THE SIR HANS KREBS LECTURE Pharmacology of vascular endothelium Delivered on 27 June 2004 at the 29th FEBS Congress in Warsaw Ryszard J. Gryglewski Jagiellonian University, Cracow, Poland Sir Hans Krebs was one of the most versatile biochem- ists of the twentieth century. Many of his sayings stay as bright as his science. My favourite quotation is: ‘‘… we all are committed to correlating biochemical events to function … the point I want to make is that it is not always immediately clear what their relevance to func- tion may be …’’ [1]. Indeed, a burning desire for imme- diate comprehension, amplified by the abomination of being just another fact collector may overcome rational cautiousness. We pharmacologists know this only too well. Sir John Vane urged his young followers: ‘‘Do simple experiments and make simple hypotheses – there are plenty of others who will come along and show how much more complicated the answer really is …’’ [2]. Keeping in mind the above advice, I present the vascular endothelium as a newly discovered target for the pharmacotherapy of arterial hypertension, athero- thrombosis and diabetic angiopathies. I intend to focus on the pharmacology of the endothelial prostacyclin ⁄ nitric oxide radical (PGI 2 ⁄ Keywords ACE-I; ASA; bradykinin; endothelial dysfunction; nitric oxide; prostacyclin; statins; thienopyridines Correspondence R. J. Gryglewski, Jagiellonian University, Kasztelan˜ ska 30, 30-116 Cracow, Poland E-mail: mfgrygle@cyf-kr.edu.pl (Received 10 February 2005, revised 13 April 2005, accepted 20 April 2005) doi:10.1111/j.1742-4658.2005.04725.x Sir John Vane named vascular endothelium ‘the maestro of blood circula- tion’. Recently, ‘the maestro’ has become a target for pharmacotherapy of atherothrombotic and diabetic vasculopathies with well known cardio- vascular drugs belonging to the families of Angiotensin Converting Enzyme inhibitors, HMG CoA reductase inhibitors or b 1 -Adrenoceptor antagonists. These drugs became upgraded to a position of the pleiotropic endothelial drugs. It is not a simple verbal change in the nomenclature. It means that these drugs apart from their well defined mechanisms of action, as indi- cated in their regular names, in addition they act in an unknown mechan- ism at the level of vascular endothelium preventing angina, myocardial infarction and stroke. Many biochemical events take place in endothelial cells. I chose for a closer inspection the nitric oxide/prostacyclin defensive system to explain the endothelial pleiotropism of the drugs in question. I tried to examine the validity of this conception according to the general rule: in vitro cognitio sed in vivo veritas. Abbreviations AA, arachidonic acid; ACE-I, angiotensin converting enzyme (and kininase 2) inhibitors; ADMA, asymmetric dimethylarginine; ASA, acetylsalicylic acid; BH4, tetrahydrobiopterin; Bk, bradykinin; BPF, bradykinin potentiating factor; CAD, coronary heart disease; CaM, calmodulin; COX-1, constitutive cyclooxygenase 1; COX-2, inducible cyclooxygenase 2; EDHF, endothelium-derived hyperpolarizing factor; EDRF, endothelium-derived relaxing factor; EETs, cis-epoxyeicosatrienoic acids; eNOS, constitutive endothelial nitric oxide synthase; FAD, flavin adenine dinucleotide; FMD, flow mediated dilatation (of brachial artery in humans); FMN, flavin mononucleotide; HMG-CoA, hydroxymethylglutaryl coenzyme A; HO-1, inducible heme oxygenase; 15-HPAA, 15-hydroperoxyarachidonic acid; HYHC, hyperhomo- cysteinemia; 6-keto-PGF 1a , prostaglandin 6-keto-PGF 1a , a stable product of decomposition of PGI 2 ; LDL, low-density lipoproteins; L-NAME, L-N(G)-nitroarginine methyl ester, a nonselective NOS inhibitor; NOHA, N ’ -hydroxy-Arg; ONOO – , peroxynitrite; ox-LDL, oxidized low-density lipoproteins; PARP, poly ADP ribosyl polymerase; PGE 2 , prostaglandin E 2 ; PGHS2, PGH 2 synthase; PGI 2 , prostacyclin; PGIS, prostacyclin synthase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SDMA, symmetric dimethylarginine; TXA 2 , thromboxane A 2 ; TXAS, thromboxane A 2 synthase; TXB 2 , thromboxane B 2 . 2956 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS NO • ) defence system. Other aspects of endothelial biology are reviewed by Nachman and Jaffe [3] with a special attention being paid to the functioning of Weibel–Palade bodies and their response to proin- flammatory or prothrombotic agents as manifested by the release of von Willebrand factor, P selectin and interleukin-8. Readers interested in the endothelial mitochondrion as a propagator of oxidative stress [4] and the mitochondrion-oriented role of reactive oxy- gen species (ROS) and hydrogen peroxide [5] are directed to studies by Keaney and coworkers [4,5]. Mitochondrial oxidases along with NAD(P)H oxidase, xanthine oxidase and uncoupled constitutive endothel- ial nitric oxide synthase (eNOS) constitute the source of endothelial ROS, which may act as modulators of tone, growth and remodelling of the vascular wall. It may well be that inflammation plays a primary role in atherogenesis, whereas oxidative stress is a secon- dary phenomenon [6]. At low concentrations, ROS may protect endothelial cells against apoptotic beha- viour [7]. Long-term treatment with antioxidant vita- mins does not influence the course of the disease or correct endothelial dysfunction in patients with atherosclerosis [8]. The great expectations for the therapeutic use of antioxidants in patients with athero- sclerosis need to be re-examined. Endothelium as the endocrine organ Why does blood not coagulate within healthy blood vessels? This question has been addressed for centuries. The warmth of the body (Plato), the lack of contact with air (James Hewson) and the vital power of blood (John Hunter) have all been claimed as reasons. The truth is that vascular endothelium secretes a bunch of antithrombotic and thrombolytic mediators that keep blood fluid within an undamaged circulatory system. Vascular endothelium is neither a ‘primitive mem- brane’, as claimed by Rudolph von Virchow, nor a ‘nucleated sheet of cellophane’, as Sir Howard Florey stated [9]. Sir John Vane named the endothelium ‘the maestro of blood circulation’ [10], which should be viewed as a peculiar dissipated endocrine organ (mass  1000 g, surface area  100 m 2 ). Among others sub- stances, endothelium releases into the passing blood – labile, lipophilic and antithrombotic local hormones like PGI 2 and NO • as well as a peptide – tissue plasmi- nogen activator. These prevent the build up of thrombi and disperse any thrombi at an early stage of their for- mation. This is why blood stays fluid within a healthy vascular bed. The inherent chemical instability of PGI 2 and NO • allows for the immediate transformation of extravasated blood into a haemostatic plug. Unfortu- nately, the same transformation may occur locally inside the circulatory system of patients with athero- sclerotic plaques or diabetic angiopathies. The endo- thelium then loses its protective properties and may even produce proinflammatory and thrombogenic agents (endothelial dysfunction). Endothelium generates many biologically active sub- stances other than PGI 2 or NO • , to mention just four regioisomers of cis-epoxyeicosatrienoic acid (EETs) produced from AA by CYP2J2 epoxygenases [11]. EETs are vasoprotective vasodilators. Some may be responsible for the activity of endothelium-derived hyperpolarizing factor (EDHF) [11], and for prevent- ing platelet adhesion to endothelium [12]. A potent vasoconstrictor, endothelin, is also produced [13], as are a vast number of mediators of haemostasis, growth factors and cytokines [14]. The outer endothelial layer of the glycocalyx houses the membrane sensors for shear stress and various types of endothelial receptors such as B 2 for bradykinin (Bk), P 2y subtypes for ADP from platelets and ATP from erythrocytes, PAF-R for platelet activating factor (PAF) from leukocytes, and PAR for thrombin [15]. The membrane-bound endo- thelial enzymes include kininase 2, also called angio- tensin 1-converting enzyme (ACE-I). Prostacyclin Prostacyclin (PGI 2 ) was discovered in 1976 during the search for biological systems that in addition to blood platelets might convert prostaglandin endoperoxides (PGG 2 or PGH 2 ) to thromboxane A 2 (TXA 2 ) [16,17]. This search was possible because newly discovered PGG 2 and PGH 2 were kindly offered to John R. Vane by the discoverer of TXA 2 , Bengt Samuelsson of the Karolinska Institutet. This search was not successful, except for the detection of minute amounts of TXA 2 made from PGH 2 by lung and spleen microsomes. Instead we found that a microsomal fraction of pig aorta transformed prostaglandin endoperoxides into an unknown, unstable substance (with a half-life of 4 min at 37 °C) that had vasodilator and platelet- suppressant properties in vitro. This substance was later named prostacyclin (PGI 2 ). Further studies revealed that PGI 2 , when administered intravenously, dissipated platelet-rich thrombi in arterial blood in vivo [18] and that this effect was augmented by theophyl- line. This latter finding confirmed that a cyclic nucleo- tide (in this case cAMP) was the second messenger of PGI 2 in platelets [19]. The common precursor for prostanoids including PGI 2 is the four double-bonds 2-carbon fatty acid – arachidonic acid (AA). It was found that the nonenzy- R. J. Gryglewski Endothelium and drugs FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2957 matic product of AA monooxygenation (15-hydro- peroxy-arachidonic acid; 15-HPAA) and other linear lipid peroxides are inhibitors of microsomal prosta- cyclin synthase (PGIS). Therefore, we hypothesized that PGI 2 deficiency resulted from an excessive non- enzymatic peroxidation of body lipids might contribute to development of atherosclerosis [20]. Consequently, we hoped to use synthetic PGI 2 as a replacement ther- apy in patients with atherosclerosis. Actually, I was the first healthy volunteer to receive an intravenous infusion of synthetic PGI 2 sodium salt. I lost the noble position of an observer during the last stage of this experiment. Still, these early trials allowed us to establish a range of therapeutic doses for PGI 2 and to observe the side effects caused by its overdos- age [21]. Eventually, PGI 2 was infused into patients with atherosclerosis of the leg arteries [22]. However, like most other powerful biological mediators, both PGI 2 (epoprostenol) [23] and its stable analogues (e.g. iloprost) [24] never became first-line drugs for the treatment of atherothrombosis, instead giving way to drugs that act as releasers of endogenous endothelial PGI 2 [25]. However, some PGI 2 analogues (e.g. tre- prostinil) are still used to treat patients with pulmon- ary arterial hypertension [26], including those with connective tissue disease [27]. The lung is a rich source of eicosanoids including leukotrienes. Various prostanoids are generated within different pulmonary compartments. Tracheal smooth muscles generate PGE 2 , contractile elements of lung parenchyma generate TXA 2 [28], whereas pulmonary endothelium secrets PGI 2 . We hypothesized [29] that pulmonary endothelium may serve as a source for circulating PGI 2 [30]. This concept was not well accepted. What kind of a circulating hormone has a half-life in blood of 3–4 min? Nonetheless, assuming PGI 2 is generated continuously by pulmonary endo- thelium, the stability of PGI 2 might be sufficient for it to be transported within the blood from the lung to atherosclerotic coronary or cerebral arteries with dysfunctional endothelium, and to save them from being occluded by platelet-rich thrombi. Pulmonary endothelium might be a good target for new specific releasers of circulating PGI 2 , although the local gen- eration of PGI 2 by the endothelium lining the vascu- lar tree is probably a more important therapeutic target, at least up to the point when the efficacy of peripheral endothelium in not seriously disturbed by an advanced atherothrombosis. Interestingly, overex- pression of pulmonary PGIS decreases the incidence of cancerogenesis in murine models of lung cancer [31]. The crude microsomal fraction of aortic homogen- ates that allowed us to discover biosynthesis the of PGI 2 from PGH 2 [16,17] contained PGIS. This enzyme was purified and characterized as a member of cyto- chrome P450 family (CYP 8A1) [32]. In endothelial cells it collaborates with a supplier of PGH 2 , i.e. with PGH 2 synthase (PGHS-2), commonly, but less pre- cisely, called cyclooxygenase 2 (COX-2). In endothelial cells COX-2 is induced by shear stress. COX-2 seems to be the major source of systemic PGI 2 in healthy humans [33]. In female mice oestrogens upregulate PGI 2 production via COX-2, and subsequently offer protection against atherothrombosis [34]. Also, intra- vascular thrombosis in rats next to hypoxia-induced hypertension is prevented by the upregulation of vascular COX-2 followed by increased generation of PGI 2 [35]. There is little doubt that, in humans and laboratory animals, the endothelial COX-2 ⁄ PGIS tandem is responsible for the generation of vasoprotective PGI 2 , whereas in blood platelets the constitutive cyclooxy- genase 1 ⁄ thromboxane A 2 synthase (COX-1 ⁄ TXAS) tandem generates vasotoxic TXA 2 . Nitric oxide radical In 1980, a series of in vitro experiments with acetyl- choline-treated aortic rings led Robert Furchgott to discover endothelium-derived relaxing factor (EDRF) [36]. Robert Furchgott likes to say that his great dis- covery arose from a number of accidental findings. Those in 1986 exploded in the grand finale, i.e. in the discovery that EDRF is nitric oxide. Actually, the idea that EDRF ¼ NO was proposed by Robert Furchgott and Louis Ignarro, independently [37]. Robert Furchg- ott is modest as only a great scholar can be. His mod- esty provokes the quotation from Louis Pasteur: ‘‘… where observation is concerned, chance favours only the prepared mind’’. The fabulous story of the discovery of EDRF(NO) was presented by Robert Furchgott [37], Louis Ignarro [38] and Ferid Murad [39,40] – three 1998 Nobel prize laureates in medicine and physiology. In vascular endo- thelium, NO • is synthetized from Arg by eNOS, which competes for substrate with tissue arginases. eNOS is a homodimeric oxidoreductase with NADPH, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), calmodulin (CaM) and tetrahydrrobiopterin (BH4) acting as cofactors. eNOS via N’-hydroxy-Arg (NOHA) generates NO • and citrulline. Physiologically, eNOS homodimer catalyses a five-electron oxidation of Arg, whereas BH4 plays a crucial role in the activation of dioxygen. In tissues, NO • is a powerful endogenous stimulator of soluble cytosolic guanylate cyclase. Thus made, cGMP is the second messenger for NO • in the Endothelium and drugs R. J. Gryglewski 2958 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS same way that cAMP is the second messenger for PGI 2 . Both cyclic nucleotides mediate the vasodilator, vasoprotective and platelet-suppressant activities of NO • and PGI 2 , respectively. However, when eNOS splits into monomers, the eNOS monomer acts as a reductase, and one-electron reduction of dioxygen leads to formation of a super- oxide anion (O 2 – ) [41]. Uncoupling of eNOS occurs as a consequence of BH4 shortage resulting from folate avitaminosis or from hyperhomocysteinaemia (HYHC) [42]. Apart from the uncoupling of eNOS, the other source of vascular O 2 – might be NAD(P)H oxidase (43). Dimerization of eNOS requires the intracellular availability of the substrate, i.e. Arg. This is ensured by the high-affinity cationic cell mem- brane transporter for Arg. Its functioning might be invalidated by homocysteine or by asymmetric dime- thylarginine (ADMA) (see below). Arg supplementa- tion in patients with atherosclerosis may be also desirable because Arg acts as a direct antioxidant. In addition, Arg promotes the secretion of insulin from pancreatic b cells and the release of histamine from mast cells – both being vasodilators. Theoretically, Arg may also produce unfavourable effects, such as generation of S-adenosyl-homocysteine from S-adeno- sylmethionine via the methylation-dependent biosyn- thesis of creatinine from guanidine acetate (44). Yet, the net therapeutic effect of Arg given orally to patients with myocardial infarction is encouraging (45) pointing to a favourable route of biotransforma- tion in these patients. Patrick Valance discovered, in human plasma, the presence of symmetric dimethylarginine (SDMA) and ADMA. Only ADMA is biologically active, i.e. it acts as endogenous inhibitor of eNOS and inhibitor of Arg membrane transporter. Clinical data on ADMA are growing. A high plasma level of ADMA is considered a novel cardiovascular risk factor. Nowadays, it is clear that ADMA contributes to vascular pathology in atherothrombotic and diabetic angiopathies, pre- eclampsia and hypertension (46). Elevated plasma lev- els of ADMA in those patients may also explain the ‘arginine paradox’, i.e. that therapeutic supplementa- tion with exogenous Arg is beneficial, although in these patients plasma levels of endogenous Arg exceed the Michaelis–Menten constant (K m ) for purified eNOS in vitro by 25-fold [47]. Prostacyclin and nitric oxide radicals A complex relationship exists between these two unsta- ble, lipophylic endothelial secretagogues. At the time when NO • still was known as EDRF it was claimed that porcine aorta endothelial cells cultured on cytodex beads, loaded into a heated column and perfused with Krebs’ buffer, when stimulated with Bk or calcium ionophore, released both PGI 2 and EDRF in a cou- pled manner [48]. Superoxide anions abolished the biological activity of the released EDRF from these cultured endothelial cells [49], and from native endo- thelium of perfused canine artery [50]. These latter findings initiated a march towards the discovery of the product of the interaction between NO • and O 2 – , i.e. peroxynitrite (ONOO – ). ONOO – is one of the most reactive nitrogen species (RNS). It arises most easily when the eNOS dimer coexists in the vicinity of a eNOS monomer – then both genders of labile free rad- icals, i.e. NO • and O 2 – arise side by side, and without any delay ONOO – is made. ONOO – is a powerful oxidant and nitrating agent that destroys the ‘macromolecules of life’, i.e. proteins (e.g. PGIS inactivation), lipids [e.g. the generation of oxidized low-density lipoprotein (ox-LDL) and iso- prostanes], and nucleic acids [e.g. DNA strand break- age with a subsequent activation of poly-ADP ribosyl polymerase (PARP)] [51]. The toxic properties of ONOO – play a major role in atherothrombotic and diabetic angiopathies. In those endothelial cells, ONOO – oxidizes the four zinc thio- late centres of dimeric eNOS. As a consequence, zinc atoms are removed and disulfide monomers of eNOS arise. The coexistence of dimeric and monomeric forms of eNOS is responsible for the further amplification of ONOO – generation by endothelial cells. This newly made ONOO – selectively nitrates Tyr430 in the enzy- mic protein of endothelial PGIS. When PGI 2 is elimin- ated from the endothelial defence system TXA 2 and PGH 2 gain the upper hand [52]. Endothelial NOS received the mischievous name of ‘the Cinderella of inflammation’ [53]. The authors had in mind that excessive stimulation of eNOS might lead to increased vascular permeability by NO • , and thus to inflammation. However, in light of the foul games played between homodimeric and monomeric forms of eNOS, ending with the generation of ONOO – which eliminates PGI 2 – the best friend of NO • – I would rather think of eNOS as ‘the Lady Macbeth of athero- thrombosis’. Endothelial pharmacology Samuel Beckett (1906–1989) wrote: ‘‘we need new par- adigms to accommodate the mess’. The paradigm of ‘pleiotropic action’ for some of cardiovascular drugs was coined to accommodate a discrepancy between their officially accepted modes of action and their R. J. Gryglewski Endothelium and drugs FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2959 additional therapeutic properties, as reported unexpect- edly, but repeatedly, by clinicians. For example, statins were introduced to the clinic with the aim of lowering blood levels of low-density lipoprotein (LDL) cho- lesterol, however, they were also found to correct symptoms of myocardial and cerebral ischaemia, inde- pendent of their capacity to inhibit hydroxymethyl- glutaryl coenzyme A (HMG CoA) reductase [54–56]. Further support for the existence of the ‘pleiotropic action’ of cardiovascular drugs was offered by the efficacy of ACE-I to protect against myocardial isch- aemia, stroke and diabetic angiopathies, as confirmed in multicentre trials that included over 25 000 patients [57], whereas the classic indication for ACE-I was the treatment of patients with arterial hypertension. The phrase ‘pleiotropic action’ is not a cognitive descrip- tion of reality. Rather, it is an attempt ‘to accommo- date the mess’. Our experimental data [58,59] pointed to the possibility that the pleiotropic action of ACE-I and statins might be explained by their stimulatory effect on the endothelial generation of PGI 2 and NO • . There are other propositions concerning the mechan- ism of endothelial actions of statins, e.g. the induction of heme oxygenase (HO-1) [60] with a subsequent anti- oxidant effect of biliverdin and CO mediation. Here, I take the opportunity to present our conception of the endothelial pharmacology emerging from clinical observations on the unexpected therapeutic effects of known cardiovascular drugs. This conception embraces not only ACE-I and statins, but also other cardiovas- cular drugs, e.g. nebivolol and carvedilol (b-adrenergic receptor antagonists) as well as ticlopidine and clopi- dogrel (antiplatelet thienopyridines). In vivo assay of endothelial function Clinicians have developed an excellent noninvasive method to measure endothelial function in humans. The method is based on the ultrasound scanning of the flow-mediated dilatation (FMD) of the brachial artery after its occlusion and reopening [61]. In prin- ciple, the FMD response is proportional to the amount of NO • released from endothelium of the vas- cular bed in question, however, an additional bio- chemical assay pointed to the release of PGI 2 , along with NO • , from the endothelium during FMD [62]. No wonder – in vitro cultured endothelial cells released EDRF(NO) and PGI 2 in a coupled manner [48]. The FMD method allowed the detection of endo- thelial dysfunction in patients with arterial hyperten- sion [62], in patients with atherosclerosis undergoing percutaneous coronary intervention with stenting [63], and in patients with type 2 diabetes [64]. In patients with chest pain, a depressed FMD of the brachial artery was a sensitive indicator of coronary heart dis- ease (CAD) [65]. FMD is impaired in tobacco smokers and in smokeless tobacco users compared with tobacco nonusers [66]. There is ample evidence for the state- ment that endothelial dysfunction occurs in patients with hypertension, atherosclerosis and type 2 diabetes, as well as in tobacco users. In vitro cognitio sed in vivo veritas (in vitro one may look for meaning, however, only in vivo is the truth to be found). This motto stimulated us to develop our own experimental model for the in vivo assay of endothelial function [18–20,29,59,67–70]. In our in vivo method it is not the vasodilator response (as in the case of FMD in humans) but rather the thrombolytic response that is used to assess endothelial capacity. Therefore, it is the endothelial release of PGI 2 that is appreciated at the first place, whereas the release of NO • remains in the background. Heparinized cats, rabbits and, most fre- quently, Wistar rats under general anaesthesia with extracorporeal circulation are used. The arterial blood superfuses (2–3 mLÆmin )1 ) a collagen strip attached to a balance. Blood returns to the venous system. Thrombus mass is recorded continuously along with arterial blood pressure (Fig. 1). Platelet-rich thrombus [70] gains a maximum mass of  100 mg within 30 min and stays unchanged for at least 4 h, unless a stimulator of vas- cular endothelium (e.g. Ach, Bk or an endotheliotropic drug) is injected intravenously. Then thrombolysis occurs (Fig. 1). Its intensity and duration correlate with plasma levels of prostaglandin 6-keto-PGF 1a (6-keto- PGF 1a ), whereas the levels of other stable prostanoids do not (Fig. 2). The participation of endothelial NO • in thrombolytic response is checked by the pretreatment of animals with l-NAME or with any other NOS inhib- itor. The participation of endogenous bradykinin in this response was checked by pretreatment with Icatibant, an antagonist of B2 receptors (Fig. 1A). In this system, thrombi were dissipated by intravenous administration of PGI 2 sodium salt or by its stable analogue (e.g. ilo- prost). NO-donors (glyceryl trinitrate, molsidomine, sodium nitropusside, NONOates) also produced throm- bolysis but their effective doses were at a range of three orders of magnitude higher than those required for PGI 2 or for its analogues. Unlike PGI 2 , NO-donors at thrombolytic doses were highly hypotensive. Angiotensin-converting enzyme inhibitors ACE-I, this name does not do justice to this class of drugs, namely captopril, enalapril, and especially per- indopril, quinapril, ramipril and many other lipophylic Endothelium and drugs R. J. Gryglewski 2960 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS ACE-I. There is no doubt that the pharmacological activity of ACE-I is associated with the elimination of cytotoxic and vasoconstrictor angiotensin 2, however, the endothelial action of those ACE-I is also executed via the local vascular accumulation of Bk, as our data clearly show (Fig. 1A) [59,67–69]. In 1965 a young Brazilian researcher, Sergio Ferreira discovered the ‘bradykinin potentiating factor’ (BPF) in the venom of Brazilian viper Bothrops jararaca [71]. At John Vane’s laboratory in London (where Sergio Ferreira was a visitor) his discovery was appreciated than it should have been. At the time, Bk was per- ceived as a mediator of pain and inflammation respon- sible for paralytic vasodilatation in the course of acute pancreatitis. The reasoning was as follows: BPF might be good for this particular viper for swift killing of its victims but for us humans – it is no good at all. So, why should we care about BPF? Perfusate from isolated guinea-pig lungs dripping over Vane’s bioassay cascade was used to study Bk [71] and angiotensin 1 [72] metabolism. Fortunately, it was soon found that various fractions of BPF given via the lungs inhibited the conversion of angiotensin 1 to angiotensin 2, and thus BPF was proved to act also as an ACE-I [73]. Inhibiting the conversion of biolo- gically inactive angiotensin 1 to hypertensive angioten- sin 2 – yes, it was an excellent principle on which to develop a new class of antihypertensive drugs [74]. Indeed, at the request of John Vane, the top industrial chemists eventually did [75], and the first orally active ACE-I (a proline derivative – captopril) was intro- duced for the treatment of arterial hypertension. The TREND trial [76] offered the first direct clinical evidence of improvement, by an ACE-I (quinapril), in endothelium-dependent vasorelaxation in patients with CAD. There then appeared a number of clinical trials pointing to the same mechanism of vascular protection by various ACE-I in patients at high risk of athero- thrombotic and diabetic vasculopathies [57]. B A BP THR THR Dose-dependent thrombolysis by perindopril µg/kg i.v. Thrombolysis by QUINAPRIL depends on the release of endogenous bradykinin and PGl 2 – only partially on NO THR THR THR 30 min mg mg 100 0 100 0 THR rat 1 rat 2 rat 3 rat 4 icatibant 100 µg/kg indomethacin 5 mg/kg L-NAME 5 mg/kg quinapril 30 µg/kg quinapril 30 µg/kg quinapril 30 µg/kg quinapril 30 µg/kg 0 100 mg Thrombogenesis thrombus weight Thrombolysis pressure transducer weight transducer carotid artery carotid artery arterial blood arterial blood collagen collagen THROMBUS i.v. drug injection jugular vein PERINDOPRIL 30. 10. 3. 30 min 100 mg 0 Fig. 1. In vivo bioassay of endothelial secretory function. Fig. 2. Effect of quinapril on prostanoid plasma levels in Wistar rats (n ¼ 7). R. J. Gryglewski Endothelium and drugs FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2961 Bk is the most potent releaser of PGI 2 from cultured endothelial cells [48], and the most potent thrombolytic agent acting via endothelial B 2 receptors in vivo [67]. In Wistar rats, exogenous Bk at thrombolytic doses is strongly hypotensive. In contrast, endogenous Bk released from vascular endothelium by low doses of ACE-I (quinapril > perindopril > captopril) evokes thrombolysis, but not a fall in blood pressure [59,68]. The principal mechanism of the thrombolytic action of ACE-I stems from their secondary nature (or rather their primary nature) of being BPF [71]. Moreover, there exist other Bk-potentiating effects of exogenous ACE-I, such as the upregulation of B2 receptors, the induction and activation of B1 receptors in the endo- thelium and the stimulation of biosynthesis of angio- tensin (1–7), which acts as an endogenous ACE-I (that is BPF) [77]. It should be added that in cultured endo- thelial cells Bk acts as a ‘minicytokin’, inducing mRNA for HO-1 and COX-2 [67]. The interaction between these two enzyme systems was claimed to amplify the generation of PGI 2 [78]. It may well be that, in addition to the immediate thrombolytic effects of ACE-I, chronic treatment with ACE-I offers an additional advantage of increasing the efficacy of the endothelial enzymic raft (COX-2 ⁄ PGIS) responsible for the biosynthesis of vascular prostacyclin along with increasing local levels of CO and biliverdin – the defensive products of endothelial HO-1. In our in vivo model for studying endothelial-medi- ated thrombolysis in Wistar rats [59,68,69] it was found that ACE-I (captopril < perindopril < quina- pril) at low nonhypotensive intravenous doses of 10– 60 lgÆkg )1 dissipated thrombi that were superfused with arterial blood. The intensity and duration of this thrombolysis were paralleled by an increase in arterial plasma levels of 6-keto-PGF 1a , and no change in plasma levels of TXB 2 and PGE 2 (Figs 1 and 2). Thrombolysis and prostacyclinaemia by ACE-I were blunted or abolished by pretreatment with icatibant (a B2 Bk receptor antagonist), by acetylsalicylic acid (ASA) at a high dose of 50 mgÆkg )1 (Fig. 3), and by the coxibs (rofecoxib > celecoxib > nimesulide) at low doses of 30–300 lgÆkg )1 . Thrombolysis by ACE-I was augmented by pretreatment with ASA at a dose of 1mgÆkg )1 (Fig. 3) or by acetaminophenen. Pretreat- ment with l-NAME delayed and flattened the throm- bolytic response to ACE-I only slightly (Fig. 1). Pharmacological analysis of the above data led us to conclude that ACE-I evoked thrombolysis by pre- venting endothelial Bk from being destroyed by cell membrane-bound ACE. Bk that appeared at the endothelial cell surface stimulated B2 receptors, which triggered the COX-2 ⁄ PGIS system to generate PGI 2 , and e-NOS to generate NO. The final thrombolytic response to ACE-I depended mainly on PGI 2 , whereas NO • served as a helper with a permissive action. The endothelial release of NO • did not appear as the conditio sine qua non for thrombolytic response to ACE-I (Fig. 1A). There is another conclusion that derives from these studies. It is as follows: effective endothelial COX-2 inhibition might be followed by thrombogenesis, whereas preferential COX-1 inhibition in platelets rein- forced the vasoprotective action of ACE-I (Fig. 3). Our data cannot be considered as a good prognostic for the clinical use of high doses of coxibs in patients with cardiovascular disorders, but they do support the idea of administrating of low doses of ASA along with ACE-I (Fig. 3). Statins In our in vivo model statins (e.g. atorvastatin and simvastatin) produce endothelium-mediated, PGI 2 - dependent thrombolysis when administered intraven- ously at doses 2–3 orders of magnitude higher than those for ACE-I [59]. In Langendorff’s preparation of guinea-pig heart, statins produce NO • -dependent vaso- dilatation of coronary vascular bed [59]. The precon- tracted bovine coronary artery rings with endothelium are relaxed by statins, partially via a NO • ⁄ PGI 2 - dependent mechanism [79]. In cultured bovine aortic endothelial cells lipophylic statins, i.e. atorvastatin, simvastatin and lovastatin (but not a hydrophilic pravastatin) at a concentration of 30 lm mobilize free cytoplasmic calcium [Ca 2+ ] i to 30–50% of that induced by Bk at a concentration of 10 nm. In the case of simvastatin and lovastatin, this effect disap- pears if their lactone rings are hydrolysed [80]. The above endotheliotropic properties of statins are hardly Fig. 3. Dose-dependent effect of aspirin (ASA) on perindopril- induced thrombolysis. Endothelium and drugs R. J. Gryglewski 2962 FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS associated with their inhibitory action on HMG CoA reductase. In genetic and pharmacological models of rat hypertension, rosuvastatin, another lipophilic sta- tin, was found to exert a beneficial pleiotropic endo- thelial effect [81]. Patients with acute coronary syndromes benefit from statin therapy [82]. Statins mobilize bone-marrow-derived endothelial progenitor cells [83] and exert a vast number of other pharmaco- logical effects that are not associated with modulation of the lipoprotein profile by statins. These unexpected effects of statins are generally described as ‘pleiotropic effects’ [84], and one of them is the endotheliotropic action of statins described by us [59,79,80]. The mode of activation of the endothelial PGI 2 ⁄ NO • system by statins is not clear. An interesting proposal was put forward by Bill Sessa [85]. Thienopyridines and some of b 1 -adrenergic receptor antagonists Here we present two groups of highly effective cardio- vascular drugs, the efficiency of which may or may not depend on their additional stimulatory action of vas- cular endothelium. Thienopyridines (ticlopidine and clopidogrel) belong to a family of antiplatelet drugs, however, in vitro they do not inhibit platelet aggregation. Their in vivo plate- let-suppressant action is executed by their labile meta- bolites. Therefore, a substantial lag period is required for the appearance of the antiplatelet action of thieno- pyridines. Only unstable metabolites of theirs are cap- able of antagonizing endogenous ADP on P2y12 purinergic platelet receptors, which when activated by ADP induce platelet release and platelet aggregation [86]. The clopidogrel metabolite exerts its antiplatelet action at IC 50 ¼ 1.8 lm [87]. There exists ample evi- dence for the high efficacy of thienopyridines (especi- ally clopidogrel) in the treatment of patients with advanced atherothrombosis of coronary or cerebral arteries, to mention only the following megatrials: clopidogrel vs. aspirin in patients at risk of ischemic events (CAPRIE) [88], clopidogrel in unstable angina to prevent recurrent events (CURE) and management of atherothrombosis with clopidogrel in high risk patients with recent transient ischaemic attacks or isch- aemic stroke (MATCH) [89]. In 1996 [90] we demonstrated that ticlopidine (10 mgÆkg )1 ) given intravenously to cats with extracor- poreal circulation evoked immediate dissipation of the platelet-rich clots superfused with their arterial blood [18]. This thrombolytic effect of ticlopidine was com- parable with that induced by PGI 2 at 0.3 lgÆkg )1 . These and other data [90] prompted us to postulate that the therapeutic efficacy of ticlopidine might be associated not only with the delayed platelet-suppres- sant effect of its unstable metabolite via blockade of P2y12 platelet receptors, but also with the instan- taneous endothelial action of the native molecule of ticlopidine showing up as an immediate, endo- thelium-mediated thrombolysis of platelet-rich clots in vivo [90]. In rats, these ‘immediate thrombolytic effects’ of thienopyridines were rather weak (EC 30 ¼ 15–30 mgÆkg )1 ). Jean-Pierre Dupin of the Bordeaux II Uni- versity decided to synthetize a series of thienopyrimi- dinones under the guidance of our pharmacological assay of their endothelium-dependent thrombolytic effects in vivo. Assessment of their structure–activity relationship revealed that the most active compound, i.e. 3[(2-trifluoromethyl-phenyl)-methyl] 1,2-dihydro- benzo[b]thieno[2,3-d]pyrimidinone-4(3H)one dissipated platelet clots in rats in vivo at a dose of IC 30 ¼ 8 lgÆkg )1 [91]. We conclude that in addition to in vivo endothelial PGI 2 -mediated thrombolysis, thienopyrimidinones and thienopyridines exert endothelial NO • -mediated coron- ary vasodilatation in perfused guinea-pig heart [92]. Mechanisms of endotheliotropic actions of these strongly lipophylic compounds remain unknown. Nebivolol and carvedilol – two b 1 -adrenoceptor antagonists – founded the ‘third generation’ of selective b-adrenolytic drugs, which are endowed with endothe- liotropic properties. Eleven years ago, Bowman et al. [93] proposed that the antihypertensive effects of nebiv- olol in man might be partially associated with endo- thelium-dependent, NO • -mediated vasodilatation. In two interesting studies Ignarro et al. [94,95] clearly demonstrated that relaxation of vascular smooth muscle by nebivolol is partially mediated by endothe- lium-dependent release of NO • and the subsequent accumulation of cGMP in smooth muscle [94], how- ever, nebivolol also inhibits vascular smooth muscle proliferation by a mechanism involving NO • but not cGMP [95]. Various routes were proposed by which the ‘third generation’ of b 1 -adrenoceptor antagonists may release endothelial NO • . Certainly adrenergic and serotoninergic receptors are not involved [96]. A fascin- ating hypothesis has been proposed [97]. Nebivolol and carvedilol stimulate the renal efflux of ATP, that releases NO • via activation of P2Y purinoceptors in glomerular endothelium. On top of the regular b 1 -adrenoceptor blockade there appears NO • -mediated relaxation of renal glomerular microvasculature. This is why nebivolol and carvedilol are so efficient in controlling arterial hypertension and improving renal circulation. R. J. Gryglewski Endothelium and drugs FEBS Journal 272 (2005) 2956–2967 ª 2005 FEBS 2963 Perspectives In endothelial pharmacology everything is new: (a) the idea that vascular endothelium may be looked upon as an organ with a secretory function; (b) considering pulmonary endothelium as a separate endocrine organ that supplies prostacyclin to the coronary and cerebral circulations; (c) a complex relationship between two endothelial mediators – NO and PGI 2 – a role for ROS and RNS in it; (d) discovering new endothelio- tropic mechanisms for old cardiovascular drugs like for ACE-I, statins or nebivolol; (e) planning new endotheliotropic chemical structures, e.g. thienopiry- midodiones; (f) discovering new biochemical mecha- nisms of action for drugs affecting endothelial function like in case of nebivolol; and (g) the interaction between basic and clinical researchers, probably one of the most efficient in the field of medicine. 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THE SIR HANS KREBS LECTURE Pharmacology of vascular endothelium Delivered on 27 June 2004 at the 29th FEBS Congress in Warsaw Ryszard J endothelial generation of PGI 2 and NO • . There are other propositions concerning the mechan- ism of endothelial actions of statins, e.g. the induction of heme