Pathophysiology of vascular endothelium and circulating platelets: implications for coronary revascularisation and treatment

International Journal of Cardiology 79 (2001) 265–275 www.elsevier.com / locate / ijcard

Pathophysiology of vascular endothelium and circulating platelets: implications for coronary revascularisation and treatment Giovanni Amoroso a , *, Dirk J. van Veldhuisen a , Rene´ A. Tio a , Mario Mariani b a

Thoraxcenter, University Hospital of Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands b Cardiothoracic Department, University of Pisa, Pisa, Italy Received 12 January 2001; received in revised form 5 April 2001; accepted 10 April 2001

Abstract Constant vasodilatation, inhibition of platelet and leukocyte adhesion, and local thrombolysis are the mechanisms through which an intact endothelial layer exerts its protective action on coronary circulation. A loss in these features is not only the first step in the development of atherosclerosis, but also a potent trigger for complications after revascularisation procedures. Percutaneous coronary interventions, particularly in the course of stenting, induce endothelial injury that can last up to months after the procedure. On the other hand, the preservation of endothelial function appears the best feature of arterial versus venous grafts after coronary bypass surgery. An early diagnosis either by invasive or non-invasive techniques has important implications for prognosis, and endothelial dysfunction can be effectively counteracted by medical treatment (ACE inhibitors, statins). Activated circulating platelets are present in the course of coronary artery disease, increasing the risk of thrombotic occlusion and / or plaque regrowth, after both percutaneous and surgical revascularisation. New antiplatelet agents are under development to reduce endothelium–platelet interaction. On the basis of the latest studies, coronary revascularisation should be integrated in a more complete treatment, which would take into account the complex processes involving the underlying atherosclerotic plaque.  2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Platelets; Coronary revascularisation

1. Endothelial physiology — an overview Vascular endothelium has been considered for years simply a physical barrier, at most responsible for passive exchange or active transport of substances, between blood and inner vascular layers. It is the largest regulatory organ present in the human body, and is in fact involved in vasoregulation, platelet aggregation, fibrinolysis, leukocyte adhesion, and vessel growth [1–3].

*Corresponding author. Tel.: 131-50-361-3544; fax: 131-50-3614643. E-mail address: [email protected] (G. Amoroso).

In the coronary circulation, endothelium guarantees continuous adaptation of flow according to myocardial needs. Endothelium-released vasoactive substances comprise both vasodilative (nitric oxide, prostacyclin and hyperpolarizing factor) and vasoconstrictive (thromboxane, prostaglandin H 2 , endothelin1 and endoperoxides) molecules [4]. In addition, endothelial cells modulate the effects of circulating vasoactive compounds. Firstly, endothelial monoaminooxidases deactivate circulating catecholamines and platelet-released serotonin, which would induce vasoconstriction [5]. Secondly, endothelial cells enhance or reduce the expression of angiotensin converting enzyme (ACE), which is able to convert inactive angiotensin-1 into active angiotensin-2, a

0167-5273 / 01 / $ – see front matter  2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 01 )00448-X

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potent local and systemic vasoconstrictive agent, and to inactivate bradykinin, a potent vasodilator [6]. Endothelial cells also preserve the internal vascular surface in an anti-adherent state. First of all, endothelium-derived nitric oxide and prostacyclin strongly inhibit platelet aggregation. Moreover, endothelium promotes continuous local thrombolysis by secreting antithrombin and thrombomodulin, and by modulating tissue-type plasminogen activator (t-tPA) and plasminogen activator inhibitor-1 (PAI-1) expression [7]. Finally, in normal conditions, endothelial cells are non-adhesive to circulating leukocytes [8]. Constant vasodilatation, inhibition of platelet and leukocyte adhesion, and local thrombolysis render an intact endothelial layer the best protective agent of the normal circulation. Endothelium also modulates cell growth and differentiation of leukocytes and smooth muscle cells [9]. Endothelium-derived growth inhibitors include nitric oxide, heparansulphates and transforming growth factor b1, while basic fibroblast growth factors, platelet-derived growth factor and, possibly, endothelin-1, promote cell proliferation. Under normal conditions cell inhibition prevails, but cell proliferation can be triggered at any moment to respond to vessel injury, both by a decrease in nitric oxide production and by the release of proliferative factors.

2. Endothelial dysfunction and coronary artery disease Unfortunately, because of its strategic position, endothelium is exposed to the detrimental effect of cardiovascular risk factors. Viral infections, hypercholesterolemia, systemic hypertension, diabetes, smoking, and autoimmune diseases all ultimately affect endothelial cells [10]. Above all, impairment of endothelial cell function is considered the first step in the development of atherosclerosis [11]: modified low density lipoproteins (LDL), whether they are oxidated, glycated (in diabetes), or incorporated in immune complexes, are probably the major promoter of endothelial dysfunction [12]. When endothelial cells are unable to completely degrade these molecules, they express surface-bound molecules and release cytokines, which favor the attraction and migration of inflamma-

tory cells in the subendothelial space. Endothelial cells also change from an anti- to a prothrombotic phenotype. Briefly, endothelial dysfunction goes through two different stages: an immediate phase (cell retraction, P-selectin exposure from Weibel-Pallade bodies, von Willebrand factor release), which does not require protein synthesis and gene upregulation, and a late phase, characterised by gene transcription and de novo protein synthesis (monocyte chemoattractant protein-1, E-selectin, interleukin-8, intercellular adhesion molecule-1 (ICAM-1), PAI-1). As reported by Ross [13], early atherosclerosis resembles a response-to-injury process. However, when the endothelial injury is chronic, the reparative events go far beyond their intended scope. Of note, in the presence of risk factors, and before the development of atherosclerotic lesions, endothelial dysfunction may be detected in the whole vascular system, affecting the coronary as well as the peripheral areas, macro- as well as microvascular circulation [14]. When shear stress alterations concur, stenotic lesions develop and localise in particular sites, for instance on coronary bifurcations, as a consequence of endothelial cell changes in shape, leading to the expression of atherogenic rather than vasoprotective genes [15].

3. Endothelial dysfunction and percutaneous coronary interventions Studies in animal models have demonstrated an enhanced deposition of von Willebrand factor [16] and expression of endothelin-1 and nitric oxide synthase [17] immediately after an intra-arterial balloon inflation, suggesting a significant endothelial injury. In the early phases after a percutaneous coronary intervention, marked endothelial denudation, retraction of endothelial cells and loose intercellular connections can be visualised. Endothelial regrowth is unlikely to be complete in less than 4 weeks [18]. As the endothelial layer overlying an atherosclerotic plaque is already dysfunctional [19], the superimposition of a mechanical damage further impairs its function. In fact, in the short-term after an angioplasty, local endothelium is unable to provide effective vasoregulation and thrombohomeostasis [20] (Fig. 1).

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Fig. 1. Endothelium–platelet interaction in the course of percutaneous coronary interventions. The mechanical injury triggers platelet adhesion to endothelial cells, the exposed subendothelial tissue, and the bare metallic surface (when a stent is deployed). Platelets and endothelial cells change towards an activated state: vasoconstriction, platelet aggregation and thrombosis, chemotaxis and cell growth are then induced through the release of soluble agents and the expression of surface receptors.

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However, coronary vasomotor abnormalities are still detectable even months later, revealing a sustained lack of functional recovery by the regenerated endothelium [21,22]. The degree of immediate and chronic endothelial dysfunction probably does not depend only on the severity of the mechanical injury. For instance, Van Beusekom et al. [23] demonstrated a loss of endothelial barrier function up to 3 months after stenting, and Caramori et al. [24] proved that, among various procedures, coronary stenting is associated with the greatest and longest-lasting impairment in endothelium-dependent vasomotility. These findings could be in part explained by the higher inflating pressures and the oversized balloons used for a satisfactory stent deployment, resulting in more severe wall damage. However, intense adhesion of leukocytes at the site of intervention has also been demonstrated in the long term after stenting. As a stent is a metal foreign body irreversibly placed in the vessel wall, it is likely to stimulate a stronger inflammatory response, which can further delay the restoration of normal endothelial function. Interestingly, a significant impairment in endothelium-dependent vasomotility can persist in distal,

and non-treated coronary segments, after successful procedures [25]. This finding can represent either a latent atherosclerotic process, or a remnant of the chronic hypoperfusion present before the intervention [26], or may be a consequence of the continuous leakage of vasoactive substances from the treated lesion [27].

4. Endothelial dysfunction and coronary artery bypass grafting Yang and Luscher [28] proved that the use of arterial grafts guarantees better long-term results after coronary bypass surgery, mainly because those conduits show a well-preserved endothelial layer, which leads to enhanced adaptability to acute and chronic flow changes, and to a preserved antithrombotic state. As a matter of fact, arterial, more than venous, grafts maintain a physiological nitric oxide and prostacyclin metabolism, both shortly and long term after surgery [29] (Fig. 2). Moreover, internal mammary artery grafts exhibit a peculiar sensitivity to stimulators of nitric oxide release, such as acetylcholine, bradykinin, histamine and substance P, or mechanical shear

Fig. 2. The role of endothelium and platelets during coronary bypass surgery. The simultaneous presence of endothelium and platelets causes a potent relaxation in the human internal mammary artery (left), prevented either by apyrase (ADP inhibitor) or L-NMMA (NO inhibitor). Indeed, in the saphenous vein (right), platelets evoke a potent and strong contraction, facilitated by the presence of endothelium. Modified from Yang et al. [71].

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stress [30], which has a positive effect on coronary vasomotion [31]. More friendly surgical handling also plays a determinant role in favor of arterial grafts, especially when maintained in-situ, as free grafts can undergo significant endothelial damage, due to preparation and storage [32].

5. Diagnostic tools for endothelial dysfunction — to see the invisible? Non-invasive assays, analysing the endotheliummediated vasomotion of peripheral areas after hyperemic flow induction, can suggest the presence of coronary endothelial dysfunction [33]. However, coronary endothelium can be directly investigated by quantifying nitric oxide release [34] or by stimulating coronary vasomotility during cardiac catheterisation [35,36]. Acetylcholine, the classic stimulus for endothelium-induced relaxation, acts via muscarinic receptors, with signal-transducing through G proteins, and mediates the release of relaxing factors nitric oxide, and, to a lesser extent, endothelium-derived hyperpolarising factor. Both these molecules cause potent vasodilatation, obscuring the direct vasoconstrictor effects of acetylcholine on smooth muscle cells [37]. A normal response is manifest as vasodilatation, whereas endothelial dysfunction, both in the presence and in the absence of overt coronary disease, is associated with reduced vasodilatation or vasoconstriction [38,39]. Given the limitations of angiography in assessing subtle diameter changes, and the direct effects of contrast dyes on vasomotility [40], intracoronary Doppler guide-wires, able to measure blood flow velocities, and 3D-ultrasound catheters, able to evaluate arterial wall architecture, have been proposed as new research tools for studying endothelial function [41]. Early diagnosis and accurate quantification of endothelial dysfunction have important implications also for prognosis. In fact, as recently demonstrated ¨ both by Suwaidi et al. [42] and Schachinger et al. [43], patients with mild coronary disease show an increased risk for cardiac events, when altered endothelium-related vasomotility is present (Fig. 3). That suggests the major role of dysfunctional endothelium

Fig. 3. Cardiovascular events (cardiovascular death, unstable angina pectoris, myocardial infarction, PTCA, CABG, ischemic stroke, peripheral artery revascularisation) during long-term follow-up, in patients with and without impaired endothelium-dependent vasomotility and mild coronary disease. A striking disproportion is present. Modified from ¨ Schachinger et al. [43].

in accelerating plaque growth and / or promoting plaque instability. Moreover, as endothelial dysfunction can extend to the coronary microcirculation, it can induce myocardial ischemia also in the absence of flow-limiting stenosis. For this reason, non-invasive perfusional techniques (positron emission tomography, singlephoton emission computed assisted tomography) have been used to detect endothelial dysfunction in patients without coronary artery disease (as in the case of hypercholesterolemia, chronic heart failure, systemic hypertension) [44,45].

6. Reversing endothelial dysfunction — how, when and why In its early stages, endothelial dysfunction can be reverted by means of a number of dietary and lifestyle changes [46,47]. While a low-fat diet has not demonstrated significant efficacy, oral supplementation with L-arginine, the nitric oxide precursor, has proven beneficial effects on endothelium-related vasodilatation in hypercholesterolemic subjects [48]. Also the use of antioxidants, ascorbic acid or vitamin

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Table 1 Reversal of endothelial dysfunction by various pharmacological treatments ( L-arginine, anti-oxidants, statins, ACE inhibitors): a synopsis of the main clinical studies Authors

Year

Treatment

Area

Population

Drexler et al. [48] Levine et al. [49] Egashira et al. [52] Treasure et al. [53] Anderson et al. [54] O’Driscoll et al. [55] TREND [56] Prasad et al. [57] Lee et al. [58]

1991 1996 1994 1995 1995 1997 1996 1999 1999

L-Arginine

Coronary Brachial Coronary Coronary Coronary Brachial Coronary Coronary Brachial

Hypercholesterolemic Coronary Hypercholesterolemic Coronary Coronary Hypercholesterolemic Coronary Mild atherosclerosis Hypercholesterolemic

Ascorbic acid Pravastatin Lovastatin Lovastatin1probucol Simvastatin Quinapril Enalapril Lisinopril

E, prevents the detrimental effect of reactive oxygen species on the availability of nitric oxide [49]. Physical training has been associated with beneficial effects on endothelial function, particularly in patients with chronic heart failure, and endothelial dysfunction clearly regresses after smoking cessation [50]. Several pharmacological approaches have also been attempted (Table 1). Three(3)-hydroxy-3methylglutarylcoenzyme A reductase inhibitors (statins) [51–55] increase coronary nitric oxide availability both by reducing the LDL-cholesterol-induced endothelial injury and by independently activating NO synthase. ACE inhibitors [56–58] support endothelial function by reducing local and systemic availability of angiotensin 2 (vasoconstrictor), and increasing that of bradykinin (vasodilator).

7. Endothelium and platelets — an unavoidable attraction In the course of atherosclerosis platelets express more P-selectin, and integrins (including activated glycoprotein (Gp) IIbIIIa receptor) on their surface [59,60]. On the other hand, dysfunctional endothelial cells express selectins, vitronectin-receptor (a v b 3 ) and integrins (vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1)). This simultaneous activation of adhesion molecules supports platelet adhesion to the vessel wall, even in the presence of dysfunctional but structurally-intact endothelium [61]. Platelets settle at first in a single layer, and then assume spreading shapes, form fibrinogen cross-

bridges between each other (through activated Gp IIbIIIa receptors), and start in situ aggregation. The latter phenomenon is facilitated by the reduced release of nitric oxide from dysfunctional endothelium. The interaction of platelets and endothelial cells stimulates cell migration and growth, by promoting the release of several active compounds. Recently, Gawaz et al. [62] demonstrated that activated platelets release Interleukin-1, and induce endothelial cells to express ICAM-1 and to secrete monocyte chemotactic protein-1 (MCP-1) (Fig. 4). The adhesion and migration of monocytes into the vessel wall is the basis for the progression and / or the instability of the atherosclerotic lesion [63].

8. Platelets and coronary revascularisation — some mutual faults Dysregulation in platelet / endothelium interaction is also at the basis of the most frequent complications following revascularisation procedures: thrombotic occlusion and plaque regrowth. Even when successful, a percutaneous coronary intervention induces plaque fracture and splitting, media disruption and wall stretching. Unfortunately these phenomena strongly stimulate platelet and coagulation system activation [64]. When subendothelial tissue is exposed, platelets adhesion is mainly mediated by the presence of von Willebrand factor diffusely covering the subendothelial collagen. Platelet membrane Gp Ib–IX–V complex is the principal receptor involved in adhesion, while Gp IIbIIIa, and other adhesion receptors, play only a subordinate role [65,66] (Fig. 1). Activated platelets

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Fig. 4. Endothelium–platelet interaction. Monocyte chemoattractant peptide-1 (MCP-1) release and surface adhesion molecule (ICAM-1) expression by endothelial cells in culture (HUVEC) is stimulated by activated platelets (black bars), but inhibited by interleukin-1 (IL-1) antagonists (grey bars). No significant effect is produced by quiescent platelets (white bars). Modified from Gawaz et al. [62].

offer their surface for catalysing the formation of thrombin from prothrombin, and, as endothelium has switched from an anticoagulant to a procoagulant phenotype (inverted t-tPA / PAI-1 ratio, enhanced von Willebrand factor and reduced thrombomodulin secretion), the coagulation cascade begins, with the subsequent formation of a firm clot [67], that can determine abrupt vessel occlusion. As the amount of platelet deposition and thrombus formation is strongly dependent on the extent of vessel injury [68], the occurrence of a coronary

dissection after balloon angioplasty was once considered a severe complication. Being able to seal dissections and, at the same time, allow wider lumen dilatations, coronary stents brought the benefits of reduced acute complications and improved long-term results. Unfortunately, as a layer of fibrinogen immediately settles onto the bare metallic surface, initiating platelet deposition, stents immediately appeared to trigger local thrombosis [69]. Further activation of the platelet and coagulation system after stenting also occurs in the systemic circulation, as demonstrated by

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overexpression of surface integrins on circulating platelets, and increased numbers of circulating prothrombin fragments F 112 . Acute thrombotic occlusion, after both balloon angioplasty and stenting, can be prevented with pharmacological aids and procedural actions. Nevertheless platelets also have a primary role in promoting restenosis. In fact, adhered platelets, in concert with dysfunctional endothelial cells, secrete chemotactic and growth factors, which in turn stimulate migration, accumulation and proliferation of smooth muscle cells and leukocytes in the intima layer. These cell lines are finally responsible for intimal hyperplasia, deposition of amorphic material and plaque regrowth [70]. Differences in endothelium–platelet interaction also determine the outcome of coronary bypass surgery. In fact, in arterial, but not in venous, grafts, platelet-derived factors, such as ADP and ATP, stimulate the endothelial production of nitric oxide [71]. Hence, only in arterial grafts, platelet aggregation is inhibited at the same sites of activation. That, together with the preserved endothelium-dependent vasodilatation, allows continuous clot flushing, and renders occlusive thrombosis a frequent complication of venous, but not arterial, grafts.

it reaches therapeutic concentrations after a single front-load oral dose [75]. The family of Gp IIbIIIa inhibitors includes a variety of newly-developed agents, which are able to selectively block the activated platelet Gp IIbIIIa receptor [76]. While the use of oral compounds is under dispute, as they have demonstrated a partial agonistic effect, eventually activating platelet aggregation, parenteral compounds (abciximab, eptifibatide, tirofiban) seem clearly effective, both in the course of acute coronary syndromes and percutaneous coronary interventions [77] (Table 2). At first abciximab, a chimeric monoclonal antibody, seemed to improve the mid- and long-term prognosis of ischemic patients, particularly when associated with coronary stenting. This molecule, more than just inhibiting platelet aggregation, also provides an effective blockade of vitronectin and Mac-1 receptors, thus widely interrupting endothelial–platelet– leukocyte interactions [78–80]. Eptifibatide, a synthetic peptide, highly specific for the Gp IIbIIIa receptor, with a shorter half-life and a reduced immunogenicity compared to abciximab, could represent a valid alternative [81,82].

10. Conclusions and clinical implications 9. Platelet inhibition — the old and the new A prophylactic treatment with aspirin guarantees a nearly 10-fold reduction of occlusive thrombi after coronary angioplasty, resulting from the inhibition of platelet prostaglandin synthesis [72]. However, aspirin has demonstrated no beneficial effect in preventing restenosis. Moreover, in the case of coronary stenting, aspirin alone does not seem sufficient to inhibiting platelet-induced thrombosis [73]. To overcome this limitation, aspirin can be associated with ADP-receptor antagonists. These compounds inhibit the platelet 2-methylthio-ADP-binding receptor, and the exposure of Gp IIbIIIa receptor. Cumulatively, these actions strongly prevent ADP-induced platelet aggregation. As a consequence, the simultaneous use of aspirin and ticlopidine lowered the incidence of stent thrombosis to 1–2% [74]. Clopidogrel, a novel derivative from ticlopidine, shows a reduced adverseeffects profile and a faster plasma availability, so that

Altered endothelial function, and activated circulating platelets are common findings in ischemic patients, even before any attempt at coronary revascularisation, mainly as a consequence of the evolving process of atherosclerosis. Nevertheless, percutaneous interventions induce mechanical injury and subsequent local inflammation, and provoke further coronary endothelial dysfunction. Endothelial function seems also jeopardised in venous, but not in in-situ arterial conduits, after coronary bypass surgery. As revascularisation procedures can also induce platelet activation, the subsequent dysregulation in endothelium–platelet interaction represents a major determinant of adverse events in patients submitted either to percutaneous interventions or surgery. Assuming that the continuous increase in coronary interventions, which we are currently facing in Western Europe, represents an effective solution for the treatment of ischemic patients, a sole mechanical

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Table 2 Differences between eptifibatide and abciximab: the two most commonly used Gp IIbIIIa inhibitors Abciximab

Eptifibatide

Brand name Supplier Molecular structure

ReoPro  Centocor / Eli Lilly Chimeric Fab fragment

Integrilin  COR / Schering-Plough Cyclic heptapeptide

Approved dose Bolus Infusion

0.25 mg / kg 0.125 mg / kg per min312–24 h

180 mg / kg 2.0 mg / kg per min372–96 h

Active site on Gp IIbIIIa Affinity for Gp IIbIIIa (KD ) Specificity for Gp IIbIIIa Plasma half-life (t 1 / 2 ) Platelet pool redistribution .50% Platelet inhibition (t)

Specific ligand site High (5 nmol / l) Low (also a v b 3 and MAC-1) Short (10–30 min) Yes Long (|24 h)

RGD recognition site Low (120 nmol / l) High (only a IIb b 3 ) Long (|2 h) No Short (3–4 h)

Clinical efficacy (trials) In ACS In PCI

?? (GUSTO IV [76]) Yes (EPIC [78], EPISTENT [79])

Yes (PURSUIT [81]) ?? (ESPRIT [82])

ACS, acute coronary syndromes; PCI, percutaneous coronary interventions; RGD, Arg-Gly-Asp aminoacidic sequence.

approach to coronary stenosis seems limiting, particularly in the light of the latest studies. It is hoped that such an approach could be integrated in a more complete treatment, which would take into account the complex processes involving the underlying atherosclerotic plaque. To optimise the immediate and long-term outcomes of revascularisation procedures and to control, if not to defeat, coronary atherosclerosis it is desirable: 1. To counteract coronary risk factors. Health-care policies encouraging low-fat diets and active lifestyles, and in particular campaigning against cigarette abuse, should be pursued, as their beneficial effects on vascular physiology will result in improved quality of life and long-term prognosis. 2. To minimise the impact of revascularisation procedures. Interventional cardiologists should make a reasonable use of coronary stents, always preferring devices with enhanced biocompatibility. Cardiac surgeons should consider the physiology of grafted conduits, preferably using in-situ arterial grafts, possibly implanted with minimally-invasive techniques. 3. To supply adequate pharmacological aids. Especially statins, but also ACE inhibitors, have

proven to restore endothelial function, and probably represent a necessary complement for effective myocardial revascularisation. GpIIbIIIa inhibitors prevent both platelet activation and endothelium–platelet interaction, helping to reduce acute and chronic vascular responses in the course of percutaneous coronary interventions.

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