells by producing basic fibroblast growth factor (bFGF), PDGF and endothelin (Figure 3) (Hannan et al, 1988; Dubin et al, 1989; DiCorletto and Fox, 1990). P H A R M A C O L O G I C A L MODIFICATION OF ENDOTHELIAL FUNCTION Endothelial dysfunction may be a cause of various diseases affecting the cardiovascular system. Hence drugs able to substitute the defective release of endothelial mediators and/or leading to an improved endothelial function may have an important therapeutic potential in patients and may even exert prophylactic effects. On the other hand, certain drugs may have side-effects of clinical relevance on endothelial cells. Nirates

Nitrovasodilators such as nitroglycerine, sodium nitroprusside (SNP) and molsidomine exert their vasodilatory effects by releasing NO from their molecule (Feelisch and Noack, 1987). Their final mechanism of action is therefore identical to that of endogenously produced NO. The sensitivity of the blood vessel wall to nitrates and nitrovasodilators is reduced in cases where endogenous NO production is increased (Pohl and Busse, 1987; Liischer et al, 1990a). This also holds true for pregnancy (Wight, submitted for publication). In human arteries devoid of endothelium, however, where the endogenous NO production is reduced, the concentration-relaxation reponse curve to molsidomine is shifted to the left, equivalent to an increased sensitivity towards this medicament (Vanhoutte, 1988). This indicates that nitrovasodilators are particularly effective at sites of reduced vascular NO formation. Furthermore, nitrates are able to reduce endothelin production under certain conditions (see above), which may comprise a new therapeutic approach. Calcium-channel blockers

Calcium antagonists do not seem to affect the release of endotheliumderived mediators (Vanhoutte, 1988); however, they facilitate their effects in vascular smooth muscle. In addition, at least in certain vascular beds, they inhibit the vasoconstrictory effects of endothelin and cyclooxygenase-dependent contracting factors (Ritz et al, 1992). In the human

543

VASCULAR TONE AND ENDOTHELIAL FUNCTION

forearm circulation, endothelin-1 induced contractions are prevented by high dosages of nifedipine and verapamil, unmasking the vasodilatory effects of the peptide (Kiowski et al, 1990). ACE inhibitors and angiotensin receptor blockers ACE is located on the endothelial cell membrane and is responsible for the conversion of the relatively inactive angiotensin I to the active angiotensin II (see Figure 2). Thus ACE inhibitors abolish or attenuate responses to angiotensin I but not to angiotensin II. As there are several substrates of ACE besides angiotensin I, ACE inhibitors may induce side-effects unrelated to the reduced levels of angiotensin II. Since ACE catalyses the breakdown of bradykinin, ACE inhibitors lead to an increase of local vascular concentrations of bradykinin, which is in turn a potent stimulator of the L-arginine/NO pathway (see Figure 2) (Palmer et al, 1987; Mombouli et al, 1991; Wiemer et al, 1991). The latter effect of ACE inhibitors may be the reason for their protective action on the cardiovascular system, as an increased local NO concentration improves blood flow, prevents platelet activation and also exerts anti-proliferative effects on the vascular wall. Chronic therapy with ACE inhibitors improves endothelial function in normotensive and particularly hypertensive rats (Clozel et al, 1990; Dohi et al, 1992a). It has recently been demonstrated that chronic therapy with an ACE inhibitor improves endothelial function in the coronary circulation of patients with angiographically documented coronary artery disease (Figure 4) (Mancini et al, 1996). Placebo

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Distinct from the ACE inhibitors are the angiotensin II receptor antagonists, which either block AT1 and AT2 receptors selectively or exhibit a balanced antagonism of both receptor subtypes (Timmermans et al, 1993). It is likely that blockade of angiotensin II receptors on endothelial cells would inhibit the angiotensin II-induced endothelin production.

Endothelin antagonists Specific antagonists of the ETA and ETB receptors, as well as combined ETA/ETB receptor antagonists, have been developed (Bazil et al, 1992; Clozel et al, 1993; L6ffler et al, 1993; Nishikibe et al, 1993; Raschack et al, 1995). These tools allow exact determination of the role of endogenously formed endothelin in various forms of cardiovascular disease. As the contractile effects of endothelin in several blood vessels are mediated through ETa and ET B receptors (Seo et al, 1994; Haynes et al, 1995), it seems that combined receptor antagonists may be required to block the unwanted effects of endothelin in patients (Haynes et al, 1996). Potential disease states in which endothelin antagonists could be effective are hypertension, cyclosporin therapy, pulmonary hypertension, atherosclerotic vascular disease, congestive heart failure and renal failure. Whether or not these substances fulfil these expectations remains to be shown. At least it seems that endothelin antagonists as well as phosphoramidone--an endothelin-converting enzyme inhibitor--are able to reduce, although not necessarily normalize, blood pressure in hypertensive rats (Yanagisawa et al, 1988; Dohi et al, 1992b; Nishikibe et al, 1993). The observation that endothelin receptor antagonists prevent the development of stroke in stroke-prone SHRs, while decreasing blood pressure only slightly, is of particular clinical interest (Nishikibe et al, 1993). In addition, it has been demonstrated that administration of bosentan, a non-selective endothelin receptor antagonist, reduces elevated pulmonary artery pressure in patients with congestive heart failure (Kiowski et al, 1995).

Sexual steroids Sexual steroids, in particular oestrogens, are vasoactive substances. (The oestrogen effects described here are limited to the so-called natural oestrogens such as 17[3-oestradiol (E2) or the conjugated equine oestrogens (CEE), used for hormone replacement therapy, and not to the synthetic oestrogens such as ethinyl oestradiol (EE2) used in contraceptive pills.) There is much evidenc in many clinical trials that documents a protective effect of oestrogens in the primary and secondary prevention of cardiovascular disease (Gruchow et al, 1988; Stampfer et al, 1991; Sullivan, 1994; Writing Group for the PEPI-Trial, 1995). The influence of oestrogens on the vascular wall seems to be exerted mainly in two ways (Gorodeski and Utian, 1994). One is through gene modulation mediated by the oestrogen receptor, which has been identified on endothelial and vascular smooth muscle cells (Colburn and Buonassisi, 1978; Karas et al, 1994).

VASCULAR TONE AND ENDOTHELIAL FUNCTION

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Second, a direct influence of oestrogens is postulated, which may be the result of post-translational modifications of enzymes. While gene modulation typically results in long-term effects, direct steroid actions can rapidly influence vascular function. Oestrogens augment or restore, respectively, the endothelium-dependent vasodilation in atherosclerotic blood vessels by increasing the local NO concentration (Gilligan et al, 1994a,b). This is caused either by inducing NOS activity (Weiner et al, 1994) or by inhibiting NO degradation, as oestrogens are potent anti-oxidants (Katsuaki et al, 1987). Besides their influence on the NO system, oestrogens stimulate the activity of prostacyclin and EDHF, while attenuating vasoconstrictory effects mediated by TXA: and endothelin (Williams et al, 1990; Polderman et al, 1993). In addition, direct influences of oestrogens on vascular smooth muscle cells have been documented, similar to the effects of calcium antagonists (Jiang et al, 1991; Collins et al, 1993; Shu-Zhong et al, 1995). Oestrogens also potentiate the action of catecholamines (Colucci et al, 1982). In conclusion, oestrogens induce vasodilation, inhibit the proliferation and migration of vascular smooth muscle cells, inhibit platelet aggregation and block the peripheral oxidation of LDL, besides influencing the lipid profile in many other ways (Krauss, 1994), exerting a profound anti-atherogenic effect. Progestogens, therapeutically used to protect the endometrium in cases of chronic oestrogen stimulation, antagonize some of the beneficial effects of oestrogens on the cardiovascular system, the amount of which is dependent on their androgenic partial effect (Gorodeski and Utian, 1994). Natural, micronized progesterone seems to have the most favourable characteristics of all progestins and therefore, in the cardiologist's view, should be preferred in hormone replacement therapy (Writing Group for the PEPI-Trial, 1995). In addition, progestogens may cause vascular spasms if injected into the coronary circulation, probably by inhibiting prostacyclin synthesis (Makila et al, 1982).

ENDOTHELIUM AS A TARGET AND MEDIATOR OF CARDIOVASCULAR DISEASE

Because of its location between the circulating blood and the vascular smooth muscle layers, the endothelium is the structure most exposed to the mechanical forces of the blood and to hormones and noxious substances circulating therein. Morphological studies have demonstrated changes in endothelial cell morphology with ageing and disease, in particular increased endothelial cell turnover and density, a marked heterogeneity in cell size, bulging of the cells into the lumen and increased fibrin and cell deposition in the subintimal space (Ross, 1993). Endothelial cell denudation, however, does not occur except in very late stages of atherosclerosis and plaque rupture. Functional alterations are almost invariably associated with these changes in endothelial cell morphology.

546

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Ageing All forms of cardiovascular disease increase in frequency with age even in the absence of known cardiovascular risk factors, suggesting that ageing per se alters vascular function. Ageing in the rat is associated with an increased formation of EDCFs (PGH2) (Koga et al, 1989) as well as with a decrease in the release, or an increased inactivation, of NO (Dohi and Ltischer, 1990; Kting and Ltischer, 1995). In humans, the increase in coronary flow induced by acetylcholine infusion decreases with age (Zeiher et al, 1993). Whether these changes are related to a dysfunction of muscarinic receptors and their signal transduction pathways or a decreased activity of NOS is still uncertain. Endothelin is able to potentiate at low and threshold concentrations the contractile effects of other mediators. With ageing, and also in hypertensive individuals, this potentiating function of endothelin is increased, indicating that this indirect amplifying effect may contribute to the increased vascular contractility as pressure rises and the blood vessel wall ages (Tabuchi et al, 1989b; Dohi and Ltischer, 1990; Yang et al, 1990b).

Hypertension Hypertension is a disorder of the circulation associated with an increase in pressure on the arterial side of the circulation, most commonly the result of high peripheral resistance, determined by the contractile state of the resistance arteries with a diameter of 200 ~tm or less. The resistance arteries are influenced by neuronal stimulation (in particular from the sympathetic nervous system), by circulating hormones and by paracrine and autocrine mechanisms within the blood vessel wall. Clinically, hypertension is associated with no or only mild symptoms. The major aim of therapy is not to correct haemodynamic abnormalities but rather to prevent the complications of hypertension, such as stroke, angina pectoris, myocardial infarction, renal failure and peripheral vascular disease. If endothelial dysfunction is to be considered as a main pathomechanism of hypertension, it is necessary to demonstrate that the activation and/or inhibition of endothelial mediators can cause a significant and persistent increase in arterial blood pressure. The vessel wall is normally in a constant state of vasodilation due to the basal formation of NO. Inhibition of vascular NO generation by L-arginine analogue causes marked and sustained increases in arterial blood pressure. Impaired endothelium-dependent relaxation elicited by acetylcholine has been demonstrated in many (Konishi and Su, 1983; Ltischer and Vanhoutte, 1986a; Watt and Thurston, 1989; Aarhus et al, 1990; Diederich et al, 1990), but not all (Tschudi et al, 1991), experimental models of hypertension involving different vascular beds. In patients with essential hypertension or renovascular and endocrine hypertension respectively, the vasodilatory effects in the forearm circulation in response to acetylcholine, but not to sodium nitroprusside, were reduced in all (Panza et al, 1990, 1993; Taddei et al, 1993) but one study (Cockcroft et al, 1994), suggesting a reduced

VASCULAR TONE AND ENDOTHELIAL FUNCTION

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formation of NO after stimulation of the muscarinic receptor. Similar findings have been obtained in the coronary circulation, particularly in the presence of left ventricular hypertrophy (Treasure et al, 1993; Zeiher et al, 1993). In the SHR, however, NOS activity is markedly increased but inefficacious, probably due to an increased deactivation of NO (Figure 5) (Nava et al, 1995). Despite this, not all hypertensive blood vessels and not all forms of hypertension are exhibiting alterations of the L-arginine/NO pathway. Impaired endothelium-dependent relaxation to acetylcholine can also be related to an increased production of EDCFs, such as PGH2, which is the case in the aorta and other vascular beds of SHRs (Figure 5) (Liischer et al, 1986; Ltischer and Vanhoutte, 1986a; Koga et al, 1989; Mayhan et al, 1989; Kato et al, 1990). These mediators are cyclo-oxygenase dependent, and therefore pre-treatment with indomethacin, a blocker of this enzyme, improves the impaired vasodilation to acetylcholine. As this is true also in the human forearm circulation (Taddei et al, 1993), one can conclude that an increased production of PGH2 or of another cyclo-oxygenase-derived contracting factor also contributes to impaired endothelium-dependent vascular regulation in humans. The role of endothelin in hypertension is quite controversial (Liischer et al, 1993). Indeed, most studies find normal plasma levels of the peptide in patients with essential hypertension (Liischer et al, 1992), but plasma endothelin concentrations are elevated in women with pre-eclampsia (Kamoi et al, 1990; Nova et al, 1991; Schiff et al, 1992) and in patients with a haemangio-endothelioma (Yokokawa et al, 1991). In most experimental forms of hypertension, the vascular response to endothelin-1 is paradoxically reduced (Dohi and Ltischer, 1991b; Deng and Schiffrin, 1992). However, the indirect potentiating effects of subthreshold endothelin concentrations appear to be augmented (Tabuchi et al, 1989b; Dohi and Ltischer, 1990; Yang et al, 1990b). Recent studies, using inhibitors of the endothelin-converting enzyme or endothelin receptor antagonists, suggest that endothelin contributes to blood pressure elevation in certain forms of hypertension in laboratory animals and humans (McMahon et al, 1991; Nishikibe et al, 1993; Haynes et al, 1996). On the other hand, experiments with transgenic laboratory animals have revealed contradictory results: endothelin-2 transgenic rats do not have high blood pressure despite high circulating endothelin-2 levels, and knock-out endothelin mice (which lack the endothelin-1 gene) are hypertensive, besides having malformations of the larynx and throat (Kurihara et al, 1994). Other vasoactive mediators are also candidates to contribute to endothelial dysfunction in hypertension. Indeed, the responses to angiotensin I and II are increased in SHRs (Tschudi and Ltischer, 1995), and in addition platelets and platelet-derived substances (ADP, ATP and serotonin), known to stimulate the formation of EDCFs (Ltischer and Vanhoutte, 1986b), may lead to increased peripheral vascular resistance and also to complications of hypertension. At this point, it is still not clear whether endothelial dysfunction in hypertension is a primary or a secondary phenomenon. Hence, most of the

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experimental studies suggest that endothelial dysfunction is a consequence rather than a cause of hypertension (Loc'kette et al, 1986; Dohi et al, 1990; Ltischer, 1994), although a few studies have found an alteration of endothelial reactivity even at an early stage of hypertensive disease. Furthermore, endothelial dysfunction could either be a generalized phenomenon occurring in all forms of hypertension and in all vascular beds, or occur in particular with certain but not other forms of hypertension (depending on the mediators involved), and certain vascular beds--such as the cerebral or coronary circulation--may be particularly affected, others being spared. Experimental evidence and clinical studies with hypertensive patients suggest distinct endothelial reactivity in different forms of hypertension and, furthermore, distinct endothelial dysfunction in different vascular beds of the same hypertensive individual, as well as different endotheliumdependent responses to different mediators in the same vascular bed. Therefore, endothelial dysfunction is likely to be a secondary event involved in the maintenance rather than the initiation of hypertension but may contribute to the vascular complications of this disease, such as myocardial infarction and stroke (Liischer, 1994). It seems that there are also gender differences in relation to endothelial dysfunction in hypertension. Pre-menopausal women with hypertension have a 'better' haemodynamic profile and show a more favourable cardiac adaptation to essential hypertension than do their male counterparts. However, these sex differences disappear after the menopause, indicating that oestrogens possibly exert a protective effect on endothelial function in pre-menopausal hypertensive women. After the menopause, however, women show an increased incidence of hypertension along with a markedly increased cardiovascular risk (Aepfelbacher and Messerli, 1996).

Hyperlipidaemia and atherosclerosis Three distinct mechanisms are currently thought to be responsible for the initiation of atherosclerotic lesions in humans: 1.

2. 3.

the accumulation of lipids and plasma-derived lipoproteins in the arterial intima as well as the adhesion, migration and accumulation of monocytes/macrophages in the subintima, which transform into lipidfilled foam cells; smooth muscle cell migration from the media into the intima and further proliferation there; accumulation of platelet and/or fibrin deposits in the intima.

Morphologically, the endothelium remains intact in the pre-stage of atherogenesis (Ross, 1986); functionally, however, pronounced alterations may already occur at this stage, as far as both endothelium-dependent relaxations and contractions are concerned. Atherosclerotic lesions develop from simple fatty streaks to fibrofatty lesions and further to fibrous plaques, which eventually rupture, leading to endothelial denudation, usually associated with more or less severe endothelial dysfunction.

550

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While short-term exposure of the coronary arteries to LDL does not cause significant alterations in endothelial function, the oxidation of LDL (to oxLDL) alters its biological properties (Kugiyama et at, 1990; Tanner et al, 1991), in particular its capability to interfere with the LDL receptor, and allows the oxLDL molecule to interact with a scavenger receptor (Figure 6) (Galle et al, 1991). This in turn alters endothelial function and influences several intracellular mechanisms by interfering with G~ protein-linked signal transduction, the mobilization of L-arginine, the activity of the NOS and/or the inactivation of NO by oxidizing substances such as superoxide (Figure 6) (Tanner et al, 1991). Although the exact mechanism has not yet been convincingly characterized, it appears that the oxidation of LDL is a crucial step (Figure 7) (Galle et al, 1991; Rosenfeld et al, 1991). In addition, several studies have demonstrated the presence of oxLDL in atherosclerotic plaques (Yla-Herttuala et al, 1989). This also could explain why anti-oxidants such as vitamins C and E and oestrogens are able to exert a protective effect in the coronary circulation, in particular at the level of the endothelial cell (Keaney et al, 1994). In contrast to hyperlipidaemia, atherosclerosis induces more pronounced impairment in endothelial function both in vitro and in vivo (Shimokawa and Vanhoutte, 1989; Zeiher et al, 1993). A consistent finding in atherosclerotic coronary arteries is the paradoxical contraction in response to acetylcholine, whereas a vasodilation can be observed in unaffected blood vessels (Ludmer et al, 1986). This abnormal reaction is prevented or reversed by oestrogens (Reis et al, 1994). It appears that, in patients, receptor-operated mechanisms activated by acetylcholine, thrombin and/or serotonin become dysfunctional at an

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earlier stage of atherosclerotic disease, whereas mechanical stimulation (shear stress) reveals substantial endothelial dysfunction only at a very late stage (Zeiher et al, 1993). If flow-dependent vasodilation finally becomes impaired, it can be demonstrated not only pharmacologically, but also during exercise, when patients with coronary artery disease exhibit a paradoxical vasoconstriction of their epicardial coronary arteries (Gage et al, 1986). These alterations in coronary vasomotion may significantly contribute to ischaemia and facilitate the occlusion of coronary arteries, particularly in the presence of atherosclerotic plaques and/or platelet activation. Experiments in the aorta and in coronary arteries of hypercholesteraemic animals (rabbit and pig) suggest that the overall production of NO is not reduced but markedly augmented; however, NO is inactivated rapidly by superoxide radicals produced within the endothelium (Shimokawa and Vanhoutte, 1989; Minor et al, 1990), finally resulting in a reduction of biologically active NO in the blood vessel wall in hypercholesterolaemia and atherosclerosis. Endothelin also seems to be of importance in atherosclerotic vascular disease, as increased endothelin levels have been demonstrated in atherosclerosis, coronary spasm and acute myocardial infarction (Miyauchi et al, 1989; Lerman et al, 1991; Stewart et al, 1991), whereas the expression of endothelin receptors is downregulated (Winkles et al, 1993). Again, oxLDL rather than native LDL stimulates endothelin-1 production by increasing endothelin gene expression and release from the porcine and human aortic endothelial cells (Figure 7) (Boulanger et al, 1992).

552

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In addition to endothelial cells, vascular smooth muscle cells, particularly those which have migrated into the intima during the atherosclerotic process, also produce endothelin. Endothelin can be released by growth factors such as platelet-derived growth factor and transforming growth factor ~3~as well as by vasoconstrictors such as arginine vasopressin (Hahn et al, 1990) from vascular smooth muscle cells in culture. Hence, several mediators involved in atherosclerosis stimulate the production of vascular endothelin. This may explain why plasma endothelin levels are increased and are positively correlated with the extent of the atherosclerotic process (Lerman et al, 1991). Furthermore, particularly unstable lesions removed from coronary arteries by atherectomy exhibit marked staining for endothelin-1 (Zeiher et al, 1994). It seems possible that local vascular endothelin contributes to abnormal coronary vasomotion in patients with unstable angina. Ischaemia and thrombin might be triggers of endothelin production in patients with acute coronary symptoms contributing to hypervasoconstriction and cellular proliferation. CONCLUSIONS The endothelium has a strategic anatomical position in the vascular wall between the circulating blood and the layers of smooth muscle cells. Endothelial mediators influencing platelet function and thrombus formation, as well as regulating activity of vascular smooth muscle cells, have a profound influence on the cardiovascular system. Under physiological conditions, the endothelium exerts a protective effect by keeping the vascular system in a vasodilated state, counteracting atherosclerosis by preventing the adhesion of circulating blood cells to the blood vessel wall and inhibiting the migration and proliferation of vascular smooth muscle cells. In hypertension, several alterations in endothelial function develop as the disease progresses, which could be involved in the increase in peripheral vascular resistance and in complications of hypertension. Endothelial dysfunction, however, is not uniform, differing in different experimental models of hypertension and in different vascular beds. Furthermore, alterations in endothelial function appear more likely to be a consequence rather than a cause of high blood pressure. Hence the degree of endothelial dysfunction may change with increasing severity and duration of hypertension. While in experimental hypertension, antihypertensive therapy may be able to reverse endothelial dysfunction, this appears to be much more difficult to achieve in the human. Nevertheless, endothelial dysfunction seems to make an important contribution to the pathophysiology of hypertension and its cardiovascular complications.

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