- Atherogenesis in Outline
- Origins of the LDL Oxidation Hypothesis
- Proatherogenic Properties of OxLDL
- Evidence That Oxidation of LDL Occurs In Vivo
- Clinical Implications
- What is the Essential Function of OxLDL Receptors That Accounts for Their Persistence Through Evolution?
- The Role of Macrophages and Scavenger Receptors in the Late Lesion
- Will Antioxidants Work in Humans?
- Where Do We Stand?
- Figures & Tables
- Info & Metrics
I want to express my thanks to Dr Sidney Smith, President of the American Heart Association, for selecting me to present the 1995 Conner Memorial Lecture. I want also to express my admiration for and gratitude to this remarkable organization, the American Heart Association, and its wonderful staff, with whom I have worked for more than 30 years. I have missed very few AHA meetings since 1959 when the American Society for Study of Arteriosclerosis became the American Heart Association Council on Arteriosclerosis. I−indeed all of us−owe a great debt to the AHA for its tireless efforts over the years to support medical research and improve medical care.
I start with the assumption that we can all agree that hypercholesterolemia−particularly hyperbetalipoproteinemia−is an important causative factor in atherogenesis and that correction of it can strikingly reduce the risk of coronary heart disease (CHD). Yet it was not so long ago that this argument had to be vigorously defended. It was only in 1983 that the National Institutes of Health (NIH) officially endorsed the position that hypercholesterolemia must be treated. That decision followed closely on the completion of the landmark Lipid Research Clinic Intervention Trial, initiated by Dr Donald S. Fredrickson and Dr Robert I. Levy and shepherded to completion by Dr Basil Rifkind.1 The following year I had the privilege of chairing the NIH Consensus Conference on Lowering Blood Cholesterol,2 which concluded unanimously that there was an unarguable cause-and-effect relationship and that lowering blood cholesterol should be an important national goal. The following year the National Heart, Lung, and Blood Institute (NHLBI) organized and spearheaded the National Cholesterol Education Program (NCEP). Actually this year marks the 10th anniversary of the NCEP, and I think we can again all agree that it has accomplished a great deal under the able directorship of Dr James I. Cleeman and his staff.3 The introduction of the HMG CoA reductase inhibitors and the astonishing successes with them in recent clinical trials4 5 has removed any lingering doubts about the efficacy of cholesterol lowering as a means not only of reducing morbidity and mortality from CHD, but also, in some studies, significantly reducing all-cause mortality. And yet, deaths from CHD continue to outnumber deaths from any other single cause in the United States. We still have a long way to go. We will undoubtedly see further decreases in CHD morbidity and mortality when we get better at treating hypercholesterolemia. But will the epidemic of coronary artery disease be wiped out by even the most intensive efforts to lower blood cholesterol levels? I doubt it.
We have all seen myocardial infarction in patients with cholesterol levels <200; we have also seen patients with heterozygous familial hypercholesterolemia and cholesterol levels >300 who somehow survive into their 70s with no clinically evident CHD. Clearly, there must be factors that modulate the impact of hypercholesterolemia on the blood vessel wall, increasing or decreasing the pace at which atherosclerosis progresses. How does LDL interact with the cells of the artery wall to initiate the atherogenic process? What is the nature of the cell-cell interactions that determine whether a lesion will progress rapidly, arrest, or undergo regression? What are the cellular and molecular mechanisms linking risk factors such as cigarette smoking and hypertension to vascular biology and atherogenesis? Questions like these are under intensive investigation, and a great deal has been learned about vascular pathobiology. As we identify the key steps in the atherogenic process, it should become possible to devise interventions that will modulate the rate of progression at any given level of LDL. In other words, we should be able to move “beyond cholesterol.”6
Atherogenesis in Outline
A number of excellent reviews are available with regard to the current views on atherogenesis.7 8 9 10 The scheme shown in Fig 1⇓ presents a current consensus on the events leading to the earliest stages of atherogenesis−the formation of foam cells and the fatty streak lesion. There appears to be general agreement that the events designated 1 through 8 are involved in generation of the fatty streak. The subsequent events−cell growth, matrix deposition, cell migration, cell death, and necrosis−are extraordinarily complex, involving multiple cytokines and growth factors and multiple, probably reciprocal, cell-cell and cell-matrix interactions.7 No attempt is made here to analyze the late stages in any detail. However, these complex interactions are being progressively elucidated, and some potential therapeutic targets have already been identified. For example, metalloproteases have been implicated in the erosion of the fibrous cap overlying a necrotic lesion,11 which appears to be the event triggering fatal thrombosis in most cases of myocardial infarction.12 Inhibition of the production or the action of such enzymes would be one logical target for pharmacological or genetic intervention.
Origins of the LDL Oxidation Hypothesis
Hypercholesterolemia is most commonly associated with an elevation of plasma LDL, and LDL is the ultimate source of the cholesterol that accumulates in developing foam cells. Yet, paradoxically, the uptake of that LDL into macrophages and into smooth muscle cells almost certainly does not occur by way of the classic Brown/Goldstein LDL receptor.13 How do we know that? First, foam cells develop in patients and in animals that totally lack LDL receptors, and their foam cells do not look very different from the foam cells developing in the lesions of patients with hypercholesterolemia but with normal LDL receptors. Second, incubation of either macrophages14 or smooth muscle cells15 with even very high concentrations of LDL does not increase the cell content of cholesterol very much. This reflects in part the fact that the native LDL receptor in macrophages and smooth muscle cells, as in other cells, downregulates when the cell cholesterol content begins to build up. For these two reasons it became necessary to postulate that the LDL in the circulation must somehow be altered before it can be a source of foam cell cholesterol. Goldstein et al14 showed that acetyl LDL, formed by treatment of LDL in vitro with acetic anhydride, was taken up much more avidly by macrophages and was taken up by way of a new receptor distinct from the LDL receptor (Fig 2⇓). Most importantly, this acetyl LDL receptor, unlike the LDL receptor, did not downregulate so that the macrophage continued to take up acetyl LDL until it became engorged with stored cholesterol esters. However, there is little or no evidence that acetylation of LDL occurs to any significant extent in vivo. Therefore, the search for other, biologically feasible modifications of LDL continued. In the early 1980s, Henriksen et al16 17 18 showed that endothelial cells and smooth muscle cells can modify LDL in vitro in such a way as to enhance its uptake by macrophages. Later studies by Steinbrecher et al19 and by Morel et al20 showed that what the cells actually do is to simply oxidize the LDL. All of the effects of the cells could be mimicked by simply incubating with copper ions as catalyst. Thus, the oxidative modification hypothesis originally rested on the finding that cell-modified (oxidized) LDL had the potential to cause foam cell formation. Before going on, I should point out that there are a number of modifications of LDL, in addition to oxidative modification, that can enhance its uptake by macrophages in vitro. These include (a) self-aggregation,21 (b) complex formation with proteoglycans,22 (c) immune complex formation,23 24 25 and (d) degradation by hydrolytic enzymes.26 27 28 29 However, none of these has been as extensively studied as oxidative modification, and space limitations make it impossible to discuss them further.
Oxidation of LDL is an incredibly complex process.30 31 Both the protein and the lipid moieties can be oxidatively attacked and each one of the lipid classes can be attacked, including sterols, fatty acids in phospholipids, cholesterol esters, and triglycerides and in fact most all of the components, major and minor, including the many antioxidants in LDL. The extent of the changes in the LDL particle induced by oxidation depends on the prooxidant conditions used and the length of time the particle is exposed to those prooxidant conditions. Therefore, there is no unique LDL particle corresponding to “oxidized LDL.”32 Instead, there is a broad spectrum of “oxidized LDLs.” Moreover, these can differ not only structurally but functionally. This is brought out nicely by the work of Berliner and coworkers,8 who showed that LDL subjected only to the mildest of oxidative stresses (minimally oxidized LDL [MM-LDL])−too little to alter its interaction with the LDL receptor or make it a ligand for the acetyl LDL receptor−nevertheless acquired important biological properties. These included the ability to stimulate release of chemokines and cytokines from endothelial cells.33 34 Some of the unsolved problems relating to the heterogeneity of oxidized LDL are discussed in more detail elsewhere.35 Until we have a systematic way of characterizing differently oxidized LDL (OxLDL) preparations, perhaps the best we can do is describe them in biological terms (ie, in terms of functionality) or by simply describing empirically (and in detail) the conditions used to prepare them.
Proatherogenic Properties of OxLDL
The ability of OxLDL to induce cholesterol accumulation in macrophages was the first proatherogenic property of OxLDL to be described16 and was the basis for the hypothesis that oxidation of LDL might be an important step in the atherogenic process. Over the following years a number of additional properties of OxLDL were described that could in principle contribute to its atherogenicity. For example, OxLDL is itself a chemotactic agent for monocytes36 and for T cells37 but not for B cells. This is consonant with the fact that lesions contain primarily monocytes and T cells. Hessler and coworkers38 showed that the cytoxicity of LDL for cultured endothelial cells was due to its oxidative modification and that this clearly could be atherogenic. Studies in Fogelman's laboratory showed that OxLDL (minimally oxidized) could stimulate the release of macrophage colony–stimulating factor (M-CSF)34 and of monocyte chemoattractant protein-1 (MCP-1)33 from endothelial cells, which would facilitate the development of fatty streak lesions by recruiting monocytes and facilitating their differentiation into tissue macrophages. The list of potentially proatherogenic properties has now grown to almost 20! We will not take the time to review all of them. Indeed, only a few have been validated as playing an active role in vivo. Suffice it to say that oxidative modification of LDL can impinge on the atherogenic process in a number of different ways. Two important questions, however, need to be asked: (1) Does oxidative modification of LDL actually occur in vivo? and (2) Is the contribution of oxidative modification sufficient such that interference with it will slow the progression of the disease?
Evidence That Oxidation of LDL Occurs In Vivo
Three lines of evidence are available both in experimental animal models and in humans to indicate that oxidation of LDL does indeed occur in vivo. They are (1) demonstration that LDL gently extracted from atherosclerotic lesions is in part oxidatively modified39 40 ; (2) demonstration by immunohistochemistry that atherosclerotic lesions contain materials reactive with antibodies generated against OxLDL41 42 43 ; and (3) demonstration of circulating antibodies reactive with OxLDL,42 implying the presence in vivo of OxLDL itself or a very similar antigen.
The most important line of evidence, namely, that intervention with antioxidants can slow the progression of the disease, is provided by a large number of studies in experimental animals. As shown in the Table⇓, a number of experimental models have been used, including cholesterol-fed rabbits, LDL receptor–deficient rabbits, cholesterol-fed nonhuman primates, cholesterol-fed hamsters, and transgenic mice (LDL receptor–deficient and apo E–deficient). The first studies were done using probucol in LDL receptor–deficient rabbits.44 45 The extent of lesions was reduced by 50% to 75%. Probucol is a remarkably potent antioxidant, more potent than vitamin E, and most of the published studies have used probucol as the antioxidant. Probucol has some additional properties that could contribute to its antiatherogenic potential. For example, it can inhibit the release of interleukin-1 under some circumstances46 and it also influences cholesterol ester transfer protein levels.47 Consequently, questions can be raised about whether the antiatherogenic effect of probucol is entirely attributable to its antioxidant activity. However, as shown in the Table⇓, positive results have also been reported with diphenylphenylenediamine, with vitamin E, and with butylated hydroxytoluene. The fact that similar results have been obtained with several different antioxidants supports the reasonable conclusion that the major effect is due to the antioxidant properties of the compounds studied.
Of 23 published studies (some papers include studies with more than one antioxidant compound) 16 have been strongly positive, 2 have been borderline positive, and 5 have been negative. It seems fair to say that the LDL oxidative modification hypothesis is strongly supported by data in experimental animal models.
It may be useful to oversimplify and use the following equation when discussing the possible importance of oxidation of LDL in atherogenesis:Rate of Lesion Progression|<|=|>|f_|<|1|>||<|[|>|LDL Concentration|<|]|>||<|+|>|f_|<|2|>||<|[|>|Rate of LDL Oxidation|<|]|>||<|+|>|f_|<|3|>||<|[|>|\mathit|<|x|>||<|]|>||<|+|>|f_|<|4|>||<|[|>|\mathit|<|y|>||<|]|>|, etc
We know already that the rate of progression of atherosclerosis is related to plasma concentrations of LDL and the quantitative relationship can be expressed by a coefficient, f1. (This coefficient may be complex; we do not want to imply simple linearity.) If the oxidative modification hypothesis is correct, the rate of progression of the disease is also related through another coefficient, f2, to the rate at which LDL undergoes oxidative modification, presumably within the arterial wall, although it may be undergoing oxidation at other sites as well. We can see that at any given concentration of LDL, the rate of progression of the disease may be fast or slow, depending on the magnitude of the second term of the equation. We have all seen patients with the same degree of hypercholesterolemia but with very different clinical courses; to some extent this may be accounted for by differences in the rate at which different patients oxidized LDL. Conversely, patients with very different LDL concentrations can come to the catheterization laboratory with similar degrees of atherosclerosis. That could in theory be a reflection of compensating differences in the rates of LDL oxidation−of balancing differences in the magnitude of the second term. Thus, as we move “beyond cholesterol” we may begin to explain some of the “paradoxical” clinical observations with respect to the correlation between plasma LDL concentration and rate of progression of atherosclerosis.
If the hypothesis formulated above is correct, there are potentially exciting implications for prevention of atherosclerosis. A combination of treatment by lowering cholesterol levels and treatment by inhibition of LDL oxidation might be additive or even synergistic. The equation shown above includes a number of additional terms representing additional components in the atherogenic process that will almost certainly be defined and may someday be susceptible to intervention. We can already identify a number of potential candidates to occupy places in that equation. Some of these are shown in Fig 3⇓. For example, Cybulsky and Gimbrone48 have identified vascular cell adhesion molecule-1 (VCAM-1) as an endothelial cell adhesion molecule to which monocytes adhere, and expression of VCAM-1 antecedes appearance of macrophages in lesions of cholesterol-fed rabbits.49 If it were possible to prevent the expression of VCAM-1 (or its function), would that limit the rate of progression of fatty streaks? As another example, MCP-1 and M-CSF are believed to play important roles in the recruitment of monocytes and in their differentiation into tissue macrophages.33 34 Again, intervention to prevent the secretion of or to inhibit the function of these cytokines might slow the progression of atherosclerosis at any given level of LDL or at any given rate of LDL oxidation. Many more examples could be given. Suffice it to say that we are moving very rapidly well beyond cholesterol.
Macrophage Receptors for OxLDL
Fig 4⇓ lists macrophage membrane proteins that have been implicated as possibly playing a role as OxLDL receptors. We will briefly discuss the evidence for their role.
The fact that acetyl LDL could inhibit the binding and uptake of OxLDL by macrophages implicated the acetyl LDL receptor as one receptor involved in OxLDL uptake.16 However, it was equally clear that uptake by this receptor could not account for all of the uptake, and this was confirmed by later studies.50 51 The acetyl LDL receptor was cloned by Kodama et al52 in the laboratory of Dr Monty Krieger, and transfected cells expressing the acetyl LDL receptor were shown to bind OxLDL specifically and to mediate its internalization.53 Thus, there is no doubt that the acetyl LDL receptor (scavenger receptor A) is one of the players involved.
Using expression cloning in Xenopus oocytes, Endemann and coworkers54 cloned a new OxLDL-binding protein, namely, the mouse homologue of human CD36. Transfected cells expressing CD36 bind and take up OxLDL.55 An estimate of the quantitative importance of this receptor comes from studies comparing monocyte/macrophages from patients totally lacking CD36 with monocyte/macrophages from normal subjects.56 The CD36-defective cells bound and took up OxLDL at only about half the rate seen in the wild-type cells. Thus, CD36 is also involved at some level.
The Fc receptor was fished out of a mouse macrophage library as an OxLDL-binding protein using expression cloning.57 However, it does not appear to make any significant contribution to OxLDL uptake by resident mouse peritoneal macrophages.54
Studies by Ramprasad et al58 have identified another OxLDL-binding protein in mouse macrophages−macro-sialin−which is the homologue of human CD68. Macrosialin was originally cloned by Smith and Koch59 on the basis of its recognition by a monoclonal antibody, FA/11. The protein was later characterized by Rabinowitz et al60 61 as a predominantly intracellular protein found in the late endosomal fraction. Only a very small proportion of the total cellular macrosialin in mouse peritoneal macrophages was found on the plasma membrane. Its biological function has not been established. It is a heavily glycosylated protein (only <50% of the mass of the mature protein is accounted for by the polypeptide backbone) and its structure places it in the family of so-called lamp proteins (lysosomal-associated membrane proteins).62 Recent studies in our laboratory62A show that only a very small percentage of the total cell macrosialin is on the surface of resident mouse peritoneal macrophages in agreement with the findings of Rabinowitz et al.61 However, significant surface expression of macrosialin was found on the cell surface of thioglycollate-elicited macrophages and of CD68 on the plasma membrane of a human monocyte/macrophage cell line (THP-1 cells) stimulated by phorbol myristic acetate. Using fluorescence-activated cell sorter analysis and monoclonal antibodies directed against CD68, it was shown that CD68 plays a functional role as an OxLDL receptor. The monoclonal antibodies inhibited 37° uptake and degradation of OxLDL by THP-1 cells by 30% to 50%.
Thus, it appears that at least three different macrophage receptors can be involved in the binding and uptake of OxLDL. How important each of them may be under in vivo conditions remains to be established. The relative levels of expression on macrophages in the atherosclerotic lesion are not known. The environment in the lesion is obviously rather different from that in the peritoneal cavity of a mouse or in a transformed cell line in culture. Even different areas within a given lesion may show large differences in patterns of cytokines and growth factor expression. We shall have to defer judgment on the relative importance of these several receptors for OxLDL until methods for assessing their function in vivo are developed or suitable gene targeting studies can be done.
What is the Essential Function of OxLDL Receptors That Accounts for Their Persistence Through Evolution?
This question has been much discussed in our laboratory and probably in all the laboratories that have interested themselves in OxLDL. At this point we are not really certain whether receptors for OxLDL stimulate the progression of lesions by helping generate foam cells or whether they possibly inhibit lesion formation by getting rid of a potentially cytotoxic substance−OxLDL. In either case, however, one cannot explain the evolutionary survival of these receptors on the basis of what they do or do not do in the atherosclerotic process. Atherosclerosis is basically a human disease, almost unheard of in animals in the wild. Moreover, because atherosclerosis begins to impair function only late in life−after procreation−one would not expect any genetic pressure either for or against processes involved in atherogenesis. Faced with this dilemma, we asked ourselves whether there might be something that shares properties with OxLDL that might account for the evolutionary survival of these receptors. In collaboration with Dr Gilbert Sambrano and Dr Sampath Parthasarathy, we tested the hypothesis that an oxidatively damaged plasma membrane might resemble an oxidatively damaged LDL.63 We chose the simplest cell model for these studies, the red blood cell, subjected it to oxidative modification by incubation with copper and ascorbic acid, and then asked whether the damaged red cell would bind to OxLDL receptors. As shown in Fig 5⇓, washed native red cells did not bind to any significant extent to freshly plated mouse peritoneal macrophages. On the other hand, oxidatively damaged red cells bound avidly. In the presence of OxLDL, however, almost all of this binding of oxidized red cells was prevented. Interestingly, acetyl LDL did not inhibit the binding at all, even at very high concentrations, nor did native LDL. Several other lines of evidence supported the interpretation that OxLDL and oxidatively damaged red cells were binding to the same receptor or receptors. In similar fashion we studied the binding of apoptotic thymocytes to macrophages and found that this also was inhibited by OxLDL but not by native LDL nor acetyl LDL.64 The inhibition in this case was less striking but highly significant−about 50%. Studies by Fadok et al65 and by Savill et al66 have previously shown that the binding of apoptotic cells is in part attributable to CD36 and, as discussed above, we now know that CD36 is able also to bind OxLDL.54 Whether or not other OxLDL receptors are involved in the binding of apoptotic cells remains to be established.
From all of these observations there seems to emerge a generalization, namely that something about the structure of OxLDL makes it a ligand for scavenger receptors whose evolutionary raison d'etre may be their function in the recognition and clearance of damaged and dying cells. What are the possible structural commonalities between OxLDL and apoptotic cells? We started our studies of the binding of oxidized red cells because we analogized between lipid-protein alterations in an LDL particle and lipid-protein alterations in a damaged cell membrane. Cell necrosis is commonly associated with an increase in free radical production and with oxidative damage; there is evidence to suggest that oxidative damage is a component of the apoptotic program.67 Oxidatively damaged red cells bind to macrophages in part because they express an excess of phosphatidyl serine (PS) on the outer leaflet of their plasma membrane.68 69 It has been suggested that this results from damage to the aminophospholipid translocase located in the plasma membrane.70 This ATP-dependent enzyme normally maintains asymmetry of the membrane with respect to PS, but when it ceases to function, the PS distributes symmetrically between the inner and outer leaflets, thus increasing markedly the expression on the outer leaflet. LDL actually contains only very small amounts of PS; the predominant phospholipid is phosphatidylcholine. However, oxidation of the sn-2 fatty acids in phosphatidylcholine converts it to a more polar form and conceivably this more polar form could play a role like that of phosphatidylserine with respect to receptor recognition. Oxidation of LDL (or of a plasma membrane) is exceedingly complex, involving oxidation of all classes of lipids and of the protein moieties. In the course of oxidation, many shorter chain aldehydes and ketones are generated that can covalently link to the protein, leading to intramolecular and intermolecular cross-linking, and the lipids can interact and covalently bond to each other. Much remains to be learned before we can identify the chemical structures responsible for the apparent similarities of OxLDL and oxidatively damaged cells with respect to macrophage receptor recognition.
The Role of Macrophages and Scavenger Receptors in the Late Lesion
What can we say about the role of these scavenger receptors in the evolution of the atherosclerotic lesion? Certainly they relate to the development of foam cells in the early fatty streak lesion. What about their role in the later evolution of the lesion? The recent work of Davies12 clearly demonstrates that the fatal terminal coronary thrombosis occurs most often overlying lesions that have a large liquid lipid core. Lesions that are primarily fibrotic, while they may cause very serious stenosis, are very seldom the site of thrombosis. The lesions with a large lipid core can have only a very thin fibrous cap overlying that lipid core and it is often the rupture of that fibrous cap that triggers thrombosis by exposing blood to the procoagulant activities inside the lesion. It has been proposed that the weakening and ultimate rupture of that fibrous cap may be the result of proteases released by activated macrophages in the shoulder of the lesion.11
What is it that determines whether a lesion evolves as a primarily fibrotic lesion or as a primarily necrotic lesion with a large lipid core? We know from recent studies by Bennett and coworkers71 and by Geng and Libby72 that cells in lesions do indeed undergo apoptosis. As suggested in Fig 6⇓, if the apoptotic cells are mostly cleared by phagocytosis before damage to the plasma membrane causes the cell contents to leak, a fibrotic lesion may develop. On the other hand, if a foam cell becomes necrotic (either because adjacent macrophages fail to engulf it or because its apoptotic program is defective), there will be a progressive accumulation of lipid in the extracellular space and the lesion may evolve to be the unstable, thrombosis-prone type of lesion. Thus, the cell-scavenging function of macrophage receptors may be playing a role both in the initiation of the fatty streak (generation of foam cells) and in the evolution of the lesion that determines whether it will be stable or unstable.
Will Antioxidants Work in Humans?
It would be unwise to extrapolate from the experimental and animal model evidence, however strong, and assume that antioxidants will work in the same way against the human disease. What can we say about the probabilities? Several points would seem to justify the extrapolation:
(1) Because antioxidants have been shown to be effective in several different animal models and in models characterized by different patterns of hyperlipoproteinemia, it is not unreasonable to assume they will work in humans. It seems unlikely that the basic pathobiology of the vessel wall is qualitatively different in the human disease. If oxidative modification is an important element in the pathogenesis in animals, it is likely to be also in humans.
(2) Oxidative modification of LDL certainly occurs in humans, as we have discussed above. It is particularly significant that OxLDL has been demonstrated in human atherosclerotic lesions.
(3) A number of epidemiological studies have shown an association between high dietary intake or high plasma levels of some of the antioxidant vitamins and lower risks of clinical CHD.
(4) A number of recent studies suggest that levels of lipoperoxides in the plasma, autoantibody titers against OxLDL, or measures of susceptibility of LDL to oxidation ex vivo correlate with either rate of progression or CHD or other risk factors for CHD.
On the other hand, the extrapolation may not be warranted for the following reasons:
(1) While the human disease may be qualitatively similar to the disease in animal models, it may differ quantitatively. Specifically, the rate at which the disease develops in animal models is so much greater than the rate at which it develops in humans that the relative contribution of LDL oxidation to the process may be very different in the two.
(2) Almost all of the published animal studies involve only the very earliest stages of atherosclerosis, so all we can say is that the antioxidants can inhibit progression of fatty streaks. Fatty streaks are benign lesions in humans. Of course, if we inhibit progression of fatty streaks, we should postpone the day when they become clinically significant. Note that this implies that the canonical 5-year clinical study may be insufficient even though it is adequate in the case of cholesterol-lowering drugs.
(3) The studies in experimental animals have involved the use of potent antioxidants at high dosages. It is possible that some threshold level of protection is needed before antioxidants can exert an antiatherogenic effect. That threshold could conceivably be higher in humans than in experimental animals. At this time we don't know whether the natural antioxidants such as vitamin E are going to be sufficiently potent to mimic what we have seen in experimental animals.
The answer will depend ultimately on carefully planned, large-scale, double-blind clinical intervention trials. One such trial has been reported, testing whether probucol might affect the progression of femoral atherosclerosis.73 Probucol is both a cholesterol-lowering agent and a powerful antioxidant. Despite a decrease in total cholesterol and in LDL cholesterol (with a rise in HDL cholesterol), the progression of femoral lesions was not significantly altered. Studies of the potential effects of beta carotene on cancer have been reported in which cardiovascular events were also recorded, and these studies have shown no beneficial effects on either.74 75 75A It is important to note that beta carotene is actually not a highly effective free radical scavenging antioxidant. In fact, beta carotene, when given in even very large doses to humans, fails to confer on circulating LDL any significant protection against ex vivo oxidative modification.76
The Finnish α-tocopherol, beta carotene study, again undertaken to test for effects on cancer, also involved a cohort of cigarette smokers given supplements of vitamin E. Vitamin E affected neither cancer incidence nor cardiovascular event rates.74 However, only 50 mg of vitamin E was given daily and such low doses, in controlled studies with normal humans or hypercholesterolemic humans, have failed to confer significant protection of LDL against ex vivo oxidation.76 77
After this lecture was presented, the first large, double-blind study specifically designed to test with adequate doses of vitamin E for effects on coronary events was reported.78 The Cambridge Heart Antioxidant Study (CHAOS) involved randomization of more than 2002 patients with angiographically proven CHD either to placebo or to vitamin E (400 to 800 IU/d). There was a statistically significant and very large decrease in nonfatal myocardial infarctions (77%) and a significant decrease in the sum of nonfatal myocardial infarction and cardiovascular deaths (47%; P=.005). These remarkable effects were obtained even though the median follow-up was only 17 months and the maximal follow-up only 32 months. Clearly, these patients had very advanced disease, and the magnitude of the effect strongly suggests an effect on lesion rupture or thrombotic tendency or both. At this point we simply cannot say with certainty whether the antioxidant effects of vitamin E contributed to the decrease in morbidity and mortality. It could, in principle, by inhibiting macrophage activity at the shoulders of lesions,53 as discussed above.
Where Do We Stand?
Despite 15 years of intensive research, many questions remain unanswered regarding OxLDL and its implications.
At a fundamental level, we are not yet able to say where in the body oxidation of LDL occurs or at what rate. We are reasonably sure it occurs in the artery wall, but it probably occurs also at any sites of inflammation where LDL is exposed to activated macrophages and to other cells that have the ability to oxidize it, such as neutrophils. Nor are we able to say with certainty which of the several enzyme systems in cells that could in theory contribute to LDL oxidation actually do so either in vitro or in vivo. While we have made reasonable progress in describing the events triggered during oxidation of LDL, we are far from being able to describe the process in detail. Consequently, we do not know exactly which properties of LDL are the most important in determining its susceptibility to oxidative modification and in evoking its proatherogenic properties.
At the level of experimental animal studies, we are reasonably sure that oxidation of LDL is a component of the early pathogenesis of the lesion (although some may legitimately challenge even that). Yet we have woefully little information about quantitative aspects. Is the susceptibility of LDL to oxidation ex vivo really a reliable predictor of the rate of development of lesions? If so, what is the quantitative relationship? Are there any more instructive markers that would allow us to predict antiatherogenic potential from a knowledge of antioxidant potential measured in any appropriate system ex vivo?
At the clinical level, we are just beginning to collect relevant intervention data. Vitamin E at doses of 400 to 800 IU/d confers very good protection of LDL against ex vivo oxidative modification,76 77 and vitamin E worked in the CHAOS trial.78 But how are we going to settle the question of whether the protective effect against clinical events was actually due to protection of the LDL against oxidative modification? Vitamin E certainly has additional biological effects, including effects at the intracellular level. Just as the “cholesterol hypothesis” was only accepted after lipid-lowering trials with a number of different agents−both diets and drugs−were completed did the medical community accept a cause-and-effect relationship between plasma cholesterol levels and clinical atherosclerosis. Now the evidence is sufficient that the Food and Drug Administration will accept a safe drug that lowers plasma LDL levels without necessarily requiring a demonstration of effectiveness in reducing clinical events.
The “cholesterol controversy” lasted for at least 50 years before we were all persuaded that plasma cholesterol levels were an important factor in CHD. Elsewhere I have tried to analyze the history of that controversy to see if it might inform us with regard to future controversies of a similar nature.79 I would not be surprised if the “antioxidant controversy” were to last at least as long and require at least as many studies before it is ultimately resolved one way or the other. Meanwhile, while waiting for the final verdict to come in, we should continue to focus on treating all of the relevant risk factors, including hypercholesterolemia. Lowering plasma LDL levels will presumably also lower levels of OxLDL.
The author acknowledges his indebtedness to colleagues, fellows, students, and staff at the UCSD Specialized Center of Research on Arteriosclerosis. Working with these wonderful people over the past 25 years has been exciting and rewarding. Let me particularly thank the current major SCOR participants: Drs Joseph L. Witztum, Peter Reaven, Wulf Palinski, Christopher K. Glass, Oswald Quehenberger, and John C. Khoo.
Presented in part at the Lewis A. Conner Memorial Lecture, 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13, 1995.
- Copyright © 1997 by American Heart Association
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Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
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Sparrow CP, Doebber TW, Olszewski J, Wu MS, Ventre J, Stevens KA, Chao YS. Low density lipoprotein is protected from oxidation and the progression of atherosclerosis is slowed in cholesterol-fed rabbits by the antioxidant N,N′-diphenyl-phenylenediamine. J Clin Invest. 1992;89:1885-1891.
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Sasahara M, Raines EW, Chait A, Carew TE, Steinberg D, Wahl PW, Ross R. Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol, I: is the extent of atherosclerosis related to resistance of LDL to oxidation? J Clin Invest. 1994;94:155-164.
- Atherogenesis in Outline
- Origins of the LDL Oxidation Hypothesis
- Proatherogenic Properties of OxLDL
- Evidence That Oxidation of LDL Occurs In Vivo
- Clinical Implications
- The Role of Macrophages and Scavenger Receptors in the Late Lesion
- Will Antioxidants Work in Humans?
- Where Do We Stand?
- Figures & Tables
- Info & Metrics