The Oxidative Modification Hypothesis of Atherosclerosis

BRIEF REVIEWS The Oxidative Modification Hypothesis of Atherosclerosis: Does It Hold for Humans? Joseph L. Witztum* and Daniel Steinberg

This review suggests that oxidation of LDL is an important, if not obligatory, event in atherogenesis. The important clinical corollary is that inhibition of oxidation can inhibit atherosclerosis independent of lowering plasma cholesterol levels. This article surveys the extensive data supporting the presence of oxidized LDL in vivo in animal models; the many studies demonstrating that inhibition of oxidation by pharmacologic and/or genetic manipulations retards atherogenesis; the data in humans that supports a role for oxidation of LDL; and the results of intervention trials with antioxidant vitamins. Limitations of these trials that may have led to inconclusive results to date are discussed, and what this may mean for the oxidation hypothesis. The oxidation hypothesis is still viable, but a great deal needs to be learned in order to design the appropriate clinical trials to properly test the importance of oxidation in the pathogenesis of atherosclerosis in humans. (Trends Cardiovasc Med 2001; 11:93–102). © 2001, Elsevier Science Inc.

Cardiovascular disease (CVD) is clearly the major cause of morbidity and mortality in the Western world. While it is a complex and multifactoral disease, there can be no doubt now that elevated plasma

Joseph L. Witztum and Daniel Steinberg are from the Department of Medicine, Division of Endocrinology and Metabolism, and the Specialized Center of Research in Molecular Medicine and Atherosclerosis, University of California, San Diego School of Medicine, La Jolla, California. *Address correspondence to: Joseph L. Witztum, University of California, San Diego, Dept. of Medicine–0682, 9500 Gilman Drive, La Jolla, CA 92093-0682, USA. Tel.: 858-534-4347; fax: 858-534-2005; e-mail: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter

TCM Vol. 11, No. 3/4, 2001

cholesterol levels play a dominant role. Indeed, one can still not generate atherosclerotic lesions in any animal model without somehow raising plasma cholesterol levels. In human populations, it is exceedingly rare to have clinically significant lesions if plasma cholesterol levels are below 150 mg/dl. However, at any plasma cholesterol above this, there appears to be an increasing risk of macrovascular disease and it is likely that a variety of different genetic and environmental effects then come into play, including factors both pro- and antiatherogenic. Multiple clinical trials have established the efficacy of lowering cholesterol for both primary and secondary prevention of CVD (Steinberg and Gotto 1999). Indeed, the principle is now so

well established that we use the surrogate end-point of lowering cholesterol to judge the efficacy of new hypocholesterolemic agents. The important issues that are being determined in current clinical trials are to define the optimal degree of cholesterol lowering and appropriate target goals for various populations. For example, it is clear that diabetic populations are at increased risk, and the importance of lowering their low-density lipoprotein (LDL) cholesterol levels is widely recognized. It should be appreciated that it was the availability of simple methods for assessing this risk factor (e.g., plasma total and LDL cholesterol levels), and the availability of highly effective hypolipidemic agents that brought us to this present state. As discussed later, this is in sharp contrast to the situation with testing the role of antioxidants in humans, in which we have neither established surrogate markers to identify individuals or populations potentially at risk for CVD because of an enhanced “oxidative stress,” nor do we have effective antioxidant therapy. The dramatic success of cholesterollowering therapy suggests that this should be sufficient to cause regression of established disease and/or halt its progression. Conceivably this might be true if plasma cholesterol levels had always been, or were chronically reduced, to below 150 mg/dl. Nevertheless, there is great variability in the expression of clinical disease in individuals with values above this, which includes the vast population of the Western world. Therefore, it is important to define those factors, both genetic and environmental, that influence both the development of atherosclerosis and the response to therapy. Atherogenesis is a chronic inflammatory process that involves a complex interplay of circulating cellular and blood elements with the cells of the artery wall (Ross 1999, Steinberg and Witztum 1990). While many factors are involved, the “oxidation hypothesis” has been a cen-

93

Table 1. Potential mechanisms by which oxidized forms of LDL may influence atherogenesis • OxLDL has enhanced uptake by macrophages leading to foam cell formation. • Products of OxLDL are chemotactic for monocytes and T-cells and inhibit the motility of tissue macrophages. • Products of OxLDL are cytotoxic, in part due to oxidized sterols, and can induce apoptosis. • OxLDL, or products, are mitogenic for smooth muscle cells and macrophages. • OxLDL, or products, can alter gene expression of vascular cells, e.g. induction of MCP-1, colony-stimulating factors, IL-1 and expression of adhesion molecules. • OxLDL, or products, can increase expression of macrophage scavenger receptors, thereby enhancing its own uptake. • OxLDL, or products, can induce proinflammatory genes, e.g. hemoxygenase, SAA and ceruloplasmin. • OxLDL can induce expression and activate PPARg, thereby influencing the expression of many genes. • OxLDL is immunogenic and elicits autoantibody formation and activated T-cells. • Oxidation renders LDL more susceptible to aggregation, which independently leads to enhanced uptake. Similarly, OxLDL is a better substrate for sphyingomyelinase, which also aggregates LDL. • OxLDL may enhance procoagulant pathways, e.g. by induction of tissue factor and platelet aggregation. • Products of OxLDL can aversely impact arterial vasomotor properties. Modified from Steinberg and Witztum (1999).

tral focus of investigation into the pathogenesis of the atherosclerotic process for almost 20 years. This hypothesis states that the oxidative modification of LDL, or other lipoproteins, is central, if not obligatory to the atherogenic process. The important corollary is that inhibition of such oxidation should reduce the progression of atherosclerosis, independent of reduction of other risk factors, such as elevated LDL levels (Chisolm and Steinberg 2000, Steinberg et al. 1989, Witztum 1994). The original interest in oxidized LDL (OxLDL) stemmed from two basic sets of observations. The first was that OxLDL was cytotoxic to endothelial and other cells and thus could directly cause damage to arterial cells (Hessler et al. 1983). The second observation was that uptake of native LDL by macrophages occurred at a sufficiently low rate to prevent foam cell formation, but uptake of OxLDL was unregulated and led to macrophage foam cell formation (Heinecke et al. 1984, Steinbrecher et al. 1984). However, it is now abundantly clear that OxLDL, with its many oxidatively modified lipids and degradation products, contributes to the pathophysiology of both the initiation and progression of the atherosclerotic lesion by many mechanisms, including its

94

proinflammatory, immunogenic and cytotoxic properties (see Table 1 and reviewed in Navab et al. 1996, Steinberg and Witztum 1999, Tsimikas and Witztum 2000). Products of OxLDL contribute to monocyte and T-cell recruitment, directly or indirectly via induction of chemokines and endothelial cell adhesion molecules. They alter gene expression of vascular cells leading to growth factor and cytokine stimulation and they are cytotoxic. Because oxidation of LDL generates many “neo-self determinants” that induce an active immune response (Hörkkö et al. 2000), there is in turn both a humoral and cellular response that affects the progression of the atherosclerotic lesion in complex ways (Libby et al. 1999). Indeed, the notion that oxidation of LDL is a driving force in atherogenesis fits in nicely with the widely held notion that atherosclerosis is a chronic inflammatory process. In this article we review the evidence supporting the oxidation hypothesis and then discuss the issue of whether or not it holds for human atherosclerosis. • Evidence for the Presence of OxLDL in vivo There is now a large body of experimental work in animal models that strongly

supports an important role for oxidative modification of LDL. This topic has been the subject of numerous reviews (Chisolm and Steinberg 2000, Heinecke 1997, Navab et al. 1996, Steinberg and Witztum 1999, Tsimikas and Witztum 2000). We will not review all of these in detail again here. However, over the past several years several new lines of evidence and important studies have appeared that have continued to add to the evidence supporting the oxidation hypothesis. The different lines of evidence are discussed in brief. Oxidation of LDL results in the generation of a variety of modifications to the lipid and protein moieties, including the covalent modification of apoB with reactive products of the decomposition of oxidized lipids, yielding malondialdehyde-, and 4-hydroxynonenal-lysine adducts, for example (Esterbauer et al. 1990). In addition, the residual oxidized phospholipid containing aldehyde terminated fatty acids, can also form adducts with lysine residues of apoB, as well as adducts with the amino head groups of phospholipids, such as phosphatidylethanolamine (Hörkkö et al. 1999). These and presumably many other such changes generate immunogenic neo-epitopes on the modified LDL. Using antibodies to such epitopes it is possible to show extensive immunostaining of essentially all atherosclerotic tissue in experimental animals and humans. LDL has been extracted from atherosclerotic tissues of both rabbits and humans and has been shown to exhibit all of the physical, immunological and biological properties of LDL oxidized in vitro (Ylä-Herttuala et al. 1989). A variety of oxidized lipids have been shown to be present in atherosclerotic plaques in animals and humans (Hulten et al. 1996, Praticò et al. 1997). Of great interest, oxidized lipids, such as HETEs, were significantly elevated in unstable atherosclerotic plaques obtained from endarterectomy samples (Mallat et al. 1999). Similarly, such oxidized lipids are also present in human plasma (Bjorkhem et al. 1999, Praticò et al. 1998). There is an increasing number of studies demonstrating that very mildly oxidized LDL can be demonstrated in plasma of animals and humans, using both physical (Sevanian et al. 1996) and immunological techniques. For example, several authors, employing antibodies TCM Vol. 11, No. 3/4, 2001

binding to oxidation-specific epitopes, have shown that minimally modified LDL (mmLDL) can be demonstrated in plasma, and levels seem to be elevated in subjects with manifestations of acute or chronic CVD (Holvoet et al. 1998, Itabe et al. 1996, Palinski et al. 1996). We have recently demonstrated that when human LDL was injected into apoE-deficient mice it progressively acquired oxidized phospholipid (OxPL) epitopes as it circulated. When the same LDL was injected into apoE-deficient mice that were also paraoxonase (PON) deficient, the rate of acquisition of OxPL epitopes was greater, consistent with a decreased rate of degradation of OxPL, presumably secondary to loss of PON activity, which degrades OxPL moieties (Shih et al. 2000). These data demonstrate not only that mmLDL is present in the circulation, but that it appears to accumulate at a greater rate in animals or subjects with increased rates of atherogenesis. OxLDL is immunogenic and this leads to autoantibody formation. We originally demonstrated that there were autoantibodies to epitopes of OxLDL in the plasma of animals and humans (Palinski et al. 1989). We and others have demonstrated an increased titer of such antibodies in animals with atherosclerosis and shown that they reflect the extent of lesion formation (Palinski et al. 1995, Tsimikas et al. 2001). Furthermore, such autoantibodies are found in plaques, in part complexed with OxLDL (Ylä-Herttuala et al. 1994). One of the reasons that there has been so much interest in OxLDL is that it has unregulated uptake by macrophages, leading to cholesteryl ester accumulation and foam cell formation. This uptake occurs by way of macrophage scavenger receptors, such as the SRAs and CD36. Wild-type apoE-deficient mice develop severe atherosclerosis. In contrast, apoE-deficient mice with gene targeted deletion of either the SRA (Suzuki et al. 1997) or CD36 (Febbraio et al. 2000) have a marked reduction in the extent of lesion development. While it is possible that there are other explanations for the inhibition of atherogenesis, these data are consistent with the more likely possibility that the beneficial effect of scavenger receptor deletion is due to decreased uptake of OxLDL and diminished foam cell formation. There are many postulated mechaTCM Vol. 11, No. 3/4, 2001

nisms by which LDL could become oxidized within the artery wall. One mechanism that has now gained strong support is that the enzyme 12/15-lipoxygenase (LO) initiates the “seeding” of LDL with hydroperoxides, leading to the subsequent initiation of lipid peroxidation. The resultant changes render the OxLDL proinflammatory and lead to its subsequent enhanced uptake by macrophages. Evidence to support this hypothesis includes the observations that incubation of LDL with isolated soybean LO leads to oxidation of LDL (Sparrow et al. 1988); that inhibitors of macrophage 12/15-LO decrease the ability of macrophages to initiate oxidation of LDL (Rankin et al. 1991); and that LDL incubated with fibroblasts transfected with LO become “seeded” with fatty acid hydroperoxides, which can then propagate lipid peroxidation under the proper conditions (Benz et al. 1995, Ezaki et al. 1995). Both mRNA and protein of 15-LO (the homologous enzyme in rabbits and humans) are found in atherosclerotic lesions of rabbits and humans, but not in normal arteries (YläHerttuala et al. 1990). Moreover, stereospecific products of the LO reaction can be found in lesions, consistent with enzymatic oxidation (Folcik et al. 1995, Kuhn et al. 1997). Treatment of hypercholesterolemic rabbits with specific inhibitors of 15-LO reduces the progression of atherosclerosis (Bocan et al. 1998, Sendobry et al. 1997). Recent studies from Colin Funk’s laboratory showed that crossing 12/15-LO-deficient mice into apoE-deficient mice caused a dramatically reduced extent of early lesions (Cyrus et al. 1999). In a further study, this protective effect has been shown to persist even at 15 months of age. Plasma levels of F2-isoprostanes, nonenzymatic breadown products resulting from lipid peroxidation of arachidonic acid, were highly correlated with the extent of lesion formation, and autoantibodies to epitopes of OxLDL were also strongly correlated both to lesion area as well as to isoprostane levels (Cyrus et al. 2001). Although it is possible that LO affected atherogenesis by other mechanisms, these studies lend strong support to the concept that a major mechanism by which LO deficiency decreased atherosclerosis was by decreasing the extent of lipid peroxidation, and specifically, the generation of OxLDL. Furthermore,

overexpression of 15-LO in endothelium led to an enhancement of atherosclerosis in LDLReceptor(R)-negative mice (Harats et al. 2000). However, in contrast is a report that macrophage-specific overexpression of 15-LO led to protection against atherosclerosis in cholesterol-fed rabbits. The 12/15-LO deletion was global, while the studies with 15-LO overexpresssion were tissue specific. Whether this explains the difference in the latter two studies is unclear, but suggests that many questions still remain to be answered regarding the role of LO. Of course, it is likely that in vivo there are many mechanisms, beside LO, by which LDL is oxidized within the artery wall (Heinecke 1997). It will be very important to determine other potential mechanisms that could be important in murine or other animal models. For any such mechanism found, it will be important, of course, to demonstrate that it is also relevant in human atherogenesis. It is likely that the ultimate design of safe and effective therapy to prevent LDL oxidation will require such detailed knowledge. Finally, the most compelling data in support of the oxidation hypothesis is the direct demonstration that treatment of hypercholesterolemic animal models with a variety of antioxidants leads to the suppression of atherogenesis. While not all such studies have shown a protective benefit, the vast majority have. Table 2 lists many of these studies including studies in rabbits, hamsters, mice and non-human primates. Most of the successful studies have used powerful synthetic antioxidants, such as probucol or probucol analogues. However probucol did not protect murine models in the initial studies reported (see Table 2) and even seemed to increase extent of lesion formation, suggesting some sort of toxicity peculiar to the mouse. Interestingly, a recent report suggested that probucol had protective effects in the proximal parts of the arterial tree in murine models, even as it exaggerated lesions in the distal aorta (Witting et al. 2000). While it is possible that all of these agents are protective by mechanisms other than their antioxidant properties, the fact that such different compounds have been successful argues against this. Recently Praticò et al. (1998) demonstrated that feeding vitamin E to apoEdeficient mice diminished atherosclerosis and in parallel diminished the aortic,

95

Table 2. Effects of antioxidants in animal models of atherosclerosis Type of study

Reference

Probucol in LDLR2/2 rabbits

Carew et al. (1987) Kita et al. (1987) Mao et al. (1991) Daugherty et al. (1991) Fruebis et al. (1994) Witting et al. (1999a) Mao et al. (1991) Fruebis et al. (1994) Witting et al. (1999a) Stein et al. (1989) Daugherty et al. (1989) Prasad et al. (1994) Shaish et al. (1995)

1 1 1 6 1 1 1 2 2 2 1 1 1

Sparrow et al. (1992) Bjorkhem et al. (1991) Mantha et al. (1993) Morel et al. (1994) Kleinveld et al. (1995) Shaish et al. (1995) Fruebis et al. (1996)

1 1 1 2 2 2 2

Parker et al. (1995) Parker et al. (1995) Tangirala et al. (1995) Zhang et al. (1997) Bird et al. (1998) Cynshi et al. (1998) Witting et al. (2000) Cynshi et al. (1998)

1 1 1 2* 2* 2* 1/2* 1 1

Probucol analogs in LDLR2/2 rabbits

Probucol in cholesterol-fed rabbits

Other antioxidants in rabbits DPPD BHT Vitamin E

Antioxidants in rodents Probucol in hamsters Vitamin E in hamsters DPPD in apoE2/2 mice Probucol in apoE2/2 mice Probucol in LDLR2/2 mice Probucol in LDLR2/2 mice Probucol in apoE2/2 mice Probucol analog in LDLR2/2 Probucol metabolite in LDLR2/2 /apoE2/2 Vitamin E in apoE2/2 Dietary antioxidants in LDLR2/2 Antioxidants in nonhuman primates Probucol Vitamin E

Witting et al. (1999) Praticò et al. (1998) Crawford et al. (1998) Sasahara et al. (1994) Verlangieri and Bush (1992)

Result

1 1 1 6

Modified from Steinberg and Witztum (1999). 1 5 positive study (atherosclerosis decreased); 2 5 negative study (atherosclerosis unchanged); 6 5 atherosclerosis equivocal; 2* 5 atherosclerosis enhanced.

plasma and urinary content of F2-isoprostanes. Importantly, there was an inverse correlation between the amount of vitamin E in plasma and the extent of lesion formation in various arterial beds. Another interesting report that may be viewed as support of the oxidation hypothesis comes from the observation that delivery of human apoE3 to LDLRdeficient mice via an adenoviral technique led to a 2–4-fold elevation in plasma apoE levels and a reduction in atherosclerosis, even though plasma lipid levels were not changed. Urinary, LDL-associated, and arterial wall iPF2a– VI levels all decreased consistent with decreased oxidant stress. Although other explanations for these findings could be offered, they are consistent with in vitro

96

findings that apoE has antioxidant properties (Miyata and Smith 1996). Another line of evidence suggesting that antioxidants decrease atherosclerosis by inhibiting oxidation of LDL comes from studies of Calara et al. (1998) in which human LDL was injected into rats. Using a monoclonal antibody that exclusively recognized human apoB, it was shown that apoB appeared in rat aortas within 6 hours. Shortly thereafter appeared oxidation-specific epitopes that colocalized with the apoB. When this experiment was repeated in rats using LDL enriched with probucol, the human apoB appeared in the aorta with the same time sequence, but this time the appearance of oxidation-specific epitopes was greatly reduced. In a recent dramatic

experiment in humans, Iuliano et al. (2000) injected iodinated LDL into patients undergoing carotid endarterectomy and demonstrated the appearance of radioactivity in macrophage foam cells in the removed specimens. No accumulation of radioactivity occurred in atherosclerotic plaques after the injection of radiolabeled albumin. In several other patients, pretreated for 4 weeks with 900 mg/d of vitamin E, there was an almost complete suppression of radiolabeled LDL uptake by macrophages. It is of course recognized that such compounds are likely to have many effects that could help protect against progression of atherogenesis. Nevertheless, not only do these data support the occurrence of OxLDL in vivo, but they also suggest that antioxidants work in part by decreasing the extent of LDL oxidation. It should also be appreciated that while we have discussed the oxidation of LDL extensively, it is also likely that other apoB-100 containing lipoproteins are also oxidized, such as IDL, that may also play similar roles. In addition, oxidative modification of other structures in the artery wall, such as cell membranes (Chang et al. 1999) and matrix proteins (O’Brien et al. 1996), may also have important biological effects. In summary, in our opinion these data provide compelling evidence that OxLDL exists in vivo and that at least in hypercholesterolemic animal models, the oxidation hypothesis is highly relevant to the pathogenesis of lesion formation.

• Does the Oxidative Hypothesis of Atherosclerosis Hold for Humans? It is clear from the above that a wealth of evidence supports a major role for oxidative modification of LDL in a variety of animal species including nonhuman primates. Does it follow that the hypothesis will apply equally to the human disease? Not necessarily, but there are good reasons to believe that it may. In brief, and as reviewed elsewhere (Steinberg 2000, Steinberg and Witztum 1999): 1. The structure and cellular composition of lesions in experimental animal models resembles very closely that of human lesions. 2. The sequence of events in the evolution of atherosclerotic plaques in animals and humans is very similar. 3. Oxidized LDL has been recovered TCM Vol. 11, No. 3/4, 2001

from human atherosclerotic lesions and shown to be comparable to LDL oxidized in vitro by structural, immunological and biological properties. 4. Autoantibodies directed against oxidation-specific epitopes of OxLDL are found in the plasma of humans and also in human atherosclerotic plaques, where they are part of immune complexes. In general the titers of such autoantibodies are increased in populations at risk for CVD and in many studies have been shown to correlate with various clinical manifestations of atherosclerotic disease and to prospectively predict an increased risk for development of carotid artery disease as well as myocardial infarction (reviewed in Ylä-Herttuala 1998). 5. In some studies the susceptibility of circulating LDL to ex vivo oxidative modification has correlated with the extent of atherosclerosis or with rates of progression of atherosclerosis in human subjects. In addition, as noted above, there are now several studies using immunological techniques that demonstrate that LDL containing oxidationspecific epitopes (e.g., mmLDL) are increased in patients with CVD. 6. In a large number of epidemiologic studies, the dietary intake or plasma levels of antioxidant nutrients has correlated negatively with the risk of clinical CVD (Enstrom et al. 1992, Riemersma et al. 1991, Rimm et al. 1993, Stampfer et al. 1993) and reviewed in (Jha et al. 1995). These data, especially the strong and consistent epidemiological studies, suggest that the oxidation hypothesis should be applicable in humans as well. However, association data can only point to cause and effect and in a country such as in the United States, where a large percentage of the population consumes megadoses of vitamins and antioxidants, such data may be hard to interpret. It is necessary that appropriately designed clinical trials be conducted to test the hypothesis and provide convincing evidence.

What do the Clinical Intervention Trials Thus Far Tell Us? The decision to proceed with clinical trials of antioxidants in human atherosclerosis was made in 1991 at a workshop convened by the National Heart, Lung

TCM Vol. 11, No. 3/4, 2001

and Blood Institute (Steinberg 1992). Epidemiologists, internists, biochemists, physiologists, pathologists and others interested in atherosclerosis attended that workshop. They reviewed the totality of the data available and decided it was sufficiently impressive to warrant clinical trials. They recommended that the first trials be done with naturally occurring antioxidants, namely, vitamin E, vitamin C and beta-carotene. This recommendation was based on the premise that these naturally occurring nutrients would pose no toxicological problems, not because they had been shown to be especially effective in experimental atherosclerosis. In fact, at the time most of the reported animal studies had utilized probucol as the antioxidant. There were no studies at all utilizing beta-carotene and vitamin C in experimental animals and only a couple of studies using vitamin E, of which only one was positive (Prasad and Kalra 1993). Note also that in that report these dietary antioxidants were lumped together and dealt with as though they shared biological properties and could be used interchangeably. We now know that this is decidedly not the case. A striking example is provided by the contrast between beta-carotene and vitamin E. The former is a potent trap for singlet oxygen but much less effective in terminating free radical chain reactions. Vitamin E, on the other hand, is an excellent terminator of chain reactions. Operationally we now know that beta-carotene either added to LDL in vitro or given in large doses to raise plasma levels does not significantly protect LDL against ex vivo oxidation (Reaven et al. 1993). On the other hand, vitamin E either added in vitro or fed is moderately effective (Reaven et al. 1995). There are also large differences in the pharmacokinetics of several nutrient antioxidants. For example, vitamin C is distributed exclusively in the aqueous phase whereas vitamin E takes up residence predominantly in lipoproteins. Because we are not certain exactly where and how LDL gets oxidized in vivo it is not possible to make meaningful comparisons of these various antioxidants. We are working by trial and error. Beta-Carotene Based on the extensive epidemiologic data correlating a high dietary intake

of beta-carotene with decreased risk of cancer, several large-scale studies were undertaken to test whether supplementation with beta-carotene would decrease cancer risk. In these studies, however, cardiovascular events were also carefully monitored and recorded. In none of the studies was there any decrease in cancer nor was there any decrease in cardiovascular events (Alpha-Tocopherol and Beta-Carotene Cancer Prevention Study Group 1994, Greenberg et al. 1996, Hennekens et al. 1996, Omenn et al. 1996). The doses used were near the maximum feasible and the numbers of patients followed were more than sufficient to have detected an effect, even if small. There was actually an increase in cancer deaths in some of the studies. Most investigators were surprised by these negative results. The presumption had been that it was the beta-carotene content of a fruit and vegetable diet that offered protection but in retrospect it may be that the protection is due to a pattern of diet, rather than to any single dietary component. Additionally, and very important, the epidemiologic data presumably represent correlations with long-term or even lifetime of exposure to a given diet. It should not be surprising if such lifetime exposure is not duplicated by an intervention starting late and lasting for only the canonical 5 years. Finally, as noted above, beta-carotene was not shown to protect LDL from oxidation when tested in ex vivo assays. Vitamin E Trials Five double-blind, placebo-controlled interventional trials have now been published (Table 3). The first, the Finnish a-tocopherol-beta-carotene trial (ATBC), was primarily designed to detect a decrease in lung cancer among heavy smokers with, in many cases, previous exposure to asbestos (Alpha-Tocopherol and Beta-Carotene Cancer Prevention Study Group 1994). The dose of vitamin E, 50 mg/d, was probably too low to give optimum protection of LDL against oxidation. In any case, over a 5.3-year follow-up there was no significant effect on cancer of the lung, although, as described in a subsequent publication (Heinonen et al. 1998), there was a significant decrease in prostate cancer. There was no significant decrease in cardiovascular events.

97

Table 3. Effects of vitamin E supplementation on CVD: results of five published double-blind, placebo-controlled intervention trials Study

n

Vitamin E/day

ATBC 29,133 50 mg (synthetic) CHAOS 2,002 400 or 800 IU (natural) GISSI 11,324 300 mg (synthetic) HOPE 9,541 400 IU (natural) SPACE 196 800 IU (natural) a b

Effect on CVD Reference None 247%a None None 246%b

Alpha-Tocopherol Study (1994) Stephens et al. (1996) GISSI (1999) Yusuf et al. (2000) Boaz et al. (2000)

p 5 0.005. p 5 0.014.

The second trial was designed specifically to test the effect of vitamin E on cardiovascular events. The CHAOS trial (Cambridge Heart Antioxidant Study) randomized patients with angiographically proved CAD to either placebo or to 800 IU/d of vitamin E (first 546 patients) or 400 IU/d (next 489 patients). Mean follow-up was only 1.4 years. The primary composite end-point—CVD death or nonfatal myocardial infarction—was reduced by 47% ( p 5 0.005). With this striking decrease in the primary endpoint one would have anticipated a decrease in total cardiovascular mortality, but none was seen. There was also no decrease in total mortality, but the short duration of the trial may have precluded the opportunity to observe any such impact (Stephens et al. 1996). In the HOPE trial (Yusuf et al. 2000) patients who had advanced symptomatic CVD were given either 400 IU vitamin E daily or an angiotensin-converting enzyme inhibitor (ramapril), neither or both. The primary end-point was a composite of non-fatal infarction, stroke or death from cardiovascular disease. Ramapril conferred significant protection but vitamin E was without effect. The GISSI trial (GISSI 1999) enrolled patients who had had a myocardial infarction within the past 3 months and they were followed over 3.5 years receiving either vitamin E (300 mg/d) or omega3-polyunsaturated fatty acids (1 g/d), both or neither. There was no vitamin E effect on the composite primary endpoint. We discuss the fifth trial, the SPACE trial, below. • Why Haven’t the Clinical Trials Been More Effective in Humans? First, the antioxidants used in these clinical trials may not be sufficiently potent or

98

the doses used may be too low. As noted above, a major problem with the conduct of such clinical trials at the present time is that we do not have reliable and validated ways to identify patients who have increased rates of lipid peroxidation and importantly, we lack reliable measures to determine if there is an adequate response to a given antioxidant intervention. While vitamin E, for example, was shown to decrease atherosclerosis in hypercholesterolemic apoE-deficient mice, the level of plasma vitamin E was increased 5-fold in treated mice, and this resulted in decreased F2-isoprostane levels. In contrast, the increase in levels of vitamin E that can be achieved in humans consuming doses of 400–800 IU/d is much less. In an important study, Meagher et al. (2001) recently determined the effects in healthy individuals on several indices of lipid peroxidation of the administration of doses of vitamin E varying from 200 to 2000 IU/d over a period of 8 weeks. They measured the urinary excretion of two different isoprostane isomers, as well as urinary 4-hydroxynonenal. Compared to a baseline period they found no decreases in any of the measures of lipid peroxidation, even in subjects taking 2000 IU/d of vitamin E, which increased plasma levels of vitamin E 5-fold. These data suggest that in otherwise healthy people even megadoses of vitamin E are insufficient to decrease basal levels of lipid peroxidation. The patients in this study were all healthy. Unfortunately we do not know if any of the subjects in the clinical trials had increased indices of lipid peroxidation or fall into the category of individuals in the study of Meagher et al. However, these data strongly support the idea that quantitative measurements of oxidative stress are needed in the selection of clinical

trial subjects, and furthermore that antioxidants that can actually lower basal or elevated levels of oxidative stress are needed in order to adequately test the oxidation hypothesis (Witztum 1998). Finally it should also be mentioned that the pharmacodynamics of antioxidants in humans may be at least quantitatively and possibly even qualitatively different than in experimental animals. In that case different antioxidants and possibly different doses might be needed to see effects in humans. A second reason is that patients studied have advanced degrees of CVD. Oxidative modification of LDL may be critical in the development of the human fatty streak lesion, as it is in animals, yet may not play an important role in the evolution of the late lesions or in the events precipitating hard clinical endpoints. It should be remembered that most of the studies in experimental animal models have been done over a relatively short time span and in this time the lesions seldom develop the evidence of chronicity seen in humans, nor do they develop unstable plaques or ruptured lesions. Indeed, lesions in humans develop over a period of decades, whereas lesions in experimental animals develop over at most a period of months. It is conceivable that because of the slowness with which human lesions progress there is more time for various repair processes to correct damage done by OxLDL (e.g., paraoxonase correction of the proatherogenic properties of mmLDL or OxLDL). Third, the rate of oxidation of LDL may be much less in humans than in mice or in markedly hypercholesterolemic animals. The metabolic rate of small mammals is extremely high and therefore the rate of generation of reactive oxygen species is also high compared to that in humans. Furthermore, there is considerable evidence that hypercholesterolemia per se promotes lipid peroxidation, both by supplying the substrate, as well as by inducing cellular mechanisms that promote enhanced oxidation (Inoue et al. 1998, Ohara et al. 1993). The degree of hypercholesterolemia in most of the animal models, and particularly in the cholesterol-fed or WHHL rabbits, and in the murine models is extreme and far in excess of that seen even in subjects with familial hypercholesterolemia. This too could contribute to the TCM Vol. 11, No. 3/4, 2001

much lower rate of oxidative stress in the artery wall in humans. So, while there is certainly qualitative evidence for oxidative modification of LDL in humans, the rate of the process may be much slower than it is in small animals and/or in the extreme degrees of hypercholesterolemia generated in the animal models in which antioxidants have proven to be beneficial. While we do have data showing that nonhuman primates with only mild hypercholesterolemia can be protected by antioxidants, those data are limited (Sasahara et al. 1994, Verlangieri and Bush 1992). Further studies in nonhuman primates or swine could be informative. Fourth, it may be that only a subpopulation will benefit from antioxidant supplementation. As noted above, it may be that not everyone will benefit from antioxidant supplementation. Indeed we could make the strong argument that only individuals with evidence of increased oxidative stress, from whatever etiology, are likely to show a beneficial effect of antioxidant supplementation, especially over a short period of time. Evidence to support this hypothesis was recently reported by Boaz et al. (2000), who reported on the effects of vitamin E supplementation to patients with end-stage renal disease on hemodialysis. It is generally accepted that these patients have a high risk of CVD and are exposed to increased oxidative stress for a variety of reasons, including the fact that free radical oxygen species are generated at the surface of the membranes used in hemodialysis (reviewed in Boaz et al. 2001). In the SPACE (Secondary Prevention with Antioxidants of CVD in End Stage Renal Disease) study (Table 3), 196 patients were randomized to receive 800 IU/d of vitamin E or placebo and followed for an average of only 1.4 years. Even though only 196 patients were randomized, the number of cardiovascular end-points was high: almost 50% of these patients had a primary end-point (myocardial infarction, ischemic stroke, peripheral vascular disease or unstable angina). Events were reduced by 54% in the vitamin E group ( p 5 0.014). Myocardial infarction was reduced by 70% ( p 5 0.016). This study strongly suggests that such a high-risk population may benefit from antioxidant intervention and further trials in such populations are clearly warranted. TCM Vol. 11, No. 3/4, 2001

In future trials it will be important to obtain measures of lipid peroxidation and to attempt to demonstrate a correlation between reduction of such parameters and improvement in outcomes. In a similar way, it has been previously shown that patients with marked hypercholesterolemia, such as those with familial hypercholesterolemia, also have increased indices of lipid peroxidation (Reilly et al. 1998), and similar studies will be important. • Summary There are a number of reasons why one would expect the oxidation of LDL to be significant in human atherosclerosis and, at the same time, there are a number of points to suggest it may not be important in all individuals, at least not to the same degree as in some experimental animal models. To date all of the studies were carried out in patients with established, welladvanced CVD. The patients in ATBC probably received too low a dose. The patients in CHAOS, HOPE and GISSI were more or less comparable in severity of CVD and the dosages were similar. However, the much larger number in the latter two trials (total of 7991) compared to the limited numbers in CHAOS (approximately 1000) means that the negative results far outweigh in significance the positive results of the CHAOS trial. The reasons for the disparity in results remain unknown. In any case, additional studies with vitamin E in unselected patients of this description (elderly patients with advanced, well-established CVD, particularly those without evidence of enhanced oxidative stress) would seem unlikely to lead to any different conclusion (Steinberg 2000). The patients studied in SPACE represented a very special subset—patients at a very high risk and patients exposed to high oxidative stress during their periodic hemodialysis. The numbers are small but the magnitude of the effect and the respectable p-values demand attention. Is this quite different result due to the larger dose of vitamin E used? Is it due to the increased oxidative stress, making it easier to see the effects of an antioxidant in a short period of time in patients at high risk? Furthermore, it is intriguing that such a dramatic effect on clinical end-points was seen in such a

short period of time. This raises the possibility that the antioxidant effects of vitamin E might be working through effects on stabilization of vulnerable plaques, improvement in oxidatively induced abnormalities in vasomotor function or disturbances in coagulation and platelet function. Clearly further studies are needed. The large numbers of subjects in GISSI and in HOPE strongly suggest that vitamin E at these doses will have no effect in the “run-of-the-mill” patient with advanced CAD. Does that mean that the antioxidant hypothesis has been proved to be inapplicable to the human disease? Far from it. The hypothesis is not that vitamin E (or any particular antioxidant) will prevent hard end-points in patients with advanced CAD, but rather that oxidation of LDL plays a role in atherogenesis. It may be that vitamin E is not the right antioxidant in humans; it may be that the initiation of treatment at such a late stage in the disease will be ineffective (although the SPACE study suggests otherwise if the correct subpopulation is selected); it may be that treatment needs to be given over a longer period of time. We cannot be sure what the negative results do mean but they do not mean that the antioxidant hypothesis is dead. What needs to be done now is to go back to experimental animals and learn more about just how oxidation of LDL (or other oxidative processes) participates in atherogenesis, which systems are responsible for LDL oxidation and which antioxidants are most effective in preventing it. We know that there are antioxidant compounds more potent than the dietary antioxidants, probucol and its analogs being examples. We need to develop reliable and validated ways to identify which populations have an increased rate of lipid peroxidation and which antioxidants effectively decrease this oxidant stress. With the exception of special groups who appear to be clearly at increased risk and to have associated enhanced degrees of oxidative stress, we believe that further clinical trials should probably be deferred until we are more confident about the correct identification of such high-risk populations, what interventions to use and the correct timing of such interventions. Meanwhile, we await with interest the results of some primary prevention trials that are in progress. These trials, initiating treat-

99

ment before there is at least clinically significant CAD, may have a better chance of demonstrating a beneficial effect in otherwise low-risk individuals. In such populations we may need to design clinical trials in which we use the development of early lesions rather than clinical events as the end-point. For example, would it be possible to use intravascular ultrasound to detect early lesions and ask whether intervention decreases the number of new lesions developing? Would it be possible to improve the sensitivity of MRI or other noninvasive methods to the point that we can detect fatty streaks and fibrous plaques? Intervention at that point might demonstrate the effect of antioxidants on early lesion development and, by inference, on later events if treatment were prolonged.

Calara F, Dimayuga P, Niemann A, et al.: 1998. An animal model to study local oxidation of LDL and its biological effects in the arterial wall. Arerioscler Thromb Vasc Biol 18:884–893.

References

Chisolm GM, Steinberg D: 2000. The oxidative modification hypothesis of atherogenesis: an overview. Free Radic Biol Med 28:1815–1826.

Alpha-Tocopherol and Beta-Carotene Cancer Prevention Study Group: 1994. The effect of vitamin E and beta-carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 330:1029–1035. Benz DJ, Mol M, Ezaki M, et al.: 1995. Enhanced levels of lipoperoxides in low density lipoprotein incubated with murine fibroblast expressing high levels of human 15-lipoxygenase. J Biol Chem 270:5191– 5197. Bird DA, Tangirala RK, Fruebis J, et al.: 1998. Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice. J Lipid Res 39:1079–1090. Bjorkhem I, Diczfalusy U, Lutjohann D: 1999. Removal of cholesterol from extrahepatic sources by oxidative mechanisms. Curr Opin Lipidol 10:161–165. Bjorkhem I, Henriksson-Freyschuss A, Breuer O, et al.: 1991. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Arterioscler Thromb 11:15–22. Boaz M, Green M, Fainaru M, Smetana S: 2001. Oxidative stress and cardiovascular disease in hemodialysis. Clinical Nephrology 55:93–100. Boaz M, Smetana S, Weinstein T, et al.: 2000. Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomised placebocontrolled trial. Lancet 356:1213–1218. Bocan TM, Rosebury WS, Mueller SB, et al.: 1998. A specific 15-lipoxygenase inhibitor limits the progression and monocytemacrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit. Atherosclerosis 136:203–216.

100

Carew TE, Schwenke DC, Steinberg D: 1987. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci USA 84:7725– 7729. Chang MK, Bergmark C, Laurila A, et al.: 1999. Monoclonal antibodies against oxidized lowdensity lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: evidence that oxidationspecific epitopes mediate macrophage recognition. Proc Natl Acad Sci USA 96: 6353–6358.

Crawford RS, Kirk EA, Rosenfeld ME, et al.: 1998. Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse. Arerioscler Thromb Vasc Biol 18:1506–1513. Cynshi O, Kawabe Y, Suzuki T, et al.: 1998. Antiatherogenic effects of the antioxidant BO-653 in three different animal models. Proc Natl Acad Sci USA 95:10123–10128. Cyrus T, Praticò D, Witztum JL, et al.: 2001. Absence of 12/15-lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein E deficient mice. Circulation 103:2277–2282. Cyrus T, Witztum JL, Rader DJ, et al.: 1999. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice [see comments]. J Clin Invest 103:1597–1604. Daugherty A, Zweifel BS, Schonfeld G: 1989. Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Br J Pharmacol 98:612–618. Daugherty A, Zweifel BS, Schonfeld G: 1991. The effects of probucol on the progression of atherosclerosis in mature Watanabe heritable hyperlipidaemic rabbits. Br J Pharmacol 103:1013–1018. Enstrom JE, Kanim LE, Klein MA: 1992. Vitamin C intake and mortality among a sample of the United States population. Epidemiology 3:194–202. Esterbauer H, Dieber-Rotheneder M, Waeg G, et al.: 1990. Biochemical, structural, functional properties of oxidized low-density lipoprotein. Chem Res Toxicol 3:77–92.

Ezaki M, Witztum JL, Steinberg D: 1995. Lipoperoxides in LDL incubated with fibroblasts that overexpress 15-lipoxygenase. J Lipid Res 36:1996–2004. Febbraio M, Podrez EA, Smith JD, et al.: 2000. Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice. J Clin Invest 105:1049–1056. Folcik VA, Nivar-Aristy RA, Krajewski LP, Cathcart MK: 1995. Lipoxygenase contributes to the oxidation of lipids in human atherosclerotic plaques. J Clin Invest 96: 504–510. Fruebis J, Carew TE, Palinski W: 1995. Effect of vitamin E on atherogenesis in LDL receptor-deficient rabbits. Atherosclerosis 117:217–224. Fruebis J, Steinberg D, Dresel HA, Carew TE: 1994. A comparison of the antiatherogenic effects of probucol and of a structural analogue of probucol in low density lipoprotein receptor-deficient rabbits. J Clin Invest 94:392–398. Greenberg ER, Baron JA, Karagas MR, et al.: 1996. Mortality associated with low plasma concentration of beta carotene and the effect of oral supplementation. JAMA 275: 699–703. Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico: 1999. Dietary supplementation with n-3 polyunsaturated fatty acids and vitamin E after myocardial infarction: results of the GISSIPrevenzione trial [see comments]. Lancet 354:447–455. Harats D, Shaish A, George J, et al.: 2000. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 20:2100– 2105. Heinecke JW: 1997. Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Curr Opin Lipidol 8:268–274. Heinecke JW, Rosen H, Chait A: 1984. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest 74:1890–1894. Heinonen OP, Albanes D, Virtamo J, et al.: 1998. Prostate cancer and supplementation with alpha-tocopherol and betacarotene: incidence and mortality in a controlled trial. J Natl Cancer Inst 90:440–446. Hennekens CH, Buring JE, Manson JE, et al.: 1996. Lack of effect of long-term supplementation with beta carotene on the incidence of malignant neoplasms and cardiovascular disease. N Engl J Med 334: 1145–1149. Hessler JR, Morel DW, Lewis LJ, Chisolm GM: 1983. Lipoprotein oxidation and lipo-

TCM Vol. 11, No. 3/4, 2001

protein-induced cytotoxicity. Arteriosclerosis 3:215–222. Holvoet P, Vanhaecke J, Janssens S, et al.: 1998. Oxidized LDL and malondialdehydemodified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation 98:1487–1494. Hörkkö S, Binder CJ, Shaw PX, et al.: 2000. Immunological responses to oxidized LDL. Free Radic Biol Med 28:1771–1779. Hörkkö S, Bird DA, Miller E, et al.: 1999. Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins. J Clin Invest 103:117–128. Hulten LM, Lindmark H, Diczfalusy U, et al.: 1996. Oxysterols present in atherosclerotic tissue decrease the expression of lipoprotein lipase messenger RNA in human monocyte-derived macrophages. J Clin Invest 97:461–468. Inoue N, Ohara Y, Fukai T, et al.: 1998. Probucol improves endothelial-dependent relaxation and decreases vascular superoxide production in cholesterol-fed rabbits. Am J Med Sci 315:242–247. Itabe H, Yamamoto H, Imanaka T, et al.: 1996. Sensitive detection of oxidatively modified low density lipoprotein using a monoclonal antibody. J Lipid Res 37:45–53. Iuliano L, Mauriello A, Sbarigia E, et al.: 2000. Radiolabeled native low-density lipoprotein injected into patients with carotid stenosis accumulates in macrophages of atherosclerotic plaque: effect of vitamin E supplementation. Circulation 101:1249–1254. Jha P, Flather M, Lonn E, et al.: 1995. The antioxidant vitamins and cardiovascular disease. A critical review of epidemiologic and clinical trial data [see comments]. Ann Intern Med 123:860–872. Kita T, Nagano Y, Yokode M, et al.: 1987. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci USA 84:5928–5931. Kleinveld HA, Hak-Lemmers H, Hectors M, et al.: 1995. Vitamin E and fatty acid intervention does not attenuate the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits. Aterioscler Thromb and Vasc Biol 15:290–297. Kuhn H, Heydeck D, Hugou I, Gniwotta C: 1997. In vivo action of 15-lipoxygenase in early stages of human atherogenesis. J Clin Invest 99:888–893. Libby P, Hansson GK, Pober JS: 1999. Atherogenesis and inflammation. In: Chien KR, ed. Molecular Basis of Cardiovascular Disease. Philadelphia, W.B. Saunders, pp. 349–366.

TCM Vol. 11, No. 3/4, 2001

Mallat Z, Nakamura T, Ohan J, et al.: 1999. The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis. J Clin Invest 103:421–427. Mantha SV, Prasad M, Kalra J, Prasad K: 1993. Antioxidant enzymes in hypercholesterolemia and effects of vitamin E in rabbits. Atherosclerosis 101:135–144.

epitopes of oxidized LDL in LDL receptordeficient mice with increased atherosclerosis. Arterioscler Thromb Vasc Biol 15: 1569–1576. Parker RA, Sabrah T, Cap M, Gill BT: 1995. Relation of vascular oxidative stress, alphatocopherol, and hypercholesterolemia to early atherosclerosis in hamsters. Arterioscler Thromb Vasc Biol 15:349–358.

Mao SJ, Yates MT, Parker RA, et al.: 1991. Attenuation of atherosclerosis in a modified strain of hypercholesterolemic Watanabe rabbits with use of a probucol analogue (MDL 29,311) that does not lower serum cholesterol. Arterioscler Thromb 11:1266– 1275.

Prasad K, Kalra J: 1993. Oxygen free radicals and hypercholesterolemic atherosclerosis: effect of vitamin E. Am Heart J 125:958–973.

Meagher EA, Barry OP, Lawson JA, et al.: 2001. Effects of vitamin E on lipid peroxidation in healthy persons. JAMA 285: 1178–1182.

Praticò D, Iuliano L, Mauriello A, et al.: 1997. Localization of distinct F2-isoprostanes in human atherosclerotic lesions. J Clin Invest 100:2028–2034.

Miyata M, Smith JD: 1996. Apolipoprotein E allele-specific antioxidant activity and effects on cytotoxicity by oxidative insults and betaamyloid peptides. Nat Genet 14:55–61.

Praticò D, Tangirala RK, Rader DJ, et al.: 1998. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in ApoE-deficient mice. Nat Med 4:1189–1192.

Morel DW, de la Llera-Moya M, Friday K: 1994. Treatment of cholesterol-fed rabbits with dietary vitamins E and C inhibits lipoprotein oxidation but not development of atherosclerosis. Am Inst Nutri 124:2123– 2130.

Prasad K, Kalra J, Lee P: 1994. Oxygen free radicals as a mechanism of hypercholesterolemic atherosclerosis: effects of probucol. Int J Angio 3:100–112.

Rankin SM, Parthasarathy S, Steinberg D: 1991. Evidence for a dominant role of lipoxygenases in the oxidation of LDL by mouse peritoneal macrophages. J Lipid Res 32:449–456.

Navab M, Berliner JA, Watson AD, et al.: 1996. The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture. Arterioscler Thromb Vasc Biol 16:831–842.

Reaven PD, Herold DA, Barnett J, Edelman S: 1995. Effects of vitamin E on susceptibility of low-density lipoprotein and lowdensity lipoprotein subfractions to oxidation and on protein glycation in NIDDM. Diabetes Care 18:807–816.

O’Brien KD, Alpers CE, Hokanson JE, et al.: 1996. Oxidation-specific epitopes in human coronary atherosclerosis are not limited to oxidized low-density lipoprotein. Circulation 94:1216–1225.

Reaven PD, Khouw A, Beltz WF, et al.: 1993. Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin E but not by beta-carotene. Arterioscler Thromb 13: 590–600.

Ohara Y, Peterson TE, Harrison DG: 1993. Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91:2546–2551.

Reilly MP, Praticò D, Delanty N, et al.: 1998. Increased formation of distinct F2 isoprostanes in hypercholesterolemia [see comments]. Circulation 98:2822–2828.

Omenn GS, Goodman GE, Thornquist MD, et al.: 1996. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 334:1150–1155.

Riemersma RA, Wood DA, Macintyre CC, et al.: 1991. Risk of angina pectoris and plasma concentrations of vitamins A, C, and E and carotene. Lancet 337:1–5.

Palinski W, Hörkkö S, Miller E, et al.: 1996. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest 98:800–814.

Rimm EB, Stampfer MJ, Ascherio A, et al.: 1993. Vitamin E consumption and the risk of coronary heart disease in men. N Engl J Med 328:1450–1456. Ross R: 1999. Atherosclerosis: an inflammatory disease. N Engl J Med 340:115–126.

Palinski W, Rosenfeld ME, Ylä-Herttuala S, et al.: 1989. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86:1372–1376.

Sasahara M, Raines EW, Chait A, et al.: 1994. 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 94:155–164.

Palinski W, Tangirala RK, Miller E, et al.: 1995. Increased autoantibody titers against

Sendobry SM, Cornicelli JA, Welch K, et al.: 1997. Attenuation of diet-induced athero-

101

sclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties. Br J Pharmacol 120:1199–1206. Sevanian A, Hwang J, Hodis H, et al.: 1996. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations. Arerioscler Thromb Vasc Biol 16:784–793. Shaish A, Daugherty A, O’Sullivan F, Schonfeld G, Heinecke JW: 1995. Beta-carotene inhibits atherosclerosis in hypercholesterolemic rabbits. J Clin Invest 96:2075–2082. Shih DM, Xia YR, Wang XP, et al.: 2000. Combined serum paraoxonase knockout/ apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis. J Biol Chem 275:17,527–17,535. Sparrow CP, Doebber TW, Olszewski J, et al.: 1992. Low density lipoprotein is protected from oxidation and the progression of atherosclerosis is slowed in cholesterol-fed rabbits by the antioxidant N,N9-diphenylphenylenediamine. J Clin Invest 89:1885– 1891. Sparrow CP, Parthasarathy S, Steinberg D: 1988. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification. J Lipid Res 29:745– 753.

Steinberg, D, Witztum JL: 1999. Lipoproteins, lipoprotein oxidation, and atherogenesis. In: Chien KR, ed. Molecular Basis of Cardiovascular Disease. Philadelphia, W.B. Saunders Co., pp. 458–475. Steinbrecher UP, Parthasarathy S, Leake DS, et al.: 1984. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci USA 81: 3883–3887. Stephens NG, Parsons A, Schofield PM, et al.: 1996. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 347:781–785. Suzuki H, Kurihara Y, Takeya M, et al.: 1997. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 386:292–296. Tangirala RK, Casanada F, Miller E, et al.: 1995. Effect of the antioxidant N,N9-diphenyl 1,4-phenylenediamine (DPPD) on atherosclerosis in apoE-deficient mice. Arterioscler Thromb Vasc Biol 15:1625–1630. Tsimikas S, Palinski W, Witztum JL: 2001. Circulating autoantibodies to oxidized LDL correlate with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol 21:95–100.

Stampfer MJ, Hennekens CH, Manson JE, et al.: 1993. Vitamin E consumption and the risk of coronary disease in women. N Engl J Med 328:1444–1449.

Tsimikas S, Witztum JL: 2000. The oxidative modification hypothesis of atherogenesis. In: Keaney JF, ed. Oxidative Stress and Vascular Disease. Norwell, Kluwer Academic, pp. 49–74.

Stein Y, Stein O, Delplanque B, et al.: 1989. Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels. Atherosclerosis 75:145–155.

Verlangieri AJ, Bush MJ: 1992. Effects of d-a-tocopherol supplementation on experimentally induced primate atherosclerosis. J Am Coll Nutri 11:131–138.

Steinberg D: 1992. Antioxidants in the prevention of human atherosclerosis. Summary of the proceedings of a National Heart, Lung, and Blood Institute Workshop: September 5-6, 1991, Bethesda, Maryland. Circulation 85:2337–2344. Steinberg D: 2000. Is there a potential therapeutic role for vitamin E or other antioxidants in atherosclerosis? Curr Opin Lipidol 11:603–607. Steinberg D, Gotto AM: 1999. Preventing coronary artery disease by lowering cholesterol levels: fifty years from bench to bedside. JAMA 282:2043–2050. Steinberg D, Parthasarathy S, Carew TE, et al.: 1989. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity [see comments]. N Engl J Med 320:915–924. Steinberg D, Witztum JL: 1990. Lipoproteins and atherogenesis. Current concepts. JAMA 264:3047–3052.

102

Witting P, Pettersson K, Ostlund-Lindqvist AM, et al.: 1999a. Dissociation of atherogenesis from aortic accumulation of lipid hydro(pero)xides in Watanabe heritable hyperlipidemic rabbits. J Clin Invest 104: 213–220.

Witting PK, Pettersson K, Letters J, Stocker R: 2000. Site-specific antiatherogenic effect of probucol in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 20: E26–E33. Witting PK, Pettersson K, Ostlund-Lindqvist AM, et al.: 1999b. Inhibition by a coantioxidant of aortic lipoprotein lipid peroxidation and atherosclerosis in apolipoprotein E and low density lipoprotein receptor gene double knockout mice. FASEB J 13:667–675. Witztum JL: 1994. The oxidation hypothesis of atherosclerosis. Lancet 344:793–795. Witztum JL: 1998. To E or not to E: how do we tell? [editorial]. Circulation 98:2785–2787. Ylä-Herttuala S: 1998. Is oxidized low-density lipoprotein present in vivo? Curr Opin Lipidol 9:337–344. Ylä-Herttuala S, Palinski W, Butler SW, et al.: 1994. Rabbit and human atherosclerotic lesions contain IgG that recognizes epitopes of oxidized LDL. Arterioscler Thromb 14:32–40. Ylä-Herttuala S, Palinski W, Rosenfeld ME, et al.: 1989. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84:1086–1095. Ylä-Herttuala S, Rosenfeld ME, Parthasarathy S, et al.: 1990. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci USA 87:6959–6963. Yusuf S, Dagenais G, Pogue J, et al.: 2000. Vitamin E supplementation and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators [see comments]. N Engl J Med 342:154–160. Zhang SH, Reddick RL, Avdievich E, et al.: 1997. Paradoxical enhancement of atherosclerosis by probucol treatment in apolipoprotein E-deficient mice. J Clin Invest 99: 2858–2866. PII S1050-1738(01)00111-6

TCM

LETTERS TCM welcomes letters on topics of interest to cardiovascular researchers and clinical cardiologists. Letters to the Editor in response to published articles are also accepted. Please submit brief letters, with a minimum of references, to: Trends in Cardiovascular Medicine Editorial Office, 655 Avenue of the Americas New York, New York 10010, USA

TCM Vol. 11, No. 3/4, 2001