The LDL modification hypothesis of atherogenesis: an update
2008; Elsevier BV; Volume: 50; Linguagem: Inglês
10.1194/jlr.r800087-jlr200
ISSN1539-7262
Autores Tópico(s)Retinoids in leukemia and cellular processes
ResumoThe accumulated evidence that oxidative modification of LDL plays an important role in the pathogenesis of atherosclerosis in animal models is very strong. The negative results in recent clinical studies have caused many to conclude that LDL oxidation may not be relevant in the human disease. Yet many of the lines of evidence that support the hypothesis have been demonstrated to apply also in humans. In this review, we briefly summarize the lines of evidence on which the hypothesis rests, its strengths, and its weaknesses. The accumulated evidence that oxidative modification of LDL plays an important role in the pathogenesis of atherosclerosis in animal models is very strong. The negative results in recent clinical studies have caused many to conclude that LDL oxidation may not be relevant in the human disease. Yet many of the lines of evidence that support the hypothesis have been demonstrated to apply also in humans. In this review, we briefly summarize the lines of evidence on which the hypothesis rests, its strengths, and its weaknesses. The defining characteristic of the fatty streak, the first visible lesion of atherosclerosis, both in animals and in humans is the “foam cell.” This cell, loaded with droplets rich in cholesteryl esters, is derived mainly from arterial wall macrophages, which originate from circulating monocytes that have penetrated into the subendothelial space. Smooth muscle cells and endothelial cells in lesions also can and do accumulate lipid droplets, but monocyte-derived macrophage foam cells predominate. This being the case, an understanding of just how arterial macrophages take up and store their load of cholesterol should shed light on the mechanisms that initiate atherogenesis. Beginning in 1979, Goldstein, Brown, and their collaborators [reviewed in (1Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis.Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar)] decided to pursue this problem, studying the metabolism of macrophages in cell culture. They noted the following apparent paradox: in patients with homozygous familial hypercholesterolemia, even in those who express absolutely no functional LDL receptors, macrophage-derived foam cells nevertheless accumulate in the artery walls just as they do in hypercholesterolemic patients that have perfectly normal LDL receptors. The implication was that LDL must be somehow altered prior to its uptake by macrophages and then taken up, not by the native LDL receptor, but rather by some alternative macrophage receptor. Indeed, they found that the rate of uptake of native LDL by normal resident mouse peritoneal macrophages was very low even at very high LDL concentrations. There was very little increase in cell cholesterol content and certainly no generation of foam cells. They then tried modifying the LDL physically, chemically, or enzymatically, looking for some form of LDL that could turn macrophages into foam cells in vitro. Several modifications worked, but the most striking was chemical acetylation. Treatment of LDL with acetic anhydride yielded a form of LDL that bound to the macrophage specifically and with high affinity, was actively internalized, and led to intracellular cholesterol accumulation. They dubbed the putative receptor the acetyl-LDL receptor. That receptor was later cloned and characterized by Kodama et al. (2Kodama T. Reddy P. Kishimoto C. Krieger M. Purification and characterization of a bovine acetyl low density lipoprotein receptor.Proc. Natl. Acad. Sci. USA. 1988; 85: 9238-9242Crossref PubMed Scopus (218) Google Scholar) in the laboratory of Monty Krieger. Because of its possible role in the LDL receptor-independent uptake of LDL and because of its broad ligand specificity, it was redesignated scavenger receptor A (SRA). However, acetyl-LDL itself is not a biological product and has never been found in vivo. The search for the biological ligand for the scavenger receptor continued. In 1981, Henriksen et al. (3Henriksen T. Mahoney E.M. Steinberg D. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: recognition by receptors for acetylated low density lipoproteins.Proc. Natl. Acad. Sci. USA. 1981; 78: 6499-6503Crossref PubMed Scopus (815) Google Scholar) discovered that native LDL simply incubated overnight with cultured endothelial cells was converted to a form (endothelial cell-modified LDL) that was recognized specifically and with high affinity by peritoneal macrophages. They proposed that this endothelium-induced modification of LDL might be the missing step that permits rapid LDL uptake and foam cell formation. Later studies showed that during its incubation with endothelial cells (and with a number of other cell types), LDL was undergoing an oxidative modification (4Hessler J.R. Morel D.W. Lewis L.J. Chisolm G.M. Lipoprotein oxidation and lipoprotein-induced cytotoxicity.Arteriosclerosis. 1983; 3: 215-222Crossref PubMed Google Scholar, 5Steinbrecher U.P. Parthasarathy S. Leake D.S. Witztum J.L. Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids.Proc. Natl. Acad. Sci. USA. 1984; 81: 3883-3887Crossref PubMed Scopus (1412) Google Scholar). This was the genesis of the so-called oxidative modification hypothesis of atherogenesis. Certainly the hypothesis has been heuristic; PubMed lists over 5,000 papers published to date under “oxidized LDL” and over 2,200 indexed under “oxidized LDL and atherosclerosis,” Over 1,000 in the latter category have been published in the past 5 years alone. So the hypothesis is very much alive. Studies in animal models of atherosclerosis continue to strongly support the hypothesis. However, a series of negative clinical trials using vitamin E or β-carotene have raised doubts about the relevance of the hypothesis to the human disease. In this review, we attempt to assess the strengths and weaknesses of the evidence for the hypothesis and suggest future directions for research. Many excellent reviews have dealt with the evidence linking oxidized LDL (OxLDL) to atherogenesis (6Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity.N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar, 7Chisolm G.M. Steinberg D. The oxidative modification hypothesis of atherogenesis: an overview.Free Radic. Biol. Med. 2000; 28: 1815-1826Crossref PubMed Scopus (671) Google Scholar, 8Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Lenten B.J. Van Frank J.S. Demer L.L. al et 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. 1996; 16: 831-842Crossref PubMed Scopus (607) Google Scholar, 9Stocker R. Keaney Jr., J.F. Role of oxidative modifications in atherosclerosis.Physiol. Rev. 2004; 84: 1381-1478Crossref PubMed Scopus (2091) Google Scholar, 10Glass C.K. Witztum J.L. Atherosclerosis. the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2615) Google Scholar, 11Boullier A. Bird D.A. Chang M.K. Dennis E.A. Friedman P. Horkko K.Gillotre-Taylor, S. Palinski W. Quehenberger O. Shaw P. al et Scavenger receptors, oxidized LDL, and atherosclerosis.Ann. N. Y. Acad. Sci. 2001; 947: 214-222Crossref PubMed Scopus (241) Google Scholar, 12Hansson G.K. Immune mechanisms in atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1876-1890Crossref PubMed Scopus (725) Google Scholar, 13Berliner J.A. Heinecke J.W. The role of oxidized lipoproteins in atherogenesis.Free Radic. Biol. Med. 1996; 20: 707-727Crossref PubMed Scopus (1272) Google Scholar). Because of space limitations, it will not be possible to cite all the primary references and so the reader is asked to consult these reviews, especially for older references. What are the major findings that form the basis for the oxidative modification hypothesis? As discussed above, they bind OxLDL with high affinity to specific plasma membrane receptors [including SRA, SRB (CD36), and lectin-like oxLDL receptor]. These receptors are not downregulated as the cholesterol content of the macrophage increases, allowing progressive accumulation of cholesterol to the point of foam cell generation. There are a number of alternative mechanisms for generation of foam cells that could also play a role, as we have discussed elsewhere (14Steinberg D. Witztum J.L. Lipoproteins and atherogenesis. Current concepts.JAMA. 1990; 264: 3047-3052Crossref PubMed Scopus (485) Google Scholar). These include uptake of aggregated LDL (via phagocytosis and facilitated by the LDL receptor), β-VLDL (probably via the LDL receptor), and LDL immune complexes (via the Fc receptor). There is as yet little or no in vivo evidence to establish that these alternative pathways actually contribute to foam cell formation. Recently, Kruth et al. (15Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein.J. Biol. Chem. 2005; 280: 2352-2360Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) have shown that macrophages activated by incubation with phorbol-12-myristate-13-acetate or macrophage-colony-stimulating factor (M-CSF) can take up native LDL from the medium by macropinocytosis, a process that internalizes large volumes of surrounding medium together with whatever solutes they contain. If the concentration of native LDL is extremely high (about 2 mg/ml), the rate of uptake by this nonspecific pathway can be sufficient to cause cholesterol accumulation and foam cell formation (15Kruth H.S. Jones N.L. Huang W. Zhao B. Ishii I. Chang J. Combs C.A. Malide D. Zhang W.Y. Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein.J. Biol. Chem. 2005; 280: 2352-2360Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Zhao B. Li Y. Buono C. Waldo S.W. Jones N.L. Mori M. Kruth H.S. Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF).J. Biol. Chem. 2006; 281: 15757-15762Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Note that the concentration of LDL needed to induce foam cell formation (2 mg/ml) is 40-fold greater than the concentration of OxLDL needed (ca. 50 μg/ml). Exactly how the contents of the macropinosome are sorted and distributed to cellular compartments is still unclear, nor has it been shown that this pathway is actually operative in vivo. Clearly it deserves further investigation. OxLDL is present in plasma and in atherosclerotic lesions, both in experimental animals and in humans (17Yla¨-Herttuala S. Palinski W. Rosenfeld M.E. Parthasarathy S. Carew T.E. Butler S. Witztum J.L. Steinberg D. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.J. Clin. Invest. 1989; 84: 1086-1095Crossref PubMed Google Scholar); antibodies against OxLDL are present in normal animal and human plasma; and both OxLDL and antibodies against it are present at higher levels in the presence of atherosclerosis. Many of these are effected by oxidized phospholipids within the OxLDL (11Boullier A. Bird D.A. Chang M.K. Dennis E.A. Friedman P. Horkko K.Gillotre-Taylor, S. Palinski W. Quehenberger O. Shaw P. al et Scavenger receptors, oxidized LDL, and atherosclerosis.Ann. N. Y. Acad. Sci. 2001; 947: 214-222Crossref PubMed Scopus (241) Google Scholar, 18Leitinger N. Oxidized phospholipids as triggers of inflammation in atherosclerosis.Mol. Nutr. Food Res. 2005; 49: 1063-1071Crossref PubMed Scopus (63) Google Scholar, 19Witztum J.L. Berliner J.A. Oxidized phospholipids and isoprostanes in atherosclerosis.Curr. Opin. Lipidol. 1998; 9: 441-448Crossref PubMed Scopus (156) Google Scholar). These oxidized phospholipid products are also the principal epitopes recognized by autoantibodies to OxLDL and the major structural feature by which scavenger receptors recognize OxLDL as a ligand. Oxidized cholesterol esters also play a role. a) OxLDL is cytotoxic for endothelial cells cultured in serum-free medium; b) It induces expression and release of monocyte chemoattractant protein-1 from endothelial cells; c) It is chemotactic for monocytes (but inhibits migration of macrophages); d) It induces endothelial expression of M-CSF; e) It increases collagen synthesis in smooth muscle cells; f) It inhibits lipopolysaccharide-induced expression of nuclear factor-κB; g) It induces apoptosis; h) It inhibits release and/or function of nitric oxide (vasospasm); i) It increases expression of vascular cell adhesion molecule-1; j) It increases tissue factor activity in endothelial cells (thrombosis); k) It induces a wide variety of proinflammatory cytokines in macrophages; and l) It is immunogenic, inducing an increase in circulating levels of antibodies against oxidation-specific epitopes (see review by J. L. Witztum in this issue). These manifold effects of OxLDL have been mostly described in cell culture settings and in only a few instances have they been confirmed in the whole animal. The findings are compatible with and lend some support to the oxidative modification hypothesis, but until they are evaluated in intact animals they remain only suggestive. In vivo veritas. Effective antioxidants include probucol, probucol analogs, butylated-hydroxytoluene, N,N'-diphenylphenylenediamine, BO-653 (an analog of probucol), and vitamin E. The fact that these several compounds, quite different in structure and metabolism but sharing an ability to trap free radicals, have all been shown to act as inhibitors of atherosclerosis considerably strengthens the oxidative modification hypothesis. Further support comes from a study in which the protective effect of vitamin E was confirmed in apolipoprotein (apo) E-deficient mice and then shown to be almost abolished when the mice were also deficient in 12/15-lipoxygenase (20Zhao L. Pratico D. Rader D.J. Funk C.D. 12/15-Lipoxygenase gene disruption and vitamin E administration diminish atherosclerosis and oxidative stress in apolipoprotein E deficient mice through a final common pathway.Prostaglandins Other Lipid Mediat. 2005; 78: 185-193Crossref PubMed Scopus (26) Google Scholar). The authors suggest therefore that the protection afforded by vitamin E and that afforded by targeting 12/15-lipoxygenase share a final common pathway. There are problems with the interpretation of some of these animal studies. For example, N,N'-diphenylphenylenediamine and butylated-hydroxytoluene have been used in only one study each. Results with vitamin E have yielded convincing results in mice but conflicting results in rabbits. Probucol was the first antioxidant to be tested and it has been the most consistently effective agent. Recently, Stocker et al. (9Stocker R. Keaney Jr., J.F. Role of oxidative modifications in atherosclerosis.Physiol. Rev. 2004; 84: 1381-1478Crossref PubMed Scopus (2091) Google Scholar) have raised the question of whether the antiatherosclerotic action of probucol is necessarily dependent on its antioxidant activity or whether it may be due to some other biological effects of the compound. Targeting of 12/15 lipoxygenase decreases the severity of atherosclerosis in three different mouse models: apoE-deficient mice (21Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice. [see comments].J. Clin. Invest. 1999; 103: 1597-1604Crossref PubMed Scopus (460) Google Scholar, 22Cyrus T. Pratico D. Zhao L. Witztum J.L. Rader D.J. Rokach J. FitzGerald G.A. Funk C.D. Absence of 12/15-lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein E-deficient mice.Circulation. 2001; 103: 2277-2282Crossref PubMed Scopus (214) Google Scholar), LDL receptor-deficient mice (23George J. Afek A. Shaish A. Levkovitz H. Bloom N. Cyrus T. Zhao L. Funk C.D. Sigal E. Harats D. 12/15-Lipoxygenase gene disruption attenuates atherogenesis in LDL receptor-deficient mice.Circulation. 2001; 104: 1646-1650Crossref PubMed Scopus (167) Google Scholar), and mice deficient in the apoB mRNA editing enzyme (24Zhao L. Cuff C.A. Moss E. Wille U. Cyrus T. Klein E.A. Pratico D. Rader D.J. Hunter C.A. Pure E. al et Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia.J. Biol. Chem. 2002; 277: 35350-35356Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Much of the effect can be attributed to the lipoxygenase in macrophages, because bone marrow transplants from 12/15-lipoxygenase-deficient mice into apoE-deficient mice affords almost the same degree of protection from lesion formation as the global knockout (25Huo Y. Zhao L. Hyman M.C. Shashkin P. Harry B.L. Burcin T. Forlow S.B. Stark M.A. Smith D.F. Clarke S. al et Critical role of macrophage 12/15-lipoxygenase for atherosclerosis in apolipoprotein E-deficient mice.Circulation. 2004; 110: 2024-2031Crossref PubMed Scopus (170) Google Scholar). Furthermore, site-specific overexpression of 15-lipoxygenase in endothelium accelerates atherosclerosis in LDL receptor-deficient mice (26Harats D. Shaish A. George J. Mulkins M. Kurihara H. Levkovitz H. Sigal E. Overexpression of 15-lipoxygenase in vascular endothelium accelerates early atherosclerosis in LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2100-2105Crossref PubMed Scopus (209) Google Scholar). Targeting of scavenger receptors ameliorates atherosclerosis in mouse models. SRA, SRB (CD36), and lectin-like oxLDL receptor are the three best-studied receptors that recognize OxLDL. Together, SRA and CD36 account for almost 90% of macrophage uptake of OxLDL (27Kunjathoor V.V. Febbraio M. Podrez E.A. Moore K.J. Andersson L. Koehn S. Rhee J.S. Silverstein R. Hoff H.F. Freeman M.W. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages.J. Biol. Chem. 2002; 277: 49982-49988Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar). In each case, knockouts in either apoE-deficient or LDL receptor-deficient mouse models have been shown to ameliorate the severity of atherosclerosis. Targeting of paraoxonase-1, an enzyme that indirectly inhibits LDL oxidation (possibly by interfering with the ability of HDL to protect LDL against oxidation) enhanced lesion formation by 50% or more in apoE-deficient mice (28Shih D.M. Xia Y.R. Wang X.P. Miller E. Castellani L.W. Subbanagounder G. Cheroutre H. Faull K.F. Berliner J.A. Witztum J.L. al et Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein oxidation and atherosclerosis.J. Biol. Chem. 2000; 275: 17527-17535Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Targeting of some of the genes coding for proteins thought to mediate the atherogenic effects of OxLDL ameliorates atherogenesis. These include, e.g., MCP-1, M-CSF, and selectins P and E. However, these knockouts would likely affect atherogenesis no matter what the details of the underlying pathogenesis, because monocyte recruitment and retention must be involved in any arterial inflammatory disease. So the findings are compatible with the hypothesis but do not directly and specifically implicate OxLDL. Shen et al. (29Shen J. Herderick E. Cornhill J.F. Zsigmond E. Kim H.S. Kuhn H. Guevara N.V. Chan L. Macrophage-mediated 15-lipoxygenase expression protects against atherosclerosis development.J. Clin. Invest. 1996; 98: 2201-2208Crossref PubMed Scopus (209) Google Scholar) overexpressed the human 15-lipoxygenase gene specifically in the macrophages of cholesterol-fed rabbits and WHHL rabbits and observed an amelioration of atherosclerosis rather than the anticipated exacerbation. In contrast, macrophage-specific deficiency of 12/15-lipoxygenase in the apoE-deficient mouse ameliorates atherosclerosis (25Huo Y. Zhao L. Hyman M.C. Shashkin P. Harry B.L. Burcin T. Forlow S.B. Stark M.A. Smith D.F. Clarke S. al et Critical role of macrophage 12/15-lipoxygenase for atherosclerosis in apolipoprotein E-deficient mice.Circulation. 2004; 110: 2024-2031Crossref PubMed Scopus (170) Google Scholar). As pointed out by Kuhn and Chen (30Kuhn H. Chan L. The role of 15-lipoxygenase in atherogenesis: pro- and antiatherogenic actions.Curr. Opin. Lipidol. 1997; 8: 111-117Crossref PubMed Scopus (90) Google Scholar), 12/15-lipoxygenase may have both pro- and antiinflammatory effects. Their results with overexpression in the rabbit and the results with lipoxygenase targeting in mouse models are not necessarily mutually contradictory. Some level of lipoxygenase may be mandatory for atherogenesis, while levels above normal may exert antiinflammatory effects. While this explanation seems to offer an attractive resolution for many of the discordant findings, it doesn't resolve the directly contradictory results on the consequence of global knockout of 12/15-lipoxygenase in the apoE-deficient mouse very recently reported by Merched et al. (31Merched A.J. Ko K. Gotlinger K.H. Serhan C.N. Chan L. Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators.FASEB J. 2008; 22: 3595-3606Crossref PubMed Scopus (328) Google Scholar) in Lawrence Chan's laboratory. In their hands, knockout increased rather than decreased the extent of lesions. The reasons for the contradictory results remain to be determined. 5-Lipoxygenase has been implicated as proatherogenic, not through oxidation of LDL, but via its other proinflammatory effects exercised in part via LTB4 (32Radmark O. Samuelsson B. 5-lipoxygenase: regulation and possible involvement in atherosclerosis.Prostaglandins Other Lipid Mediat. 2007; 83: 162-174Crossref PubMed Scopus (70) Google Scholar). The enzyme and its products are present in lesions and pharmacologic inhibition can reduce lesion size (33Aiello R.J. Bourassa P.A. Lindsey S. Weng W. Freeman A. Showell H.J. Leukotriene B4 receptor antagonism reduces monocytic foam cells in mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 443-449Crossref PubMed Scopus (223) Google Scholar). However, Cao et al. (34Cao R.Y. Amand T. St Grabner R. Habenicht A.J. Funk C.D. Genetic and pharmacological inhibition of the 5-lipoxygenase/leukotriene pathway in atherosclerotic lesion development in ApoE deficient mice.Atherosclerosis. 2009; (In press)Abstract Full Text Full Text PDF Scopus (41) Google Scholar) reported that neither global knockout nor pharmacologic inhibition of 5-lipoxygenase affects atherogenesis in the apoE-deficient mouse. Oxidative stress may be involved much more broadly in atherogenesis. Reactive oxygen species are produced in endothelium, in smooth muscle cells, and adventitia. A large body of evidence documents their potential importance in vasomotor activity, smooth muscle cell growth, expression of adhesion molecules, apoptosis, activation of metalloproteinases, and, of course, lipid oxidation (35Harrison D. Griendling K.K. Landmesser U. Hornig B. Drexler H. Role of oxidative stress in atherosclerosis.Am. J. Cardiol. 2003; 91: 7A-11AAbstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar). Any or all of these oxidation-related processes may accompany oxidation of LDL or occur independently of it. Recent studies in Freeman's laboratory failed to confirm the ameliorating effect of CD36 and SRA knockouts cited above (36Moore K.J. Kunjathoor V.V. Koehn S.L. Manning J.J. Tseng A.A. Silver J.M. McKee M. Freeman M.W. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice.J. Clin. Invest. 2005; 115: 2192-2201Crossref PubMed Scopus (317) Google Scholar). The authors suggested that the contradictory results might be due to differences in genetic background. Other possible explanations have been discussed in detail by Witztum (37Witztum J.L. You are right too!.J. Clin. Invest. 2005; 115: 2072-2075Crossref PubMed Scopus (73) Google Scholar). Most of the large clinical trials of antioxidants have been done using vitamin E or β-carotene. Meta-analysis of the data from these large studies (n = ca. 80,000) shows no benefit at all with regard to cardiovascular outcomes (38Kris-Etherton P.M. Lichtenstein A.H. Howard B.V. Steinberg D. Witztum J.L. Antioxidant vitamin supplements and cardiovascular disease.Circulation. 2004; 110: 637-641Crossref PubMed Scopus (325) Google Scholar). There is no doubt that at the doses used (50–400 mg/d), vitamin E offered no protection against coronary heart disease in a general population. Do these disappointingly negative results mean that the oxidative modification hypothesis is irrelevant in the human disease? Not necessarily. As discussed elsewhere (39Steinberg D. Witztum J.L. Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted to date refute the hypothesis?.Circulation. 2002; 105: 2107-2111Crossref PubMed Scopus (454) Google Scholar), vitamin E may be the wrong antioxidant in humans, the dosage may have been too low, treatment may have been started too late in life, or antioxidant treatment may be beneficial only in some subset of patients subject to unusual oxidative stress (see below). After all, the hypothesis is not that any antioxidant, at any dosage, in any individual will necessarily be effective. The hypothesis is that oxidative modification plays a quantitatively significant role in pathogenesis. Of course, the implication is that one day an effective antioxidant intervention will be found that will slow the progress of the disease. It would be a mistake to ignore the strong scientific base of the hypothesis and assume prematurely that the human disease is categorically different from that in experimental animals, including the nonhuman primate (40Sasahara M. Raines E.W. Chait A. Carew T.E. Steinberg D. Wahl P.W. 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: 155-164Crossref PubMed Scopus (189) Google Scholar). The major exception in this category of trials in a general population was the very first trial, the Cambridge Heart Antioxidant Study. Stephens et al. (41Stephens N.G. Parsons A. Schofield P.M. Kelly F. Cheeseman K. Mitchinson M.J. Brown M.J. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS).Lancet. 1996; 347: 781-785Abstract PubMed Scopus (1977) Google Scholar) followed just over 2,000 men with angiographically established coronary heart disease randomized to placebo or to 400–800 units of vitamin E daily. There was a statistically significant 47% reduction in the primary combined end point of cardiovascular death or nonfatal myocardial infarction, mainly due to the latter. It is not clear why this study yielded a positive result in contrast to the several larger studies that followed. The dosage of vitamin E (400–800 units/d) was higher and the natural form of the vitamin rather than the racemic mixture was used. Otherwise the protocols were much the same. A secondary prevention trial (3 year follow-up) using a succinylated analog of probucol (succinobucol) was reported recently. The drug (AGI-1067) had been shown to inhibit atherogenesis in animal models (42Sundell C.L. Somers P.K. Meng C.Q. Hoong L.K. Suen K.L. Hill R.R. Landers L.K. Chapman A. Butteiger D. Jones M. al et AGI-1067: a multifunctional phenolic antioxidant, lipid modulator, anti-inflammatory and antiatherosclerotic agent.J. Pharmacol. Exp. Ther. 2003; 305: 1116-1123Crossref PubMed Scopus (88) Google Scholar), but it failed to show any decrease in its primary composite end point of cardiovascular death, resuscitated cardiac arrest, nonfatal myocardial infarction, nonfatal stroke, hospitalization for unstable angina, or coronary revascularization above that seen with statin therapy alone (43Tardif J.C. McMurray J.J. Klug E. Small R. Schumi J. Choi J. Cooper J. Scott R. Lewis E.F. al P.L. L'Allier, et Effects of succinobucol (AGI-1067) after an acute coronary syndrome: a randomised, double-blind, placebo-controlled trial.Lancet. 2008; 371: 1761-1768Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Data to evaluate antioxidant effect were not presented. While vitamin E probably confers no benefit in general populations, there is some evidence that it may benefit patients under increased oxidative stress. For example, the SPACE trial was carried out in patients with end-stage renal disease undergoing hemodialysis, patients known to be under oxidative stress. This study randomized 196 patients to 800 mg/d vitamin E or placebo (44Boaz M. Smetana S. Weinstein T. Matas Z. Gafter U. Iaina A. Knecht A. Weissgarten Y. Brunner D. Fainaru M. al et Secondary prevention with antioxidants of cardiovascular disease in endstage renal disease (SPACE): randomised placebo-controlled trial.Lancet. 2000; 356: 1213-1218Abstract Full Text Full Text PDF PubMed Scopus (930) Google Scholar). Pooled vascular events were reduced by 54% and myocardial infarction by 70%, both at P < 0.02. An extensive literature establishes that patients in this category are subject to increased oxidative stress, in part due to prooxidant conditions accompanying dialysis and in part due to the disease itself. Another study in patients with end-stage renal failure using N-acetylcysteine as the antioxidant also showed a significant 40% decrease in the combined primary end point of cardiovascular events (45Tepel M. der M.van Statz Giet, M. Jankowski J. Zidek W. The antioxidant acetylcysteine reduces cardiovascular events in patients with end-stage renal failure: a randomized, controlled trial.Circulation. 2003; 107: 992-995Crossref PubMed Scopus (343) Google Scholar). N-acetylcysteine has also been shown to significantly suppress atherosclerotic lesion progression in uremic apoE-deficient mice (46Ivanovski O. Szumilak D. Ruellan T.Nguyen-Khoa, N. Phan O. Lacour B. Drueke B.Descamps-Latscha, T.B. Massy Z.A. The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.Kidney Int. 2005; 67: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Levy (47Levy A.P. Application of pharmacogenomics in the prevention of diabetic cardiovascular disease: mechanistic basis and clinical evidence for utilization of the haptoglobin genotype in determining benefit from antioxidant therapy.Pharmacol. Ther. 2006; 112: 501-512Crossref PubMed Scopus (28) Google Scholar) has shown that the haptoglobin 2-2 genotype is associated with inferior antioxidant protection. In diabetics, this haptoglobin genotype is associated with a 2- 5-fold increased risk for cardiovascular disease. His group has recently reported a trial of vitamin E (400 U/d versus placebo) in a group of 1,434 diabetic patients with the haptoglobin 2-2 genotype (48Milman U. Blum S. Shapira C. Aronson D. Anbinder R.Miller-Lotan, Y. Alshiek J. Bennett L. Kostenko M. Landau M. al et Vitamin E supplementation reduces cardiovascular events in a subgroup of middle-aged individuals with both type 2 diabetes mellitus and the haptoglobin 2–2 genotype: a prospective double-blinded clinical trial.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 341-347Crossref PubMed Scopus (236) Google Scholar). The study was terminated at approximately 500 days, because the first interim analysis showed highly significant protection in the treated group. The primary composite end point of cardiovascular death, nonfatal myocardial infarction, or stroke was reduced by 47% in the treated group (P = 0.01). In a later study, again in a cohort of diabetics with the haptoglobin 2-2 genotype, it was shown that adding vitamin E to statin therapy reduced risk by over 60% compared with statin-only treatment (49Blum S. Milman U. Shapira C. Bennett R.Miller-Lotan, L. Kostenko M. Landau M. Keidar S. Levy Y. Khemlin A. al et Dual therapy with statins and antioxidants is superior to statins alone in decreasing the risk of cardiovascular disease in a subgroup of middle-aged individuals with both diabetes mellitus and the haptoglobin 2–2 genotype.Arterioscler. Thromb. Vasc. Biol. 2008; 28: e18-e20Crossref PubMed Scopus (22) Google Scholar). These are striking results suggesting that oxidative modification does indeed occur in the human disease but that the benefit of vitamin E (and perhaps the other natural antioxidants) is only apparent in populations under unusual oxidative stress. Diabetic patients with the 2-2 haptoglobin phenotype only represent a few percent of the population, but with these encouraging data in hand, it will be important to look for other subgroups that may benefit and to look for other antioxidant interventions that might be more generally effective. The benefits of antioxidant treatment in these subgroups should be further tested as a means of preventing vacular events in these types of patients and others that may be uncovered by further research. In any case, these studies provide evidence that oxidative stress may indeed be involved in the human disease and that alternative approaches to antioxidant therapy in the general population may be discovered: different antioxidant agents, different doses, different treatment schedules (including treatment earlier in life). How are we going to resolve some of the conflicting data reviewed above? First, it is essential to go back to studies of the animal models in which antioxidant treatment works and ask hard questions about the detailed mechanisms involved. If oxidation of LDL is an obligatory step in pathogenesis, where and how does it come about? Which enzyme system(s) are responsible? The sometimes contradictory results of gene targeting experiments in mouse models need to be rationalized. The fate of OxLDL in vivo needs to be studied, and the in vivo relevance of the biological effects observed in vitro needs to be explored. Once some of these fundamental questions are answered, we will have a basis for choosing the best antioxidant intervention regimen. Does LDL oxidation occur at all intravascularly? At or under the endothelium? Is it possible that OxLDL found in plasma represents in part LDL that has acquired oxidized lipids, especially oxidized phospholipids, from apoptotic cell membranes by an exchange process? Does macrophage uptake of OxLDL protect (at least initially) by sequestering OxLDL, a potentially cytotoxic substance? Can oxidation take place at other than vascular sites? Can we devise a test for the rate at which LDL oxidation is occurring, a test that could be used to evaluate effectiveness of a potential antioxidant intervention in vivo? Chain-breaking antioxidants like vitamin E may not be the right antioxidants in humans. Once we have the answers to questions like these, we should be able to design more meaningful clinical trials based on solid basic science. Second, the alternative pathways for generating foam cells should be intensively explored in animal models. For example, if LDL could be altered somehow so as to reduce its tendency to aggregate, would that ameliorate atherosclerosis? Do knockouts or inhibitors that interfere with macropinocytosis ameliorate atherosclerosis? Finally, we must keep an open mind about the still imperfectly understood pathogenesis of this disease. It may very well be that multiple pathways are involved and to different extents at different stages of the disease. For example, oxidation of LDL could play a role in initiation in the generation of foam cells but become less important in the later stages of plaque evolution. Perhaps clinical studies need to be done in younger people, inhibiting at the fatty streak stage. At present that would be difficult to do, but with improvements in noninvasive imaging techniques, it should be possible in the near future.
Referência(s)