Cardiovascular disease still accounts for the majority of morbidity and mortality in Western countries. Most forms of cardiovascular disease involve atherosclerotic vascular changes in the coronary, cerebral, renal and peripheral circulation system leading to angina pectoris and myocardial infarction, stroke, renal failure and claudication.
The endothelium is in a strategic anatomical position within the blood vessel wall, located between the circulating blood and vascular smooth muscles. It can sense mechanical and respond to hormonal signals from the blood. Of particular importance is the fact that the endothelium is a rich source of a variety of mediators of both vasodilating and vasoconstricting, predominantly paracrine, substances which modulate the contractile state and proliferative responses of vascular smooth muscle cells, platelet function, coagulation as well as monocyte adhesion (1). Thus, 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, adhesion of platelets and monocytes as well as proliferation and migration of vascular smooth muscle cells, which are all events known to occur in atherosclerosis.
Endothelial function is impaired in certain cardiovascular conditions including atherosclerosis (2) and in the presence of risk factors such as diabetes (3), smoking (4), hypercholesterolemia (5,6), aging (2), menopause (7) and hypertension (8).
Hypertension is an important risk factor for the development of cardiovascular disease (9). It is associated with an increase in pressure on the arterial side of the circulation system, mostly due to elevated peripheral resistance, determined by the contractile state of the resistance arteries with a diameter of 200 mm or less. The resistance arteries are influenced by neuronal stimulation (in particular from the sympathetic nervous system), by circulating hormones, and paracrine and autocrine mechanisms within the blood vessel wall.
Normally the vessel wall is in a constant state of vasodilation due to the basal formation of nitric oxide (NO) by endothelial cells (10). On the other hand, the vascular endothelium might be involved directly to increase peripheral resistance, via an enhanced release of constricting factors and/or a decreased release or enhanced breakdown of relaxing factors. Furthermore, the endothelium may contribute to the vascular complications of hypertension as it becomes dysfunctional.
THE PHYSIOLOGIC FUNCTION OF THE ENDOTHELIUM
The endothelium is a highly active endocrine organ (Fig. 1). Endothelial cells release numerous vasoactive substances [i.e., NO, endothelin (ET)-1, prostacyclin and endothelium-derived hyperpolarising factor (EDHF)] that regulate vascular smooth muscle and circulating blood cells. In response to many stimuli, such as increased shear forces exerted by increased blood flow, the endothelium releases NO, which is a potent vasodilator that also inhibits cellular growth and migration (1,11,12). In addition, NO possesses antiatherogenic and thromboresistant proprieties by preventing platelet aggregation and adhesion (13).
NO is formed from L-arginine by oxidation of its guanidine-nitrogen terminus and is rapidly inactivated by free radicals, so that its half-life is only a few seconds (14). The catalyzing enzyme NO synthase (NOS) is constitutively expressed and exists in several isoforms in endothelial cells, platelets, macrophages, vascular smooth muscle cells, and the brain (1).
ETs have been implicated in the pathogenesis of several, mainly cardiovascular, disorders in view of their powerful vasoconstrictor and growth-promoting properties (15). The three members of the family, ET-1, ET-2, ET-3, are 21-amino acid residue peptides that are produced in a variety of tissues, where they act as modulators of vasomotor tone, cell proliferation, and hormone production (16)(Fig. 1). Because of degradation by endopeptidases in the plasma, lung, and kidney, circulating ET-1 has a short half-life (about 4-7 min) (17). Correspondingly, the mRNA half-life of preproendothelin is approximately 15 min, indicating that vascular cells can rapidly adjust ET production as required for the regulation of vasomotor tone (16). ET-2 is produced predominantly in the kidney and the intestine, but the cells of origin are still not clear. ET-3 is associated with neuronal cells and has been found in high concentrations in the brain.
Stimuli for the release of ETs include hypoxia and shear stress, as well as endotoxin, adrenaline, angiotensin II, vasopressin, insulin, thrombin, transforming growth factor β and interleukin-1β (18). Prostacyclin and NO inhibit ET production via a cyclic (c)GMP-dependent mechanism (19). Atrial natriuretic peptide also inhibits both basal production of ET-1 and that stimulated by angiotensin II and thrombin (20).
Under most conditions, the production of ET appears to require de novo protein synthesis (19). After expression of the ET gene, a first transcript of 6836 bp is formed which leads to the production of preproendothelin, a peptide with 203 amino acids (15). Post-translational cleavage yields the 39 amino acid peptide big ET-1 that undergoes an additional cleavage between the nondibasic bond of Trp21-Val22 to form mature ET-1 (16). ET-1 synthesis is catalysed by at least two ET converting enzymes (ECEs), ECE-1 and ECE-2 (21,22). Cloning of ECE-1 was first reported in bovine adrenal cortex (22), but ECE-1 mRNA expression was also demonstrated in various other organs, including the heart, lung, and the kidney (23). Interestingly, the pH optimum of ECE-1 is 6.8, whereas that of ECE-2 is in the acidic range (pH 5.5) (21). ECE-2 is the predominant form in neuronal tissue (21), but expression has yet not been shown in humans. Both ECEs are membrane-bound proteinases that show structural similarity with a cytoplasmatic N-terminus, a single transmembrane domain and a large extracellular C-terminus, containing the catalytic domain. ECE-1 is a zinc metalloprotease displaying 40% similarity to neutral endopeptidase 24.11 that is present at the cell surface and on intracellular vesicles (24). Two subtypes of ECE-1 have been identified, ECE-1a and ECE-1b which are both encoded by the same gene by alternative splicing; they differ only in their N-termini (25).
Endothelins exert their biological effects via activation of specific receptors (Fig. 1). These membrane-bound receptors consist of seven transmembrane domains and are coupled to G proteins. Two types of ET receptors have been cloned, ETA and ETB, in mammalian tissues (26,27).
Endothelin can stimulate the release and action of NO via a distinct endothelial receptor (ETB receptor). This explains why ET causes a transient vasodilation at lower concentrations, which precedes its pressor effect (28).
Moreover in pathological conditions, the endothelium can also produce other endothelium-derived contracting factors (EDCFs) which are mainly cyclo-oxygenase-dependent prostanoids [thromboxane (TX)A2 and prostaglandin (PG)H2] or superoxide anions.
EXPERIMENTAL MODELS OF HYPERTENSION
Inhibitors of NO production cause endothelium-dependent contractions of isolated arteries (29), decrease blood flow (30) and induce pronounced and sustained hypertension when infused intravenously or given orally in vivo(31).
Alteration in endothelium-dependent relaxation in hypertension is not uniform and depends on the model of hypertension as well as the vascular bed studied (Fig. 2). In some vascular beds of hypertensive rats such as the aorta, mesenteric, carotid and cerebral vessels, endothelium-dependent relaxation is impaired (1,32). In contrast, in coronary and renal arteries of spontaneously hypertensive rats (SHR), endothelial function does not seem to be affected by high blood pressure (33). On the other hand, depending on the animal model and the vascular bed, endothelium-dependent contractions have been documented. Since cyclo-oxygenase inhibitors and TX receptor antagonists can inhibit this response, the most likely contractile factors are TXA2 and/or PGH2(34).
Acute, pharmacologically induced elevation in blood pressure causes an increased release of NO and a drop in blood pressure, followed by a decreased production of NO, suggesting that high blood pressure upregulates NO production and vice versa. The mechanism by which high blood pressure leads to an increased production of NO remains elusive. It is known that the release of NO by endothelial cells can be altered by changes in blood flow (35) and that mRNA and protein for endothelial NOS (eNOS) can be induced by mechanical forces (36). It is likely that other mechanical factors as well as shear stress, such as blood pressure itself and pulsatile stretch, contribute to this phenomenon (37).
The activity of cNOS is higher in mesenteric resistance arteries obtained from SHR compared with age-matched normotensive rats (38). Moreover, the concentration of the oxidative product of NO, nitrate, measured by high-performance liquid chromatography (HPLC) and capillary electrophoresis, is higher in the hypertensive rats as compared with their normotensive controls (38). In contrast, prehypertensive young SHR exhibit similar nitrate levels to age-matched normotensive controls. These observations demonstrate that the basal release of NO is increased in rats with spontaneous hypertension and that this increased production is directly related to the increased blood pressure of the animals.
Further studies demonstrated that the level of cGMP in mesenteric resistance arteries is similar in SHR and in Wistar-Kyoto (WKY) rats (38). Moreover, the NO-dependent vasodilator tone, assessed by the blood pressure effects of L-nitroarginine methylester (L-NAME), is not higher in hypertensive rats, as would be expected in a situation where the production of NO is increased. The capacity of vascular smooth muscle cells of hypertensive rats to respond to NO, on the other hand, must be fully maintained as organic nitrates lower blood pressure in a similar fashion in both strains of rats and relaxations to sodium nitroprusside are enhanced in this condition (39). These studies indicate that the endogenously produced NO is increased in spontaneous hypertension, but is not able to properly raise cGMP levels in the vascular smooth muscle cells of these animals. Hence, it appears that, in essential forms of hypertension, an additional unknown event takes place that blunts the hemodynamic actions of NO. The hypertrophied and fibrotic intimal layer of hypertensive vessels may represent a physical barrier for NO. The chemical environment determines the fate of NO. In particular, oxidative stress has been proposed to play a role in the pathogenesis of some cardiovascular diseases including hypertension (40-42). Recent experimental evidence using a porphyrinic microsensor for direct measurement of NO has demonstrated that, in the presence of superoxide dismutase, NO release from isolated resistance vessels is improved in the stroke-prone SHR. Thus, a higher production of oxidative radicals such as superoxide anion by a dysfunctional NO-synthase or a diminished activity of superoxide dismutases may account for an increased degradation of NO (43). However, NO production might be heterogeneously affected in different forms of hypertension. Indeed, in Dahl salt-sensitive rats, endothelium-dependent relaxations are impaired, but not those sensitive to sodium nitroprusside (44). In contrast to spontaneous hypertension, in salt-sensitive hypertension no release of vasoconstrictor prostanoids can be demonstrated (44). This suggests that a decreased NO production could contribute to the pathogenesis of this form of hypertension.
Similar experimental findings suggest that ET may be differently involved in different forms of hypertension. In fact, in some animal models of hypertension, such as the deoxycorticosterone acetate (DOCA)-salt hypertensive rat, ET-receptor blockade causes marked reduction in blood pressure, which is also associated with regression of vascular hypertrophy (45). Thus, ET-1 secretion is augmented in cultured endothelial cells from DOCA-salt hypertensive rats (46). Accordingly, ET antagonists lower blood pressure in salt-depleted monkeys (47). However, the effects of ET antagonism in other experimental models of hypertension, notably SHR, are less clear. In the SHR, both circulating and vascular ET as well as ET tissue content of the renal medulla are reduced (48). In contrast, in the stroke-prone SHR, the ET axis is activated and ET antagonism significantly reduces blood pressure and prevents cardiac and vascular hypertrophy (49). As in Dahl salt-sensitive rats, ET levels are increased and ET antagonists lower blood pressure; this indicates that the ET system is particularly activated in severe, salt-sensitive hypertension (50). In one-kidney, one-clip (low-renin) hypertension (51) and two-kidney, two-clip acute renal failure the circulating and tissue ET-1 systems are activated (52). At variance, the ET expression is augmented only in the late phase of two-kidney, one-clip Goldblatt (high-renin) hypertension resembling true renovascular hypertension and activation of the renin-angiotensin system in humans (53). In contrast, 2 weeks administration of angiotensin II increases the production of ET in the blood vessel wall of the rat (31). Most interestingly, selective ETA receptor antagonism reduces blood pressure and, in particular, vascular hypertrophy (31) and endothelial dysfunction under these experimental conditions (54). These data strongly suggest that ET antagonists may be of particular value in conditions of increased activity of the renin-angiotensin system. This is consistent with additional hypotensive effects of ET antagonists in hypertensive dogs already treated with an angiotensin-converting enzyme (ACE) inhibitor (55). However, the discrepancy between the beneficial effects of selective ETA receptor blockade in angiotensin II-induced hypertension and the lack of effects in the two-kidney, one-clip model is difficult to reconcile, but may be due to a lesser activation of the tissue (rather than circulating) renin-angiotensin and ET systems.
In NO-deficient hypertension induced by L-N-monomethyl-arginine (L-NMMA) or L-NAME, ET production is enhanced, but the peptide is only involved in the early, and not the chronic, phase of hypertension (56). Recently, a role of ET has also been suggested in fructose-fed hypertensive rats exhibiting hyperinsulinemia and insulin resistance, as chronic combined ET blockade reduces blood pressure in this experimental model of hypertension (57). Interestingly, hepatic overexpression of preproendothelin-1 in rats also resulted in elevation of blood pressure that was reduced by an ETA antagonist (58).
To further elucidate the role of ET, transgenic and gene knock-out rats have been developed. ET-2 transgenic rats exhibit elevated ET plasma levels, but do not develop hypertension (possibly because of the activation of compensatory vasodilator mechanisms) (59). Surprisingly, ET-1 gene knock-out mice are actually hypertensive (60). It is likely that the small increase in blood pressure in ET knock-out mice is related to hypoxia and, in turn, to activation of the sympathetic nervous system in these animals. The finding that ET knock-out rats have profound malformations of the throat indicates that the peptide may be importantly involved in the development of these organs (60).
Other vasoactive mediators are also candidates for explaining endothelial dysfunction in hypertension. Indeed, the responses to angiotensin I and II are increased in SHR (61) and, in addition, platelets and platelet-derived substances (ADP, ATP, serotonin), known to stimulate the formation of EDCFs (32), may lead to increased peripheral vascular resistance and also to complications in hypertension.
Hypertension in humans
Role of nitric oxide: Experiments in humans have demonstrated a diminished basal and stimulated NO production (8,62). The decrease in forearm blood flow induced by L-NMMA (reflecting basal NO formation) is smaller in hypertensive than in normotensive patients (63). L-NMMA infusion, however, provides only indirect evidence and no analytical determination of basal NO production. Forte et al. demonstrated that indeed plasma levels of NO are reduced in patients with essential hypertension (64)(Fig. 3). The stimulated release of NO as assessed by the vasodilator effects of acetylcholine in the forearm circulation of patients with essential, renovascular or endocrine hypertension is reduced in all but one study (62,64). The reasons for the negative results in the study by Cockcroft et al. (65) is most likely due to the low dosages of acetylcholine infused and/or heterogeneity in endothelial dysfunction in different patients. Similar findings have been obtained in the coronary circulation, particularly in the presence of left ventricular hypertrophy (2).
In patients with essential hypertension, the impaired response to acetylcholine in the forearm circulation can be improved by indomethacin, suggesting that cyclooxygenase-dependent vasoconstrictor prostanoids also contribute to impaired endothelium-dependent relaxation in hypertensive patients (8). Moreover, besides EDCFs such as TXA2 and PGH2, oxygen-free radicals can play an important role in endothelial dysfunction in hypertension (66).
NO plays also an important role in renal function. Indeed, the kidney is extremely sensitive to NO inhibitiACE on as very low doses of L-arginine analogues, which do not affect blood pressure, diminish diuresis, natriuresis and renal plasma flow (67). Hence, in some forms of hypertension, minimal alterations in the renal production of NO, which do not alter endothelium-dependent relaxation, lead to systemic hypertension due to a change in the management of body fluids by the kidney. Moreover, it has been recently shown that renal failure is also associated with an accumulation of an endogenous inhibitor of NO synthesis, asymmetrical dimethylarginine (68), which could also explain the increase in peripheral resistance and hypertension observed in these patients.
Role of ET
Whether or not ET is involved in hypertension is still controversial. Because of its vasoconstrictor action and its effects on vascular hypertrophy, ET-1 has also been implicated in the pathogenesis and/or the maintenance of hypertension. However, whether ET production is altered in human hypertension remains elusive (18). Although few studies found increased plasma levels of ET in hypertensives, many others found no difference compared with controls. Interestingly, patients with ET-secreting hemangioendotheliomas show increased plasma ET levels and were hypertensive (69). Plasma ET concentrations are also elevated in women with pre-eclampsia (70). Increased ET levels in African-Americans who often present with severe and salt-sensitive (low-renin) hypertension, point to the fact that severity of the blood pressure increase as well as salt-sensitivity (as suggested by the experimental models, see above) are important denominators for the activation of the ET system in hypertension (71). However, circulating plasma ET may not reflect local levels of the peptide, as in the blood vessel wall ET is primarily released abluminally (72). Recent studies, using inhibitors of the ECE or ET receptor antagonists, suggest that ET does contribute to blood pressure elevation in certain forms of hypertension in laboratory animals and in humans (73,74).
Infusion of exogenous ET does increase blood pressure in experimental animals and in humans (75). In healthy subjects, ET-1 receptor antagonism as well as infusion of the inhibitor of ECE phosphoramidone increase forearm blood flow and lower blood pressure indicating a role for ET-1 in the regulation of vascular tone (28,76). In essential hypertensives, ET-1 induces an increase in blood pressure and systemic vascular resistance, while cardiac index and natriuresis are reduced (77). Moreover, the vasoconstrictor response to ET is increased in the human hand vein circulation in patients with essential hypertension (78). Interestingly, normotensive offspring of hypertensive parents exhibit enhanced plasma ET responses to mental stress indicating that genetically determined activation of the ET system is already present at this early stage of disease (79). Furthermore, ET-1 gene expression is enhanced in small arteries of patients with moderate to severe hypertension, whereas expression is similar in control subjects and untreated mild hypertensives (80). In line with these studies, chronic therapy with the ETA/ETB receptor antagonist bosentan does indeed lower blood pressure in patients with essential hypertension to a similar degree as the ACE inhibitor enalapril (73)(Fig. 4).
Antihypertensive therapy and endothelial function
Endothelial dysfunction is a common feature of several pathological processes including hypertension, hyperlipidemia and atherosclerosis. Drugs that could improve endothelial function or enhance alternative pathways for the alterations in the release of endothelial mediators may have a potential advantage in the treatment of these pathological conditions. Several antihypertensive agents can prevent and reverse impaired endothelium-dependent relaxations in large conduit arteries as well as in resistance arteries of hypertensive rats (44,81). Since diuretics, calcium antagonists, ACE inhibitors and angiotensin II (AII) receptor antagonists improve or normalize endothelial dysfunction in hypertensive rats, the blood pressure lowering properties of these agents appear to be involved in this effect. However, additional pressure-independent effects of the various drugs cannot be ruled out and are described for each class of pharmacological agents.
ACE inhibitors: ACE is mainly located on the endothelial cell membrane where it transforms angiotensin I into angiotensin II and breaks down bradykinin, a potent stimulator of the L-arginine and cyclo-oxygenase pathways (14). Therefore, ACE inhibitors not only prevent the formation of a potent vasoconstrictor with proliferative properties, but also increase the local concentration of bradykinin and, in turn, the production of NO and prostacyclin (82). This latter effect may contribute to the protective effects of ACE inhibitors by improving local blood flow and preventing platelet activation. Accordingly, pretreatment of human saphenous vein and coronary artery with an ACE inhibitor enhances endothelium-dependent relaxation to bradykinin (83). Decreased degradation of bradykinin could therefore explain the improved endothelial function observed with ACE inhibitors in normotensive and particularly in hypertensive rats (81). In addition, the compounds stabilize the B2-receptor (84). However, the improvement of the endothelial function by ACE inhibitors in L-NAME-induced hypertension suggests that they may also enhance other endothelium-dependent mechanisms (i.e., enhanced release of EDHF), since the activity of the enzyme NOS is inhibited in this experimental model (34,85). The mechanism used by ACE inhibitors also seems to require some time to develop and cannot be reproduced in acute conditions, again suggesting that another mechanism other than ACE inhibition, which is rapid, is involved.
In contrast to the striking improvements obtained in experimental models of hypertension, data of studies in hypertensive patients are still controversial. ACE inhibitors seem to improve endothelial function in subcutaneous arteries (86), epicardial arteries (87) and the renal circulation (88). In the forearm circulation, on the other hand, treatment with captopril and enalapril (89) or cilazapril (90) failed to improve vasodilation to a muscarinic agonist, while lisinopril selectively improves the vasodilating response to bradykinin without restoring NO bioavailability (91). The reasons for this discrepancy between results obtained in experimental models of hypertension and studies in hypertensive patients are not clear at the present time. This discrepancy may originate from the fact that endothelial dysfunction may be treated at a much later stage in patients than in animal models of hypertension. Alternatively, duration of therapy and differences in tissue selectivity may account for the discrepancies of the effects of ACE inhibitors on endothelial function observed between animal models and hypertensive patients. Interestingly, in patients with coronary artery disease, 6 months treatment with the ACE inhibitor quinapril improved endothelial function of epicardial coronary arteries (87).
AII receptor antagonists: The recently developed AII receptor antagonists may have advantages, as they block the AT1-receptor only, leaving the AT2-receptor unchanged, which is implicated in antiproliferative effects of angiotensin II (92). These new drugs are also not associated with cough, a side effect of ACE inhibitors generally attributed to the diminished breakdown of bradykinin. However, if indeed the concomitant stimulation of the L-arginine NO pathway by bradykinin also proves to be an important property of ACE inhibitors, AII receptor antagonists would lack this beneficial effect.
In the experimental model of angiotensin II-induced hypertension, losartan enhanced endothelial-dependent relaxation to acetylcholine and prevented the increase in tissue ET-1 content, suggesting that a AT1 receptor blocker can modulate tissue ET-1 in vivo(93). In addition, losartan also blocked the angiotensin-induced production of oxygen-derived free radical in this model (94).
In aortic rings obtained from SHR, prolonged antihypertensive treatment with losartan reverses endothelial dysfunction not only by enhancing NO-dependent relaxation but also by reducing formation of cyclo-oxygenase-dependent EDCFs (95). Similar results were also found with captopril as an antihypertensive agent (95).
The effects of AII antagonists on endothelial dysfunction in human hypertension are still unknown. As expected in normotensive and hypertensive men, the intrabrachial infusion of an AT1-receptor blocker (losartan) inhibits the angiotensin II-induced vasoconstriction (96).
Endothelin antagonists: In recent years a large number of ET-receptor antagonists have been developed (Table 1). As discussed above, they have a modest antihypertensive efficacy in DOCA-salt hypertensive rats, although they have a more profound effect in preventing vascular hypertrophy (45). These antagonists are also able to prevent L-NAME-induced hypertension acutely and early on in the chronic phase of blood pressure elevation but eventually a similar increase in blood pressure is noted despite ETA/ETB blockade. This is surprising, if one considers the negative feedback exerted by NO on ET-1 release that is blocked in this model (97,98).
As studies in humans have demonstrated that ETA and combined ETA/ETB receptor blockade increases blood flow in the forearm (76) as well as in the human skin microcirculation, ET must contribute to the regulation of the cardiovascular system in humans (99). Indeed, intravenous infusion of the ETA/ETB receptor antagonist TAK-044 in humans with preserved left ventricular function lowers peripheral vascular resistance and blood pressure, and increases cardiac output and heart rate (100). Similarly, intravenous infusion of the ETA/ETB receptor antagonist bosentan in patients with coronary artery disease lowers blood pressure under acute conditions (101). Bosentan does exhibit a pronounced antihypertensive activity in patients with essential hypertension similar to that exerted by the ACE inhibitor enalapril (Fig. 4)(73). This strongly suggests that ET is indeed involved in human hypertension and may provide a new therapeutic approach to treat this condition. Of particular interest in this context is the fact that ET antagonists are particularly efficacious in reversing vascular functional and structural changes in experimental hypertension.
However, it is still a matter of debate whether an ETA receptor or combined ETA/ETB receptor antagonists are the best way to block the effects of the ETs in human disease. So far experimental data do not provide evidence in favor of combined ETA/ETB or selective ETA receptor blockade in hypertension, except for the surrogate parameter endothelial dysfunction that is only ameliorated by selective ETA receptor blockade (54). Thus, head-to-head comparative, large-scale studies with combined ETA/ETB and selective ETA receptor antagonists are needed to further delineate the best approach of pharmacologically blocking the ET axis in human hypertension.
ECE inhibitors may also prove to be valuable therapeutic agents, although evidence from clinical trials is not yet available.
Endothelial dysfunction occurs in cardiovascular disease and involves an enhanced release of EDCFs such as TXA2/PGH2, superoxide anions and ET-1 as well as a decreased release and/or enhanced breakdown of NO. Endothelial dysfunction may be particularly clinically relevant, if it is further aggravated by other risk factors such as hypercholesterolemia, smoking and diabetes.
However, further large-scale clinical trials are required to prove whether reversing endothelial dysfunction offers a new therapeutical approach for the benefit of patients with cardiovascular disease.
Acknowledgements: Swiss National Foundation (T.F. Lüscher Nr. 32.52069.97), G. Noll (Nr. 32.52690.97) and the Swiss Heart Foundation.
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A Spirapril symposium held in Vienna, Austria, August 25, 1998
The symposium and the publication of this supplement were supported by an educational grant from ASTA Medica AG, Frankfurt am Main, Germany.