endothelial dysfunction and atherosclerosis · endothelial dysfunction and atherosclerosis e21...

European Heart Journal (1997) 18 {Supplement E), E19-E29

Endothelial dysfunction and atherosclerosis

P. M. Vanhoutte

Institut de Recherches Internationales Servier, Couzbevoie, France

The endothelium mediates a number of responses (relaxa-tion or contraction) of arteries and veins from animals andhumans. The endothelium-dependent relaxations are due tothe release, by endothelial cells, of potent non-prostanoidvasodilator substances. Among these, the best characterizedis endothelium-derived relaxing factor (EDRF), which isbelieved to be nitric oxide (NO). Nitric oxide is formed bythe metabolism of L-arginine by the constitutive NO syn-thase of endothelial cells. In arterial smooth muscle, therelaxation evoked by EDRF is explained by the stimulationby NO of soluble guanylate cyclase that leads to theaccumulation of cGMP. In a number of animal bloodvessels and in human coronary arteries, the endothelial cellsrelease a substance that causes hyperpolarization of the cellmembrane (endothelium-derived hyperpolarizing factor,EDHF). The release of EDRF from the endothelium can bemediated by both pertussis toxin-sensitive (a2-adrenoceptoractivation, serotonin, aggregating platelets, leukotrienes)and insensitive (adenosine diphosphate (ADP), bradykinin)G proteins. In blood vessels from animals with regenerated

and reperfused endothelium, and/or atherosclerosis, there isa selective loss of the pertussin toxin-sensitive mechanism ofEDRF release, which favours the occurrence of vasospasm,thrombosis and cellular growth. The available informationfrom isolated human blood vessels or obtained in situconcurs with the conclusions reached from studies withisolated animal tissues. In addition to relaxing factors,the endothelial cells can produce contracting factors(endothelium-derived contracting factors; EDCFs) whichinclude superoxide anions, endoperoxides, thromboxaneA2 and endothelin. From animal studies it can be con-cluded that the propensity to release EDCFs is maintained,or even augmented, in diseased blood vessels. The switchfrom a normally predominant release of EDRFs to that ofEDCFs may play a crucial role in atherosclerosis.(Eur Heart J 1997; 18 (Suppl E): E19-E29)

Key Words: Atherosclerosis, endothelial dysfunction,EDRF, EDHF, EDCFs, G proteins.

Introduction

The normal endothelium contributes to the local regu-lation of vasomotor tone and the maintenance of anon-thrombogenic surface, acts as a selective barriercontrolling permeability and transport of solutes andmacromolecules to help metabolize many factors circu-lating in the blood or generated locally, and helps tocontrol the proliferation of underlying vascular smoothmuscle cells and to regulate the adhesion and extravasa-tion of neutrophils, monocytes, and lymphocytes. Theseproperties are due to the ability of endothelial cells tosense humoral and haemodynamic stimuli. Three basicmechanisms underlie these properties: the secretion ofendothelium-derived factors; the expression at the cellmembrane surface of binding proteins, adhesive mol-ecules, and metabolizing enzymes (converting enzymes);and shape changes. The discovery by Furchgott andZawadzki1'1 of the role played by the endothelial cells inrelaxation of isolated arteries in response to acetylcho-line initiated a major scientific inquiry into the pivotalrole of the endothelium in contributing to the normal

Correspondence: Professor Paul M. Vanhoutte, I.R.I.S., 6, Placedes Pleiades, 92415 Courbevoie Cedex, France.

physiological function of the vascular wall. It soonbecame obvious that endothelium-dependent responsesare mediated by the release of several diffusible sub-stances (endothelium-derived relaxing factor (EDRF)and endothelium-derived contracting factor (EDCF))from the endothelial cells. Under normal conditions,relaxing factors, especially nitric oxide (NO), are pre-dominantly released by endothelial cells. This reviewfocuses on how the secretion by endothelial cells ofvasoactive substances controls the changes in the toneof the underlying vascular smooth muscle cells and onwhy dysfunction of the endothelial cells may underlieor accompany atherosclerotic processes. It updatesprevious overviews'.[2-14]

Endothelium-derived vasoactivefactors

Endothelium-derived nitric oxide

Nature, synthesis and releaseThe labile diffusible (half-life of a few seconds), non-prostanoid substance that mediates the endothelium-dependent relaxation to acetylcholine described by

0195-668X/97/0E0019+11 S18.00/0 © 1997 The European Society of Cardiology

E20 P. M. Vanhoutte

Agonists

Endothelialcell

solubleguanylate cyclase K+

Figure 1 Release of cndothelium-derived factors. Endothe-lial receptor (R) activation induces an influx of Ca2+ into thecytospasm of the endothelial cell. When agonists activate theendothelial cells, an increase in inositol phosphate (IP3) maycontribute to the increase in cytoplasmic Ca2+ by releasing itfrom the sarcoplasmic reticulum (SR). Following interactionwith calmodulin, Ca2 + activates nitric oxide synthase (NOS)and leads to the release of endothelium-derived hyperpolariz-ing factor (EDHF). The increased Ca2+ also accelerates theformation of prostacyclin (PGI2) from archidonic acid (AA)by cyclooxygenase. NO causes relaxation by activating theformation of cGMP from GTP. Prostacyclin causes relaxa-tion by activating the formation of cAMP from ATP. EDHFcauses hyperpolarization and relaxation by opening K+ chan-nels. Any increase in cytosolic Ca2 + (including that induced bythe ionophore A23187) causes the release of relaxing factors.(Reproduced from Boulanger and Vanhoutte1341 and modifiedfrom Vanhoutte et al.ll3>.)

Furchgott and Zawadzki'11, has been identified asfree radical NO[4I5"18]. Nitric oxide is formed fromthe guanidine-nitrogen terminal of L-arginine by anenzyme called NO synthase, which is constitutive innormal endothelial cells (NO synthase III[I9'21]). Theactivation of this NO synthase depends on the intra-cellular concentration of calcium ions (Ca2+) in theendothelial cells, is calmodulin-dependent and requiresreduced nicotinamide adenine dinucleotide phosphate(NADPH) and 5,6,7,8-tetrahydrobiopterin (BH4) foroptimal activity'22'. Nevertheless NO can also be pro-duced without an increase in intracellular Ca2+, andshear stress may modulate the level of NO throughphosphorylation of NO synthase123241. The enzyme canbe inhibited competitively by L-arginine analogues, such

as NG-monomethyl-L-arginine or NG-nitro- L-arginine(Fig. 1). In addition to the constitutive NO synthasepresent in endothelial cells, two other isoforms of thisenzyme are currently known: a neuronal (type I) and aninducible (type II) form, which is Ca2+-independent andcan be observed in numerous cell types (includingvascular smooth muscle) after exposure to endotoxin,tumour necrosis factor and other cytokines. NO diffusesto the vascular smooth muscle cells and relaxes themby stimulating a cytosolic enzyme, soluble guanylatecyclase, that leads to an increase in cGMP1251. The latterincrease is associated with inhibition of the contractileapparatus (Fig. 1). The production of NO is a majorcontributor to endothelium-dependent relaxations inlarge isolated arteries, including coronary, systemic,

Eur Heart J. Vol. 18. Suppl E 1997

Endothelial dysfunction and atherosclerosis E21

Endothelial cells -

ACh Histamine AVP

Aggregating platelets

Figure 2 Some of the neurohumoral mediators that cause the release of endothelium-derivedrelaxing factors (EDRFs) through activation of specific endothelial receptors. In addition, EDRFscan be released independently of receptor-operated mechanisms by the calcium ionophore A23187(not shown). A, adrenaline (epinephrine); AA, arachidonic acid; ACh, acetylcholine; ADP,adenosine diphosphate; a, a-adrenoceptor; AVP, arginine vasopressin; B, bradykinin receptor;ET, endothelin receptor; H, histamine receptor; 5-HT, serotonin and receptor; M, muscarinicreceptor; NA, noradrenaline (norepinephrine); P, purinergic receptor; T, thrombin receptor; VP,vasopressine receptor. (Reproduced with permission113'.)

mesenteric, pulmonary and cerebral arteries. Its signifi-cance in vivo is suggested by the observations thatinhibitors of NO synthase cause vasoconstriction inmost vascular beds and an increase in systemic arterialpressure, both in animals and humans1'3'16'.

The endothelial cell secretes NO not only towardthe underlying vascular smooth muscle but also in thelumen of the blood vessel. Under normal conditions, thepresence of oxyhaemoglobin in the erythrocytes imme-diately neutralizes NO, which only has a physiologicalrole at the interface between the endothelial cells andthe blood content. Thus, NO inhibits the adhesion ofplatelets and leukocytes to the endothelium. It acts(synergistically with prostacyclin) to inhibit plateletaggregation14'6-8'16-261. NO also inhibits the growth of thevascular smooth muscle cells'27'28'.

The release of NO is modulated by physical andhumoral stimuli. Among the physical stimuli, the shearstress exerted by the blood on the arterial wall is one ofthe main factors regulating the local release of NO.Indeed, flow-induced vasodilatation is endothelium-dependent in vivo'291. Bioassay studies show that anincrease in flow, or the introduction of pulsatile flow,stimulate the release of NO and prostacyclin from theendothelium of the perfused arteries'30'. Vasoconstric-tion of perfused arteries induced by the elevation ofintraluminar pressure can be prevented by removal ofthe endothelium and is, in part, dependent on a reducedrelease of NO'311.

G proteins and endothelium-dependent release of NOSeveral neurotumoral mediators cause the release ofNO through activation of specific endothelial receptors

(Fig. 2). The endogenous substances stimulating thisrelease are either circulating hormones (catecholamines,vasopressin), autacoids generated within the vascularwall (bradykinin, histamine) or mediators released byplatelets (serotonin, adenosine diphosphate; ADP) orformed during coagulation (thrombin). The receptorsfor these substances are coupled to the production ofNO (NO synthase) by different G proteins (guaninenucleotide-binding proteins) (Fig. 3). In isolated porcinecoronary arteries, direct activation of G proteins evokesendothelium-dependent relaxations, mediated by therelease of NO, which are blocked by inhibitors of NOsynthase. Pertussis toxin (ADP-ribosylates) has revealedtwo different pathways for endothelium-dependentrelaxations mediated by NO: toxin-sensitive (G,) andinsensitive (Gq)'

33'. In porcine endothelial cells, a2-adrenoceptors, serotonin receptors and thrombinreceptors are coupled to pertussin toxin-sensitive Gi2

proteins, whereas ADP or bradykinin receptors mediatethe production of NO by activation of pertussistoxin-insensitive Gq protein134 361.

Pertussis toxin-sensitive endothelium-dependentrelaxations in porcine endothelial cells, involve several asubunits identified as Gi2 and Gj|-Gj3'37' and the major asubunit involved in the release of NO is Gi2'

38]. Gjproteins may activate KCa channels139'. In isolated por-cine blood vessels, the production of NO through the Gprotein pathway may be related to a tyrosine kinasepathway140'.

Pertussis toxin-insensitive endothelium-dependentrelaxations evoked by activation of bradykininreceptors are coupled either to small G proteins (morelikely to be involved in the release of NO than

Eur Heart J, Vol. 18, Suppl E 1997

E22 P. M. Vanhoutte

CONTROL ENDOTHELIAL CELLSerotonin

UK 14 304 I Endothelin BradykininADP, ATP

Phospholipase C

— EDRF-NO

Inhibition of: EDRF-NO

fSmooth muscle

contraction

7Platelet

aggregation

TExpression

adhesion-moleculesEndothelinproduction

Smooth muscleproliferation

OxidationofLDL

Monocyte andplatelet adhesion

Figure 3 Postulated signal transduction processes in a normal endothelialcell. Activation of the cell causes the release of endothelium-derived relaxingfactor-nitric oxide (EDRF-NO), which has important protective effects in thevascular wall; a, a-adrenoceptor; 5-HT, serotonin and serotonin receptor; ET,endothelin receptor; B, bradykinin receptor; P, Purinergic receptor; G, Gprotein; LDL, low-density lipoprotein. (Reproduced with permission1 '.)

endothelium-derived hypopolarizing factors EDHF), orby Gq proteins1411.

In addition, using cultured endothelial cells, it ispossible to distinguish between a G protein-dependenttransient production of NO, in response to a rapidchange in flow, and a G protein-independent productionof NO, in response to a smooth transition in shearstress'42^.

NO and platelet aggregationFrom the physiological point of view, the substancesproduced during platelet aggregation are importantreleasers of NO. This conclusion is based on the findingsthat in various species, including humans, platelet aggre-gation induces endothelium-dependent relaxations andthat the presence of endothelial cells substantially inhib-its the vasoconstriction induced by thromboxane A2 andplatelet-derived serotonin. There are two major media-tors of the endothelial response to platelets: serotoninand ADP, which act on 5-HT,D-receptors and P2y

receptors, respectively (Fig. 3). The endothelial action ofthrombin and platelet products is crucial for the protec-tive role played by the normal endothelium againstunwanted coagulation (Fig. 4). Therefore, local plateletaggregation, with the associated release of serotonin andADP, as well as the production of thrombin (because ofthe local activation of the coagulation cascade), leads toa major local release of NO, which diffuses toward theunderlying vascular smooth muscle, induces its relaxa-tion and thus dilatation of the artery. This reaction helpsto eliminate the micro-aggregate. The release of NOtoward the blood vessel lumen also inhibits plateletadhesion at the endothelium-blood interface and (in

synergy with prostacyclin), exerts a major feedback onplatelet aggregation, thereby eliminating the imminentdanger of vascular occlusion. In addition, the endothe-lial barrier prevents the platelet-derived vasoconstrictorsubstances (thromboxane A2 and serotonin) from reach-ing the smooth muscle. If the endothelial barrier hasbeen removed, for example as a result of trauma, there isa breakdown in the feedback control of platelet aggre-gation by NO (and prostacyclin). Aggregation proceedswith the continuous release of serotonin and thrombox-ane A2, both of which, in the absence of the endothelialbarrier, have unrestricted access to smooth muscle.Hence, the smooth muscle contracts and the bloodvessel closes down to constitute the vascular phase ofhaemostasis (Fig. 4)[4'81143].

Prostacyclin

Prostacyclin is formed following the activation of phos-pholipase A2, cyclooxygenase and prostacyclin syn-thase, primarily in endothelial cells but also in the mediaand adventitia in response to shear stress, hypoxia, andseveral mediators that also release NO. Its releasedepends mainly upon Ca2+ release from intracellularstores (Fig. 1)[44). In cultured human endothelial cells,shear stress induces the release of prostacyclin, which ismediated by a pertussis toxin-sensitive G protein'451.Prostacyclin causes relaxation of vascular smooth mus-cle by activating adenylate cyclase and increasing theproduction of cAMP. In most blood vessels, the contri-bution of prostacyclin to endothelium-dependent relaxa-tion is negligible, and its effect is essentially additive to



Aggregatingplatelets

Endothelialcells pGI2 A NO NO

Vessellumen

Aggregatingplatelets

Smooth musclecells

Figure 4 Interaction between platelet products, thrombin andendothelium. If the endothelium is intact, several of the sub-stances released from the platelets (in particular, the adeninenucleotides (ADP and ATP) and serotonin) cause the release ofEDRF and prostacyclin (PGI2). The same is true for anythrombin formed. The released EDRF will relax the underlyingvascular smooth muscle, opening up the blood vessel, and thusflushing the microaggregate away; it will also be released towardthe lumen of the blood vessel to prevent platelet adhesion to theendothelium and, synergistically with prostacyclin, inhibit plate-let aggregation. In addition, monoamine oxidase (MAO) andother enzymes will break down serotonin, limiting the amount ofthe monoamine that can diffuse toward the smooth muscle.Finally, the endothelium acts as a physical barrier that preventsthe access to the smooth muscle of the vasoconstrictor plateletproducts serotonin and thromboxane A2 (TXA2). These differentfunctions of the endothelium play a key role in preventingunwanted coagulation and vasospastic episodes in blood vesselswith a normal intima. If the endothelial cells are removed (e.g. bytrauma), the protective role of the endothelium is lost locally,platelets can adhere and aggregate, and vasoconstriction follows;this contributes to the vascular phase of haemostasis. +, activa-tion; — , inhibition. (Reproduced with permission1131.)

that of NO. The two substances act synergistically toinhibit platelet aggregation (Fig. 4)[26-46].

Endothelium-derived hyperpolarizing factor

Electrophysiological studies in various arteries, includ-ing the human coronary artery, demonstrate that acetyl-choline and other endothelium-dependent dilators causeendothelium-dependent hyperpolarizations and relaxa-tions that are due to a diffusible EDHF, different fromNO and prostacyclin147'481. The chemical identity ofEDHF is not known and several EDHFs may exist.In some blood vessels, epoxyeicosatrienoic acids(EETs) formed from arachidonic acid by the action ofcytochrome P-450, may correspond to EDHF[48~511.

The hyperpolarization of smooth muscle cellsinduced by EDHF is mediated by an increased move-ment of potassium ions. The type of K+ channelinvolved has not been definitively established, but thesechannels are more likely to be calcium-dependent ratherthan ATP-dependent K+ channels'48'52'531. As for NO,the release of EDHF requires an increase in theintracellular Ca2+ concentration of the endothelial cells(Fig. 1).

The contribution of hyperpolarization inendothelium-dependent vascular relaxation variesaccording to the size of the arteries and is prominent inresistance vessels'49'541. In large arteries, both mediatorscan contribute to endothelium-dependent relaxations,but the role of NO predominates under normal circum-stances. In these arteries, if the synthesis of NO is


E24 P. M. Vanhoutte

Stretch

ACh AA ADP 5-HT I Hypoxia

I I I 1 1

Endothelium

Figure 5 A number of physicochemical stimuli, neuro-humoral mediators, and the calcium ionophore A23187can evoke endothelium-dependent contractions in certainblood vessels, presumably because they evoke the releaseof endothelium-derived contracting factors (EDCFs). AA,arachidonic acid; ACh, acetylcholine; ADP, adenosinediphosphate; 5-HT, serotonin and receptor; M, muscar-inic receptor: P, Purinergic receptor. (Reproduced withpermission132'.)

inhibited, EDHF can mediate near normal endothelium-dependent relaxations'48'551. In diseases such as athero-sclerosis, when the production and/or the activity of NOis reduced, this preservation may be important for theregulation of vascular tone.

Endothelium-derived contracting factors

Under certain conditions, the endothelium can initiatevasoconstriction through the release of diffusible sub-stances'56'571. Endothelium-dependent contractions canbe explained either by the withdrawal of the release ofEDRFs or by the production of diffusible vasoconstric-tor substances (EDCFs)1412'58'591. The EDCFs identifiedto date include superoxide anions (which presumably actby scavenging NO'601), endoperoxides, vasoconstrictorprostanoids such as thromboxane A2, and the peptideendothelin-1 (Fig. 5). The latter is more likely to play arole in the long term modulation of vascular tone andstructure than in moment-to-moment changes in thedegree of contraction of vascular smooth muscle'61"641.Circulating levels of endothelin-1 are low under physio-logical conditions and cause vasodilatation at lowerconcentrations by activation of endothelial ETB recep-tors associated with the release of NO, prostacyclin andEDHF. At higher concentrations, endothelin-1 pro-vokes sustained contractions by activation of ETA (andin some blood vessels, ETB) receptors on the vascularsmooth muscle cells'651. The stimuli for endothelium-dependent contraction include hypoxia, physical stimulisuch as pressure and stretch, and several neurohumoralmediators (Fig. 4).

Endothelial dysfunction inatherosclerosis

As in several other diseases, the endothelial cells becomedysfunctional in atherosclerosis'4"8'1''. This dysfunctionexpresses itself as an impairment in endothelium-dependent relaxations either due to a reduced release, oraction, of EDRFs and/or a greater propensity to evokeendothelial-dependent contractions.

Regenerated endothelium

In human resistance vessels, among variables includingage, lipid levels, and blood pressure, age remainsthe most significant predictor of impairment ofendothelium-dependent vasodilatations'661. The normalageing process induces a turnover and regeneration ofendothelial cells resulting in an abnormal function. Thenormal life span of an adult human endothelial cell isestimated to be about 30 years. After this time cells arereplaced by regenerated endothelium. These regeneratedcells have lost some of their ability to release EDRF,in particular the ability to respond to platelet aggrega-tion and thrombin. This conclusion is based onin vivo animal studies in which regeneration and thecharacteristics of endothelium-dependent responses weremonitored following the removal of the coronaryendothelium'33'67'681. The regenerated endothelium wasno longer able to prevent aggregating platelet-inducedcontraction, and responded poorly to serotonin andother substances that use the pertussis toxin-sensitivepathway controlling the release of EDRF (Fig. 3). Incultured regenerated endothelial cells, G, proteinsare expressed normally, but they have a reducedactivity'34'691. The loss of the pertussis toxin-sensitiveresponse is selective and does not apply to endothelium-dependent responses induced by ADP or bradykinin.The area of regenerated endothelium becomes a prefer-ential site for triggering exaggerated vasoconstriction inresponse to serotonin or ergonoviner

,[70]

Mechanism(s)No impairment of the expression of G; protein a sub-units was observed in regenerated endothelial cells ofporcine coronary artery, in endothelial cells withimpaired release of NO'69'711. Therefore, the impairedrelease of NO, after activation of the pertussis toxin-sensitive pathway in regenerated endothelial cells, couldbe explained by an altered function of the G, protein'341.

Hypercholesterolaemia and atherosclerosis

Animal dataIn experimental animals, hypercholesterolaemiainduced by high-fat and/or high-cholesterol dietsimpairs endothelium-dependent relaxations in the



rabbit'4'810-72'731. By contrast, endothelium-independentrelaxations to nitroglycerin, sodium nitroprusside, oradenosine are normal or only slightly impaired. Aprogressive deterioration of endothelium-dependentrelaxations is also observed in genetically hyperlipidae-mic rabbits'74'. Endothelium-dependent relaxationsare reduced also in the coronary microcirculation ofhypercholesterolaemic animals and primates'75'76'. Inrabbits, using in vivo ultrasound assessment, impair-ment of endothelium-dependent vasodilating responsesprecedes the appearance of echographic atheroscleroticfindings'771.

Clinical dataEndothelium-dependent relaxations are also impairedin humans with atherosclerosis and/or hyper-cholesterolaemia'78"89'. In isolated coronary arteriesfrom heart transplant recipients, endothelium-dependentrelaxations are reduced in atherosclerotic segments incomparison with segments without atherosclerosis'90'911.Coronarographic studies show that coronary arterieswith atherosclerotic lesions constrict to injections ofacetylcholine, whereas acetylcholine produces eithervasodilation or no change in the coronary diameter innormal patients'921. Studies with inhibitors of theL-anginine-NO pathway suggest that acetylcholine-induced coronary dilatation in humans is mediatedmainly by NO'93"951. These arteries are capable of dilat-ing in response to nitroglycerin. The paradoxical con-striction of atherosclerotic arteries to acetylcholinesuggests the occurrence of endothelial dysfunction witha loss of the dilator effect of NO to offset the directconstrictor action of acetylcholine on vascular smoothmuscle.

The endothelial dysfunction occurs in theabsence of intimal thickening and may be an early andpathogenic event in atherosclerosis'961. Abnormal cor-onary responses to acetylcholine are linked directly tothe risk factors for coronary artery disease, andthe degree of vasoconstriction (or vasodilatation) toacetylcholine varies linearly with the level of plasmacholesterol'86'971. Endothelial dysfunction occurs pre-dominantly at coronary branching points, whichmay explain the propensity of these sites of disturbedflow to develop atherosclerosis'981. Elevated lipo-protein(a) is associated with the impairment ofendothelium-dependent vasodilatation even whenatherosclerotic lesions are not recognizable byangiography'"1. In patients treated with lipid-loweringagents, the improvement in coronary vasodilatorresponse to acetylcholine is related to the degree ofprotection of low density lipoproteins (LDLs) fromoxidation"001.

In peripheral blood vessels, hypercholesterolae-mia and other risk factors for atherosclerosis are associ-ated with endothelial dysfunction, as illustrated byabnormal responses to endothelium-dependent vasodila-tors. The latter can be detected before the developmentof vascular lesions and even during the first decade oflife'101'102'. The endothelium is also dysfunctional in

microvessels of patients with atherosclerosis, both in thecoronary and peripheral circulations'103'104'.

Endothelium-dysfunction is generalized to vas-cular beds other than those with clinically overt athero-sclerosis. Ultrasound techniques demonstrated a relationbetween brachial vasodilator response and coronaryvasodilator response'1051.

Mechanism(s)Because of the predominance of NO in endothelium-dependent relaxations particularly in large arteries,many studies have focussed on potential alterations ofthe L-arginine-NO pathway to explain the reducedresponse to acetylcholine and other endothelial agonistsin atherosclerosis. Possible interferences include areduced intracellular availability of L-arginine, altera-tions in signalling mechanisms, modification of theexpression or the activity of NO synthase, or increaseddestruction of NO'4'106'. A reduced availability ofL-arginine seems unlikely, as endothelial cells containthis substrate in concentrations a thousand times greater(millimolar range) than those that are optimal for con-stitutive NO synthase activity (micromolar range).L-arginine supplementation can normalize endothelium-dependent relaxation to acetylcholine in the micro-circulation and the aorta of rabbits with an enrichedcholesterol diet'107-108'. In hypercholesterolaemic patients,L-arginine improves the response to acetylcholine incoronary microvessels but not in large coronaryarteries"03'.

In the early stage of the atherosclerotic process,the endothelial dysfunction appears to be limited to theG; protein pathway, which leads to NO formation (Fig.6). Thus, the ability of regenerated endothelial cells,chronically exposed to high cholesterol levels, to ADP-ribosylate pertussis toxin, is reduced'37'. Consequently,in coronary arteries from hypercholesterolaemic pigs,endothelium-dependent relaxations evoked by agentsthat activate G, protein (serotonin, a2-adrenoceptor ago-nists, aggregating platelets, thrombin) are depressed,whereas those induced by ADP, bradykinin, or thecalcium ionophore A23187 are preserved'33'35"37'68'73'74'.Oxidized LDLs induce a similar selective endothelialdysfunction in vitro for stimuli activating the G, proteinpathway, whereas at higher concentrations, they alsoinhibit endothelium-dependent responses evoked byreceptor-independent stimuli (A23187)'4'106'1091. Thisselective dysfunction of the pertussis toxin-sensitiverelease of NO could be related to a direct inhibitoryeffect of lysophosphatidylcholine on the function of G;proteins and/or to an inhibition of oxidized LDLs onthe expression of Gj proteins'110'1"1, which is alsodecreased in endothelial cells from atherosclerotichuman coronary artery'"2'. This impairment does notconcern smaller coronary microvessels, where, even innormal conditions, the level of the expression of Gjprotein is low"13'. In addition, the decrease in theprotective action exerted by the endothelium may beenhanced by the increased production of endothelin-1 byoxidized LDLs"14'"5'.


E26 P. M. Vanhoutte

MODULATED ENDOTHELIAL CELL

SerotoninUK 14 304 I Endothelin

BradykininADP, ATP

Hyper- fcholesterolemia _-/Modified LDL

ROS

Adenylatecyclase

cAMP Phospholipase C

NFkB <«— EDRF-NO

Transcriptional activation

fIncreasedmonocyteadhesion

i tRelease ofmonocyte

chemotacticprotein (MCP-1)

Release ofmacrqphage

colony stimulatingfactor (M-CSF)

Expressionof tissue

factor

Production ofother

cytokines

Figure 6 Postulated signal transduction processes in a dysfunctional endothelial cell.There is impairment in Gi2 protein-dependent signalling with a subsequent decrease inthe release of endothelium-derived relaxing factor—nitric oxide (EDRF-NO). Further-more, the resulting increase in cAMP activates the transcription factor NFKB, whichinduces proatherogenic activity in the endothelial cell. ROS, reactive oxygen species;R, membrane bound receptors; a, a-adrenoceptor; 5-HT, serotonin receptor; ET,endothelin receptor; B, bradykinin receptor; P, Purinergic receptor; G, G protein;LDL, low-density lipoprotein. (Modified from Flavahan and Vanhoutte1 '.)

Conclusion

The most important mechanism in the reduction inendothelium-dependent responses is a lower release ofNO. Furthermore, as the disease progresses and theartery thickens and stiffens, it becomes increasinglydifficult for NO to reach smooth muscle that is still ableto relax. Endothelial dysfunction is probably a funda-mental initial step in the progression of atherosclerosis.This hypothesis argues that ageing and prolonged expo-sure to shear stress, coupled with risk factors such ashypertension, smoking and stress, accelerate endothelialaging and hence the process of endothelial regeneration.Consequently, increasingly larger and larger sections ofthe endothelium become unable to resist platelet adhe-sion and aggregation and respond less well to thrombinformation. The feedback effect of NO, together withprostacyclin, on platelet aggregation decreases steadily,whereas vasoconstrictor factors (serotonin and throm-boxane A2) are released in increasingly greater amountstogether with growth factors such as platelet-derivedgrowth factor (PDGF), which is probably responsiblefor initiating the characteristic morphological changes inatherosclerosis'4-8'10-"-35-36-43'.

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