European Journal of Clinical Investigation (1991) 21, 355-359

ADONIS

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This paper was previously published in issue 2 0 588-592. Due to a serious error, it is republished here in its correct form.

Arterial wall hypoxia following thrombosis of the vasa vasorum is an initial lesion in atherosclerosis J . F. MARTIN, R. F. G. BOOTH & S. MONCADA, Wellcome Research Laboratories, Beckenham, Kent and Department of Medicine, King’s College School of Medicine and Dentistry, Denmark Hill, London, UK

Received 9 October 1989 and in revised form 19 February 1990

Abstract. Pressure on the outside of arteries can cause physical and biochemical changes in the vessel wall of rabbits which are characteristic of atherosclerosis. It is hypothesized that occlusion of the vasa vasorum causes ischaemia of the arterial media which results in smooth muscle cell proliferation and cellular accumulation of cholesteryl esters. Hypoxia increases mRNA for platelet-derived growth factor in arterial wall cells and increases the activity of acyl CoA : cholesterol acyltransferase (ACAT). Such a mechanism may explain many of the anatomical, actuarial and environmental risk factors for atherosclerosis. Hypoperfusion may follow thrombosis of the vasa vasorum. Keywords. Arteries, atherosclerosis, hypoxia, thrombosis, vasa vasorum. Introduction

For over 100 years it has been believed that atherosclerosis begins at the luminal surface of the artery. Whilst Virchow 111, later supported by Ross & Glomset [2],held contrary views to Rokitansky [3] and later Duguid [4], the essential argument was whether the initial lesion in atherosclerosis was luminal injury or luminal encrustation of thrombus. All, however, believed that the endothelium was involved in the initiation of the lesion. We will present evidence that atherosclerosis may be a disease of the outer layers of arteries, and we will propose that luminal changes occur subsequent to this initial pathology. The lesions of human atherosclerosis may be separated into three major forms: the fatty streak, the fibrous plaque and the advanced plaque. Fatty streaks are composed almost exclusively of macrophages and lymphocytes and are almost devoid of smooth muscle cells [2,5,6]. Many foam cells and much extracellular lipid are also present. Fibrous plaques are composed

* Present address: Group Head of Biology, Roche Products Ltd, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AY, UK. Correspondence: Prof. J. F. Martin, Head, Cardiovascular Research, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, UK.

predominantly of smooth muscle cells of varying morphology, many of which have become lipid-laden foam cells containing large quantities of cholesterol and cholesteryl ester [2,5,6]. The cells are surrounded by lipid, collagen, elastic fibres and proteoglycan. Advanced plaques are composed of complex layers of smooth muscle cells and macrophages which appear to have been altered as a result of haemorrhage, calcification, cell necrosis and mural thrombosis [2]. The evolutionary interrelationships between the three lesions is unclear at present. In both fatty streaks and fibrous plaques an intact endothelium is invariably present and although endothelial denudation is a frequent occurrence in complex plaques [7] no study has been able to demonstrate such loss at the initiation of the lesion. Animal models of atherosclerosis are of two general types: those involving direct damage to the endothelium [8] and those maintaining elevated levels of plasma cholesterol [9]. Both models produce lesions which have some of the characteristics of early human atherosclerotic lesions. However, each model assumes either endothelial denudation or hypercholesterolaemia to be primary events in human atherosclerosis. If such changes do not occur initially in man, then the relevance of such models to the natural history of atherosclerosis is questionable. A new model of atherosclerosis

We have recently developed a new model of atherosclerosis in which several of the elements of early human atherosclerosis are found under an anatomically intact endothelium [lo]. A light, flexible, biologically inert silastic collar is placed around a carotid artery of the rabbit. It touches the artery circumferentially at two points 1.5 cm apart. Blood flow through the artery is not affected by the collar. The contralateral artery acts as a control in the same animal. After 7 days there is proliferation of smooth muscle cells within the arterial intima in the area of the collar and when rabbits are fed a short-term, high-cholesterol diet, foam cells are also present but only at the site of the collar. Between 7 and 14 days a progressive 355

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accumulation of extracellular lipid and collagen occurs. Outside of the collar region, no smooth muscle cell proliferation occurs and no foam cells accumulate. Most interestingly, cholesteryl ester accumulates within the lesion (and not elsewhere) even when rabbits are fed a normal, low-cholesterol diet where blood cholesterol levels are 2-3 mmol I-'. The lesion is covered by an ultrastructurally normal endothelium which produces normal amounts of prostacyclin and which demonstrates the capacity to evoke endothelium-dependent relaxation. Therefore, this model produces a focal proliferative lesion which possesses several of the features of human atherosclerosis. Although extrapolation from an animal model to human disease may sometimes be misleading, the similarity of the anatomy and physiology of the vessel wall between the species allows the construction of a novel hypothesis about the origin of certain types of human atherosclerosis. In the model described, the lesion is produced by light pressure on the outside of the vessel which will almost certainly occlude the vasa vasorum within the adventitia. Some of the first observations on nutrition of the artery wall were made by Geiringer who demonstrated that in those species in which the thickness of the aorta exceeds 0.5 mm the media is supplemented by vasa vasorum in its outer layers [l I]. In vessels that have 29 or fewer lamellar units or fibromuscular layers there are no vasa vasorum found in the aortic media [12]. Although the demand for oxygen by the arterial wall is relatively modest, the thickness of certain vessels could result in the media being on the borderline of hypoxia. Direct measurements of oxygen tension within both the femoral arterial wall and thoracic aortic wall of dogs have demonstrated that it is highest in the adventitia and lowest in the media before increasing at the luminal surface [I 3,141. Several investigators have interfered with flow through the vasa vasorum of the thoracic aorta in dogs using either ligation of the vessels which supply them [I 51 or occlusion of the vasa vasorum with thrombin [16,17]. Each of these procedures resulted in medial necrosis and a marked thickening of the intima with a proliferation of smooth muscle cells and production of elastin and collagen fibres. Occlusion of the vasa vasorum would cause hypoxia of medial smooth muscle cells. It has been demonstrated that exposure of cultured endothelial cells or monocytes to hypoxia results in a significant increase in their production of mRNA for platelet-derived growth factor (PDGF) [18]. PDGF is a growth factor for smooth muscle cells and is a potent chemoattractant for leucocytes [191. Therefore, medial ischaemia, including ischaemia of the endothelium of the vasa vasorum, may cause proliferation of smooth muscle cells accompanied by migration of monocytes through the luminal endothelium towards the area of ischaemia. Moreover, the increase in cholesteryl ester within atherosclerotic lesions may be similarly explained. Intracellular cholesterol is stored within the cell in the

form of cholesteryl ester as a result of esterification with fatty acyl CoAs, a reaction catalysed by the enzyme acyl CoA :cholesterol acyltransferase (ACAT). In smooth muscle cells this enzyme increases in activity after exposure of the cells to hypoxia and results in an increased capacity of these cells to store cholesteryl ester [20]. Therefore, hypoperfusion through the vasa vasorum could cause the cellular and biochemical changes in our model (Fig. 1). A similar origin might underlie some forms of human atherosclerosis. This proposed origin might explain a number of parameters which are associated with atherosclerosis: anatomical distribution of arteries affected, age, maleness, diabetes, hypertension, stress and inflammation of the artery. These will be considered individually. Parameters associated with atherosclerosis

Specific arteries are known to be at risk from atherosclerosis; thus, the abdominal aorta is more at risk than the thoracic aorta. The coronary arteries and the femoral arteries are also known to be at particular risk, while the brachial arteries are usually spared. The abdominal aorta of humans is unusual as it is approximately 0.7 mm thick and only has 28 lamellae. It is surprising that such a thick artery has an avascular media which is approximately 40% greater than the avascular zone of the thoracic or abdominal aorta in nine other species [2 I]; indeed, others have speculated that the avascular media may contribute to the greater susceptibility of this artery to atherosclerosis although no mechanism was proposed [21]. The femoral arteries are known to be dependent upon a medial vasa vasorum while the brachial arteries, being relatively thin, do not need such a supply. Hypoperfusion through the vasa vasorum in the femoral arteries would result in medial hypoxia, whereas such hypoxia could not occur in the brachial artery which does not need vasa vasorum. The coronary arteries are unusual in humans since in a species comparison it was demonstrated that when their thickness exceeded 0.35 mm, medial vasa vasorum were required [I 11. If this is because the coronary arteries are more metabolically active and hence have a high demand for oxygen, then they would also be more susceptible to medial hypoxia, and by extrapolation to atherosclerosis. The observation that atherosclerosis occurs at branches in the arterial system in areas of low shear or turbulence can also be explained by this hypothesis. In areas of normal blood flow, the proliferative lesion occurring as a result of hypoxia might cease to progress or reverse as a result of normal repair processes. However, if the lesion causes protrusion of the vessel wall into the lumen in an area of turbulence, rheological problems would be exaggerated and endothelial damage would result. The progression of the lesion might then be encouraged by blood-borne mitogens as suggested by Ross & Glomset 121.

ARTERIAL WALL HYPOXIA

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Figure 1. A schematic representation of the hypothesis that atherosclerosis may be initiated by hypoperfusion following

thrombosis of vasa vasorum. (1) Under normal conditions the oxygen tension in the outer media of many human arteries is maintained via blood in the vasa vasorum. (2) Thrombosis of a vas vasis causes hypoxia of the outer media inducing the release of cytokines from adventitial and vasa vasorum-related leucocytes. (3) Smooth muscle cells divide and migrate or migrate and divide. Monocytes enter the intima from the lumen of the vessel or from adjacent vasa vasorum. ACAT is upregulated. (4) Intimal proliferation results from the combined effect of proliferation of smooth muscle cells, the entry of monocytes and the formation of foam cells from macrophages in the presence of upregulated ACAT. (5) (a) If the lesion protrudes into the lumen and is buffeted by the haemodynamic forces in the flowing blood because of a susceptible anatomical site, endothelial damage occurs; (b) alternatively, the lesion can heal ‘ad integrum’ by re- or neovascularisation of the adventitia. (6) Platelets and leucocytes may finally adhere to the subendothelium releasing PDGF and other cytokines that are responsible for the formation of the advanced pkdq ue .

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Hypertension is a risk factor for atherosclerosis [22]. In contrast to the expected result of an increased flow through the vasa vasorum in hypertension there is a decreased flow [23,24]. Decreased flow through the vasa vasorum would aggravate medial hypoxia. Diabetes is complicated by atherosclerosis and peripheral neuropathy. The latter is associated with abnormalities of both small blood vessels [25] and of platelets [26] which would predispose to thrombotic occlusion. Plugging of the vessels supplying blood to the nerves (vasa nervorum) with platelets and fibrin has been demonstrated in diabetes [27]. If vasa nervorum can become occluded, then similarly vasa vasorum may become thrombosed in diabetes, giving rise to medial hypoxia. A similar phenomenon may occur in syphilitic aortitis where endarteritis obliterans commonly involves the vasa vasorum; superimposed atherosclerosis is always present [28]. If platelet and fibrin plugs were to cause thrombosis of vasa vasorum in diabetes then similar occurrences might also give rise to non-diabetic atherosclerosis. It is well described that megakaryocytes (the platelet precursor cell) are larger [29] and that changes in factor V11 and fibrinogen are risk factors for myocardial infarction and presumably therefore atherosclerosis [30]. The mechanism by which platelets and coagulation factors are involved in vascular disease may therefore not only operate at the luminal endothelial surface, but also at the level of the endothelium of the vasa vasorum. There is already some indication that the endothelium from the vasa vasorum may be different from luminal endothelium [3 11. Furthermore vasa vasorum may also differ from other arteries in that they might fill during diastole. (The coronary artery, which fills during diastole, may be considered embryologically as a vas vasis.) Psychosocial stress and type A personalities are risk factors for the development of atherosclerosis. A number of studies have demonstrated that increased sympathetic activity can exacerbate the development of atherosclerosis in animal models while surgical or chemical sympathectomy can reverse its development [32,33]. Catecholamines increase vascular oxygen consumption [34,35] and moreover, activation of the sympathetic innervation of the vasa vasorum through stimulation of the stellate ganglion reduces blood flow through vasa vasorum of the thoracic aorta by approximately 40% [36]. Therefore, increased sympathetic activity, possibly associated with stress, may result in medial ischaemia. Finally, each of age, maleness and smoking are major risk factors for the development of atherosclerosis [37,38]. It is, therefore, interesting to note that arterial Po2declines with age [39,40] and that the value of arterial Po2 has been reported to be higher for young females than for males [39]. The effects of smoking in decreasing the oxygen carrying capacity of blood are well known [41]. Each of these factors may, therefore, exacerbate the risk of medial hypoxia.

Concluding remarks We have presented a new proposal for an origin of atherosclerosis. This proposal is consistent with many of the risk factors which are commonly recognized to be associated with the development of atherosclerosis. Hypercholesterolaemia, which has been demonstrated in a multitude of careful and elegant epidemiological studies to be a major risk factor, has not been discussed. Our view is that once the atherosclerotic lesion has been initiated, through an event which results in medial ischaemia, an elevated plasma cholesterol concentration will provide a superimposed risk for the lesion progressing to a more advanced stage. Acknowledgments John Martin is British Heart Foundation Professor of Cardiovascular Science, King's College School of Medicine and Dentistry, London SE5 9PJ, UK.

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Arterial wall hypoxia following thrombosis of the vasa vasorum is an initial lesion in atherosclerosis.

Pressure on the outside of arteries can cause physical and biochemical changes in the vessel wall of rabbits which are characteristic of atheroscleros...
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