Atherosckrosis, 89 (1991) 97-108 G 1991 Elsevier Scientific Publishers ADONIS 002191509100139Q
Arterial wall oxygenation, oxyradicals, and atherosclerosis D.W. Crawford Atherosclerosis
and D.H. Blankenhorn
UniL~ersity of Southern California,
201 I Zonal A~~ernre, HMR 804, Los Angeles, CA YO03.3 (U.S.A.
(Received 9 July, 1990) (Revised, received 11 February, 1991) (Accepted 12 April, 1991)
The oxygen supply of inner media and thickened intima of atherosclerosis prone arteries depends largely on diffusion from the endothelium. Conditions which increase wall thickness and oxygen diffusion or reduce oxygen transmissibility produce hypoxia and steep PO, gradients within the wall. Cerebral injury and myocardial reperfusion studies indicate that intermittent hypoxia and steep PO, gradients lead to oxyradical formation and tissue damage. Products of lipid and sterol peroxidation are found in atherosclerotic plaques and can be generated by arterial wall cells in culture. It is likely that peroxidation occurs directly within the arterial wall. Sufficient oxyradical generation occurs during normal oxygen metabolism that local scavenger mechanisms are required to avoid tissue damage. Experimental hypertension, hyperlipemia and balloon injury produce medial hypoxia with steep PO, gradients and redistribution of the pattern of arterial wall antioxidant enzymes. This suggests that minor deviations from normal arterial wall anatomy and function can lead to oxyradicals which can be directly injurious and can amplify the atherogenic potential of lipoprotein infiltration.
Key words: Arterial wall oxygenation; Oxyradicals; Atherosclerosis
Normal oxygen utilization in mammalian tissue requires an enzymatically controlled four-electron reduction to form water. If oxygen accepts less than four electrons it forms highly reactive
Correspondence to: Dr. D.W. Crawford. Atherosclerosis Research Institute, University of Southern California, 2011 Zonal Avenue, HMR 804, Los Angeles, CA 90033, U.S.A.
free radicals (oxyradicals), with one or more unpaired electrons [Il. One electron reduction of molecular oxygen yields the superoxide anion radical; two electron reduction, hydrogen peroxide (a strong oxidant rather than a true free radical); and three electron reduction, the hydroxyl radical [21. Superoxide anion radical, hydrogen peroxide, the hydroxyl radical and their propagation products all can produce chemical damage to intact tissue as well as unsaturated fats and a variety of proteins [Z]. Under normal cir-
98 cumstances oxygen metabolism produces only small amounts of these products which are quickly quenched by antioxidant defense mechanisms. Abnormal oxygenation such as intermittent hypoxia or hyperoxia alternating with hypoxia can accelerate oxyradical formation to overcome local protective mechanisms and produce local tissue damage 121.Another important source of oxyradicals is the respiratory burst of activated phagocytic cells, monocytes, macrophages and eosinophils. The anatomy of arterial walls determines the distribution of oxygen. In large arteries the endothelial surface is in contact with highly oxygenated blood but the media is largely avascular and dependent on oxygen diffusion from endothelium and adventitial vasa vasorum. Important deviations from normal structure and function which alter oxygen distribution patterns include: (1) thickening which increases diffusion distances, (2) abnormal composition which reduces diffusion rates, and (3) increased metabolic rate which increases demand. These factors also cause hypoxia of thickened intima in large arteries which is avascular. In 1944 Hueper proposed, on the basis of an extensive review of the literature, that atherogenesis involved recurring hypoxia due to persistent interference with oxidative metabolism of the vascular wall [31. Later evidence indicates that medial hypoxia due to abnormally long diffusional distances from endothelium and adventitial vessels might produce failure of lipid transport and severely decreased oxidative enzyme activity [4-61. In 1977 Fridovich suggested that hypoxia might lead to leakage of oxyradicals from the respiratory chain and hypoxia followed by reoxygenation would increase the probability of peroxidation reactions. Fridovich also pointed out that hypoxia might attract granulocytes which could release oxyradicals during a stimulated respiratory burst [7l. The central theme of this review is that abnormalities of oxygen distribution in the arterial wall produced by increased wall thickness, abnormal wall composition, and/or increased metabolism contribute to lipoprotein oxidation and atherogenesis through the formation of oxyradicals. We review evidence that factors causing arterial wall hypoxia also favor oxyradical formation. Our hy-
pothesis is that since the endothelium is in an environment of high oxidative injury potential protected under ordinary circumstances by high concentrations of antioxidants and deeper layers of the wall including the inner media and thickened intima exist at hypoxic PO, there are intervening zones where conditions favor oxyradical production. We further hypothesize that hypoxic media and thickened intima can produce small molecular precursors of oxyradicals which can diffuse toward the endothelium and encounter increasing levels of PO, in areas with little antioxidant protection. Thus the basic structure of arteries dictates oxygen distribution patterns which foster production of oxyradicals where antioxidant defense mechanisms may be inadequate. As a result minor deviations from normal arterial anatomy or function can initiate oxyradical reactions within the arterial wall which can be directly injurious and which can amplify atherogenie effects of systemic risk factors. In particular, arterial wall oxyradicals can influence the fate of infiltrated low density lipoproteins (LDL). Physical mechanics of arterial wall oxygenation by radial diffusion
Oxygen is supplied by radial diffusion from the vessel lumen to arteries of all species and supplies from other sources (adventitial vessels and vasa vasorum) vary according to species and size of vessel IS]. Actual measurements of arterial wall oxygenation have been made in rabbits and dogs where the vessels studied contain no vasa vasorum and oxygenation is from lumen or adventitial vessels. Similar but not identical anatomical features are found in atherosclerosis prone human arteries and animal arteries with induced hypertension, lipid infiltration, or balloon injury [9,10]. Target vessels for atherosclerosis typically have thicker walls and may have variable penetration of adventitial vasa vasorum into the media. However, the inner media and thickened intima of target vessels must be supplied by diffusion over relatively large distances from luminal blood [Ill. Mathematical models relating arterial wall dimensions and composition to PO, levels are useful in understanding quantitative aspects of wall oxygenation. PO, provides the pressure for diffu-
99 sional transport of oxygen with movement from higher PO, to lower levels. Arterial wall anatomy dictates that there is an area of lowest PO, designated P, within the arterial wall and that there can be a zone with marginal oxygen supply in abnormal arteries [12,13]. P,, also known as the zero oxygen flux region, can be located in relation to the central vessel axis in Eqn. 1 which is based on radial diffusion and which estimates PO, along radii through the arterial wall (Eqn. 1): P=P,-(W/DS)[r,2(ln
P is the PO, in the arterial wall at any radial distance (r) from the central axis of the lumen, P, is PO, at or near the endothelium at a distance re from the center of the lumen, W is the oxygen consumption of the arterial wall, D is the oxygen diffusion coefficient of the arterial wall, and S is the solubility coefficient for oxygen [14-161. DS (D x S) is the transport capacity (oxygen transmissibility) of the arterial wall. The ratio W/DS (oxygen consumption/transport capacity) is an important determinant of the location of P,, the zero flux point. The balance between W/DS inside and outside of P, determines the location of P, which can be anywhere within the wall. If there is limited adventitial blood supply with very low PO,, P, may occur at the adventitia . Equation 1 can be used with experimental measurements to calculate W/DS or to calculate distance from endothelium of P, if W/DS is known. The major underlying assumptions for the model are constant conditions for arterial blood PO,, adventitial blood PO,, wall dimensions, oxygen consumption, oxygen solubility, and oxygen diffusion coefficient. The model also assumes a circular arterial wall. All these assumptions are never quite true in normal or diseased arteries but the equation yields valid estimates over distances involved in atherosclerotic lesions. The model expressed in Eqn. 1 implies a smooth curvilinear decrease in PO, from the endothehum to P,. The importance of the model in relation to atherogenesis is that the many conditions leading to atherosclerosis also produce steep gradients of PO, within the arterial wall. As a
consequence, heterogeneous PO, within the arterial wall can result from relatively minor failures of normal function as well as a variety of disease states. Arterial wall PO, measurements support the concept of radial diffusion Amperometric measurements of arterial wall PO, in vivo or ex vivo are made with cathodes maintained at a constant voltage with reference to a nearby calomel or silver/ silver chloride electrode. Early in-vivo measurements of local PO, within the arterial wall made use of very large (125 pm) bare-tipped platinum wire for measurements in the dog femoral artery . An “oxygen current” appeared to decrease progressively from adventitia to intima, and then rise abruptly to an arterial level. Similar overall in vivo results were obtained in the normal [lS] and injured, hyperlipidemic rabbit abdominal aorta by Heughan et al. . These showed large local discontinuities in PO, values over only a few pm which appear to have been artifactual in light measurements made with improved techniques. The art of measurement of tissue PO? was advanced when Whalen  designed a glass microelectrode in which the gold cathodal surface is recessed inside a sharply beveled, 2-3 pm open tip. Schneiderman and Goldstick examined the geometry of microcathode tips and showed that oxygen diffusion produced by the very small oxygen consumption of the electrode itself is confined within the tip recess [21,22]. These fragile electrodes perform adequately [231 with very little tissue damage  if introduced into the arterial wall with a vibrating micromanipulator . Results obtained with these electrodes indicate constant oxygen consumption W and transmissibility DS over distances of 100 to 200 Frn. Although the sharp discontinuities in PO1 found with cruder early electrodes were artifacts, it is should be noted that within a thickened intima with high oxygen consumption and low transmissibility radial PO, values may fall sharply. Schneiderman et al.  removed and artificially supported normal rabbit thoracic aortas to study the transmural distribution of oxygen tensions. They found that PO, fell in a smooth curve
pm Fig. 1. Typical in vivo PO, values of a normal dog femoral artery. Abscissa represents distance in pm from bloodstream into the depths of the wall. Ordinate, the PO, measured in torr. The area marked by A is the blood-intimal boundary, B is the medial-adventitial junction. The rise in PO, at C indicates the presence of adventitial blood vessels. Hence, the PO, values on the right of C are in surrounding extravascular tissue.
from arterial blood levels to a lowest value about one-third of the way to the adventitia and then rose slowly due to perfusion from adventitial vasa vasorum. Jurrus and Weiss  perfused normal and atherosclerotic specimens of rabbit aorta and found smooth transmural oxygen tension distributions which also provided a striking demonstration of effects of abnormal wall thickness. In diseased vessels 1000~cl,rnthick with surface oxygen tension 150 torr (mm Hg) at the intimal surface, P, occurred only 200 Frn beneath the endothelium where 0, tension was 0 torr. The relationships expressed in Eqn. 1 have been tested with in vivo measurements by one of us in normal rabbit aorta and femoral artery [16,28]. These vessels have no medial vasa vasorum, and demonstrate a smooth curvilinear drop from arterial blood PO, to the adventitia. The dog femoral artery has extensive adventitial vasa vasorum, some of which may penetrate outer media, but otherwise shows the same oxygen pattern (Fig. 1). Our estimates of in vivo oxygen consumption (56-142 nl,/ml . set) are very close to reported values obtained with various vascular wall tissues in a respirometer (20-230 nl,/ml . set) . We have also studied hypertension and balloon injury in rabbits. Hypertension depresses PO, throughout the inner intima and media of the abdominal aorta. In 8 normal rabbits the difference between PO, in arterial blood and that at P, was 34 I~I3 torr, in 9 rabbits with mild hypertension the difference was 58 + 15 [281. Our results are compatible with increased arterial wall
oxygen consumption in hypertensives reported by others which we discuss later. Hypertensive aortae also showed glycosaminoglycan accumulation as has also been seen in hypoxic cultured arterial endothelial cells . Glycosaminoglycans can be expected to alter 0, transmissibility CDS) and are also known to bind lipoproteins  and so these ground substance components may combine the effects of altering oxygen distribution gradients and slowing lipoprotein turnover rates to increase the probability of lipoprotein peroxidation. Balloon injury of the endothelium resulted in growth of vasa vasorum and reduced the extent of hypoxia , a result compatible with the protective effects of capillary ingrowth described by Geiringer . Atherogenic effects of factors altering radial arterial wall oxygen diffusion Changes in endothelial PO,
Arterial blood PO, determines the PO, at the endothelium and has direct effects on POZ throughout avascular zones of the arterial wall. Arterial blood PO, does not vary much in humans without pulmonary or cyanotic congenital heart disease but in experimental animals alteration of arterial PO, by changing atmospheric PO, has been shown to influence atherogenesis. For instance, hypoxia has been reported to enhance, and hyperoxia to reverse arteriosclerosis produced by cholesterol feeding in rabbits [32,33]. Although general arterial PO, is normally constant, temporal changes in arterial wall oxygenation do occur when the oxygen carrying capacity of blood is reduced by carbon monoxide inhalation during smoking. Schneiderman and Goldstick have demonstrated that this can cause inner wall hypoxia and increase the heterogeneity of arterial wall oxygenation . Inhalation of carbon monoxide in amounts comparable to that inhaled by cigarette smokers enhances the development of atherosclerosis in cholesterol-fed rabbits . An interesting but untested possibility is that intermittent inhalation of carbon monoxide or airborne agents impeding lung function can lead to a hypoxia followed by reoxygenation in atherosclerosis prone human arteries. Also, despite constant general arterial PO,, spatial distur-
101 bances of blood flow, such as separated flow flow stasis can lower PO, in the intima adjacent arterial wall [36,37]. Regions of separation and stasis appear to be preferred for the development of plaques .
and and flow sites
Changes in arterial wall oxygen consumption The magnitude of the radial fall in PO, from the intima and the position of P,, the region of lowest PO, and zero oxygen flux, are altered significantly by changes in arterial wall oxygen consumption . If the oxygen consumption is increased above normal the radial distribution of arterial wall oxygenation becomes more heterogeneous and P, is likely to be close to the region of highest oxygen consumption. Oxygen consumption of the arterial wall is generally increased in cholesterol fed animals [39-411. Hypertension also increases arterial wall oxygen consumption. Seidel and Strong have shown a clear increase in oxygen consumption in aortic rings from rats with spontaneous, renal, and DOCA-salt hypertension [421. In vivo measurements of POz through the wall of hypertensive rabbit aortae are compatible with increased oxygen consumption. There is hypoxia and greater radial divergence of PO, between intima and media than in controls 1281. Hypoxia of intima and media appears due to combination of longer diffusional distances and increased medial oxygen consumption. The effects of hypertension and hyperlipidemia may be additive. Cozzi et al.  showed that combined hypertension and hyperlipidemia led to maximum plaque formation associated with a seven-fold increase in oxidative metabolism. Foam cells increase oxygen consumption. In 1980, Brown et al. [441 suggested that energy dependent cyclic cholesteryl ester hydrolysis and re-esterification in macrophage derived foam cells could utilize excessive amounts of oxygen and lead to hypoxic foam cell necrosis. Bjornheden and Bondjers 1451 have presented data compatible with this concept. They found increased oxygen consumption in arterial segments from cholesterol fed rabbits with 3 times higher oxygen consumption in isolated foam cell fractions than in smooth muscle cells. Using reported values for foam cell O2 consumption we calculate using
Eqn. (1) that profound hypoxia can exist within 100-200 pm atherosclerotic foam cell lesions. P,, at a radial distance into the wall rr might well occur within a thickened intimal or foam cell lesion. The increased metabolic activity reported by Cozzi et al.  was principally located in foam cells.
Changes in wall thickness and oxygen transmissibility Increased thickness of avascular oxygen consuming arterial wall and decreased transmissibility for oxygen (DS) both accentuate the radial reduction of PO, and decrease the lowest level of arterial wall PO,. Age, hypertension and atherosclerosis all increase the thickness of the intima of large human arteries. Hypertension and atherosclerosis also produce focal changes in fat, fibrous tissue, and ground substance which alter DS and favor more patchy oxygen distribution patterns. Oxygen diffusion coefficients were measured by Kirk and Laursen 1461 in aortic intimasubintima and media from middle aged and older human cadavers. Coefficients tended to increase with age. The average coefficient for 50 intimal specimens was slightly lower (0.84 x 10-5 cm2/sec) than that for 50 medial specimens (0.96 x lo-” cm2/sec). Unfortunately the presence and extent of disease in individual specimens was not given. Additional data are needed because normal variation in oxygen transmissibility could account for differences in arterial susceptibility to atherosclerosis.
Source of lipid and sterol oxidation products in the arterial wall Lipid and sterol peroxidation products have been isolated from the arterial wall. Included are malondialdehyde (MDA)  which is formed during the peroxidation of unsaturated fatty acids and sterol oxides which may be produced independently or concomitant with lipid oxidation . Some reports of lipid and sterol oxidation products in human aorta and plasma [49-521 may be incorrect. Tissue and plasma lipids and sterols can undergo auto-oxidation and photo-oxidation during storage and analysis unless adequate precautions are taken. The widely used thiobarbi-
102 turic acid reactive test (TBAR) may not accurately estimate the amount of oxidized lipid. Measurement of UV-absorbing conjugated dienes to estimate unsaturated fatty acid peroxidation may record products unrelated to lipid peroxidation . The problem of oxidation artifact has been reviewed by Halliwell . Despite problems, there is now good evidence that peroxidation products do accumulate within the arterial wall in-vivo. Mowri et al.  extracted lipid droplets from atherosclerotic plaques of Watanabe Heritable Hyperlipidemic (WHHL) rabbits under argon gas and found peroxidized lipid. A technique independent of postmortem oxidation artifact was used by Haberland, Fong and Cheng  to demonstrate LDL peroxidation within the arterial wall. Monoclonal antibodies were raised to LDL modified by MDA and used for immunocytochemical analysis of the aortae of WHHL rabbits. MDA-altered protein co-localized with extracellular deposition of apolipoprotein B-100 in atherosclerotic lesions but was not found in normal aorta or in plasma from WHHL rabbits. Further, Rosenfeld et al.  demonstrated cell-associated as well as extracellular staining of spontaneous aortic lesions in WHHL rabbits with antibodies directed against three different epitopes from the oxidative modification of LDL. In transitional lesions staining for LDL and oxidative epitopes was often located in the vicinity of active foam cells. Sources outside of the arterial wall must be considered. Using the TBAR test Sulyok et al.  found that cholesterol feeding produced lipid peroxidation in both the liver and plasma of rabbits. A general oxyradical scavenger reduced both peroxidation and hyperlipidemia and they suggested a causal role for peroxidation in hyperlipidemia in the rabbit . Kosykh et al. found that the commercial cholesterol used as dietary supplement contained about 5% of oxidized cholesterol derivatives. Purification reduced both the increase in plasma cholesterol and hepatocyte cholesteryl ester from cholesterol feeding in rabbits. Although these studies indicate potential extra-arterial sources it seems unlikely these can be the sole source of sterol oxides associated with LDL within the arterial wall. Peroxidative alteration of LDL causes very rapid removal from the
circulation by hepatic receptors which minimizes the exposure time of the arterial wall . Also, arterial cells can produce peroxidation products in cell culture. Cell culture studies have demonstrated that oxyradicals can be produced by cellular elements of the arterial wall. They have also demonstrated cytotoxic and pharmacologic effects of lipid and sterol peroxidation which relate to induction of early atherosclerosis [60-621. It is clear that oxyradicals in cell culture can be cytotoxic and can alter the smooth muscle and endothelial cell production of growth factors [61,63,64]. Cell culture studies also demonstrate the effects of changing oxygen environment on oxyradical production. Electron paramagnetic resonance measurements indicate free radical formation in cultured bovine aortic endothelial cells subject to reoxygenation after a period of hypoxia . In these studies superoxide and hydroxyl radicals were identified by selective use of scavenger enzymes. Production of oxyradicals was effectively blocked by oxypurinol suggesting that the enzyme xanthine oxidase was the source. Induction of cerebral and myocardial oxyradical injury by steep PO, gradients
Hypoxia, cyclic changes in PO,, and patchy variation in oxygen tension have all been implicated as damaging to the brain, spinal cord and myocardium [66,67]. Manning et al. and others have shown that coronary artery occlusion and reperfusion leads to release of oxyradicals [67,68]. Herbacznska-Cedro and Gordon-Majszak [691 found evidence of the production of lipid peroxidation products in myocardial regions remote from the site of coronary occlusion and speculated that regional differences in perfusion led to oxyradical production. Demopoulos et al. who studied regional nervous system ischemia and spinal cord injury, noted that the solubility of 0, in fat is 700% greater than in an aqueous environments. Tissues with irregular fat deposits can therefore have remarkable heterogeneity in the amount of dissolved oxygen at the same PO, [7Ol. Meerson et al.  commented on heterogeneity of dissolved oxygen in myocardial fat in a study of lipid peroxidation with myocardial infarction.
Since temporal and spatial variability in oxygenation lead to oxyradical production and lipid peroxidation in the brain and heart, we believe that similar events may occur in the arterial wall but at a slower rate. Small molecular precursors of oxyradicals (hypoxanthine, catecholamines) could form in hypoxic regions and diffuse into closely adjacent oxygenated areas. We also wonder whether fat in superficial foam cell layers may serve as an oxygen reservoir to potentiate local peroxidation. Oxyradical scar’engers are needed to protect normal tissue; diseased tissues may require greater protection
Oxyradical reactions important in many normal processes , have potential for tissue damage when oxyradical production is high relative to local defense mechanisms. Several reviews in the last ten years have discussed the protective importance of tissue antioxidants including non-enzymatic scavengers of oxyradicals [72-741. The major antioxidant enzyme systems are (1) superoxide dismutase, which catalyses dismutation of the superoxide anion, (2) catalase, which inactivates hydrogen peroxide, and (3) the glutathione peroxidases, which inactivate a variety of organic peroxides. The role of antioxidant defense is most clearly seen in pulmonary tissue. Local oxygen tension can be manipulated by adjusting atmospheric concentrations and the potential for damage here is normally high because of high alveolar PO,. If rats are exposed to gradually increasing atmospheric PO, the activity of superoxide dismutase in lung tissue is increased and animals are partially protected against oxygen toxicity [75,76]. In similar experiments Rister and Baehner  studied hyperoxic guinea pig lung polymorphonuclear leukocytes and alveolar macrophages and found induction of superoxide dismutase. They also found a subsequent decrease in catalase and glutathione peroxidase activity which they attributed to the action of the superoxide dismutase in producing hydrogen peroxide from superoxide anion. Liu et al. [781 compared rabbit alveolar macrophages with peritoneal macrophages which exist at a low PO, and found that superoxide dismu-
tase activity was considerably higher in alveolar macrophages. When atmospheric PO, is varied the tissues most likely to be in equilibrium are alveolar lining, other pulmonary parenchyma, endothelium and intima with PO, levels in normal intima only slightly lower than in alveolar lining. Hyperoxia can induce and hypoxia suppress antioxidant enzyme activity in other tissues but results are variable possibly because enzyme regulation may depend heavily on local PO,. Changes in atmospheric oxygen partial pressure are not fully transmitted to all tissues and so studies of whole organ antioxidant activity may have limited meaning for the arterial wall. The superoxide dismutase activity in solid tissue of hypoxic mice is suppressed compared to the activity in normal animals. Guarnieri et al.  reported that hypoxia decreased the activity of both superoxide dismutase and glutathione peroxidase in hypoxic rat hearts. Further evidence of the importance amount of enzyme activity relative to the degree of oxidizing stress is the finding that superoxide dismutase delivered in liposomes to cultured porcine endothelial cells prevented hyperoxic injury . Working with the hypothesis that atherosclerosis involves oxidative modification of LDL, Carew et al. 1811were able to show that probucol, a nonenzymatic antioxidant, retarded the progression of atherosclerosis in Watanabe rabbits independent of effect on plasma cholesterol levels. This was also found by Kita et al. . Antioxidants in the arterial wall are altered by atherogenic stress
Antioxidant activity has been studied less in arteries than in other tissues. Henriksson et al. [831 found an increase in the activity of superoxide dismutase from whole aortae of cholesterolfed atherosclerotic rabbits, which they took as presumptive evidence of oxyradical generation. We have studied the microscopic location of arterial wall superoxide dismutase, catalase, and glutathione peroxidase activity in hypertensive and early atherosclerotic aortae of NZW rabbits [84861. In the normal aorta very little superoxide dismutase, catalase, or glutathione peroxidase activity was demonstrable and all that was present
104 was in the intima. In hypertensive animals increased staining, especially of superoxide dismutase and glutathione peroxidase, was localized in intima and subintima. In hyperlipemic animals increased staining of superoxide dismutase and glutathione peroxidase was demonstrable throughout the entire wall. In the hypertensivehyperlipidemic animals, there was intense staining in the intima of lesions and in the media below lesions, and again superoxide dismutase and glutathione peroxidase activity predominated. It is of interest that the adventitia of the atherosclerotic vessels (PO, should be higher here than in the media) stained intensely. This suggests that in the arterial wall as in the lung antioxidant enzyme activity may be regulated by the local PO,. It also suggests that oxygen poor sub-endothelial regions are relatively depleted of antioxidant protection. In any event these findings indicate a redistribution of arterial wall antioxidant enzymes with atherogenic stress. Although causal linkage with changes in local PO, have not been shown there is depletion of protective enzyme activity in arterial wall areas where steep oxygen gradients are predicted. Steep PO, gradients have been shown to produce tissue injury in heart and brain; similar gradients in the arterial wall should also be injurious, in particular where protective enzymes have been depleted. Arterial wall oxyradicals influence cyte-macrophages interactions
Conditions of oxygen distribution determined by wall anatomy allow minor deviations from normal anatomy and function to produce oxyradicals which can potentiate the atherogenic effects of lipoprotein infiltration. Small amounts of superoxide anion radical and derived hydrogen peroxide are normal products of mitochondria [72,87]. H,O, production increases with increasing oxygen partial pressure [87,88]. The activity of oxyradical .generating enzymes systems including xanthine oxidase, cyclooxygenase, and lipoxygenase appear to be increased as well as the rate of auto-oxidation reactions catalyzed by transition metals [89,90]. The relatively high PO, of the endothelium makes it a potential source of oxyradicals. At the other extreme, low (but not
zero> PO, levels can cause uncoupling of the respiratory chain with production of superoxide anion radical and H,O, . PO, levels low enough to cause uncoupling appear possible in thickened subintima and underlying media. They are also theoretically possible in intimal foam cell lesions. Lipoprotein uptake by monocyte-macrophages plays a central (possibly dominant) role in both human atherosclerosis and its counterpart in experimental animal models [91-941. Macrophages cannot accumulate significant amounts native LDL by way of the LDL receptor  but will take up LDL which has been modified by cellular elements in the arterial wall. Heinecke et al. found evidence of superoxide production in cultured monkey and human aortic smooth muscle cells when reduction of cytochrome c was inhibited by the enzyme superoxide dismutase . In the presence of transition metal, these cell cultures also produced modification of LDL resulting in enhanced uptake by macrophages . Morel et al.  presented evidence of LDL peroxidation in cultures of bovine aortic smooth muscle cells and human umbilical vein endothelial cells. Exposed LDL gave a positive thiobarbituric acid test and was toxic to cultured fibroblasts. Malondialdehyde (a product of lipid peroxidation in living systems) modifies LDL to cause cholesteryl ester accumulation in monocyte-macrophages by scavenger receptors [47,98]. In addition, 4-hydroxynonenal which is a relatively stable lipid peroxidation product in oxidized LDL has also been shown to induce phagocytic LDL uptake in macrophages . Oxidized beta-VLDL is also more avidly taken up by macrophages than native beta-VLDL [ 1001. Oxyradical modified LDL have strong secondary effects which can amplify atherogenesis. Oxidatively modified LDL are chemotactic to human monocytes in culture and may participate in recruitment of macrophage precursors to the arterial wall [loll. Complexes of LDL and proteoglycan also are taken up avidly by macrophages in culture with formation of foam cells. Production of proteoglycans is increased under hypoxic conditions and in the arterial wall hypoxic conditions also foster oxyradical formation. Thus, hypoxic conditions may participate in foam cell lesion formation [30,102,103]. Further, entrapment of
105 LDL by proteoglycans or connective tissue in the arterial wall can enhance the probability of modification by oxyradicals because of prolonged residence time . Last, it should be noted that oxidatively modified LDL can damage both fibroblasts  and endothelial cells . Recommendations
for future studies
Intramural arterial wall PO, should be measured in vessels of the thickness of diseased human coronary artery or aorta because current theory is based on measurements in vessels approximately one tenth as thick. Improved electrode technology may be required. More information on oxygen consumption, diffusion, and solubility coefficients in the various layers of the arterial wall would be extremely useful. Healthy, hypertensive, and atherosclerotic vessels all should be studied. Studies of atherogenic mechanisms with cultured arterial wall cells should be conducted with PO, levels in a range which realistically simulates arterial wall conditions. Atmospheric PO, is now typically used for cell cultures but is never encountered in normal or diseased vessels. Direct measurements and mathematical models predict that effects on oxyradical reactions of levels between 90 and 10 torr should be studied in arterial wall cell systems. This recommendation is supported by the finding that cultured glial cells have shown remarkable differences in accumulation of peroxidation products depending on whether cultures are exposed to atmospheric air or PO, levels of brain parenchyma 11071. In addition multilayered culture systems  might be used to study effects of steep gradients in PO, between 90 and 20 torr. References
7 8 9 10
14 15 16
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