The Discovery That Changed the Direction of Heart Disease Research: An Interview with Dr. Daniel Steinberg
by Richard A. Passwater, Ph.D.
Daniel Steinberg, M.D., Ph.D. is a Professor of Medicine at the University of California at San Diego. A few years after receiving his Ph.D. degree with distinction from Harvard, Dr. Steinberg started his brilliant research career at the National Heart, Lung and Blood Institute in 1951. In 1968, he became the head of the Division of Metabolic Disease in the School of Medicine of the University of California at San Diego.
Dr. Steinberg is a former editor of the Journal of Lipid Research and former Chairman of the Council on Arteriosclerosis. He is a member of the National Academy of Sciences and has published over 400 scientific articles.
In recent columns, I have been interviewing the worlds leading scientists dealing with the role of antioxidant nutrients in protecting against heart disease. The interviews have dealt with the actions of free radicals and antioxidants, the atherosclerotic process, and epidemiology. All, of this information was to provide you with the background knowledge you needed to understand the research of Dr. Daniel Steinberg. Dr. Steinberg's research changed the direction of heart disease research to a more productive approach that has most scientists very excited. The public health benefits of Dr. Steinberg's research are very obvious. This additional knowledge above what we understand about dietary fats and cholesterol could save millions of lives.
Passwater: Dr. Steinberg, your breakthrough research has inspired the heart disease research community. As the acknowledge leader in the oxidized-LDL hypothesis of heart disease, you were asked by the National Heart, Lung and Blood Institute to convene a congress of the prime researchers in this field that you created to evaluate whether there was enough justification to conduct prospective clinical trials. What did this congress conclude?
Steinberg: The Workshop I chaired for the National Heart, Lung and Blood Institute had the task of evaluating "Antioxidants in the Prevention of Human Atherosclerosis". The Workshop was held in September 1991, in Bethesda, Maryland. A distinguished group of over 30 specialists in various aspects of the problem reviewed all of the evidence available up to that time. Their conclusion was that there was compelling evidence supporting a key role for oxidative modification of low-density lipoprotein (LDL) in experimental atherosclerosis.  LDL carries cholesterol from the liver to cells throughout the body.
At that time, there were only four reported studies in animals--all of them in rabbits--but in the intervening year and a half, two studies have been completed using primates, and two different antioxidant compounds have been utilized successfully. The panel also reviewed the epidemiologic data compatible with the oxidative modification hypothesis, and their final recommendation was that studies utilizing naturally-occurring antioxidant vitamins (e.g. vitamin E, B-carotene and vitamin C) should proceed. [2-7]
Supplements of the natural antioxidants carry little risk, if any, and further studies would be unlikely to importantly alter the design protocol of such intervention trials. In part because of the consensus at this Workshop, at least two clinical trials are already underway and two or three more are in the active planning stages.
Passwater: What was the "eureka" event that suggested to you that lipid peroxidation could modify LDL to start the atherosclerotic process?
Steinberg: By 1979, it was generally accepted that most of the cholesterol accumulating in early lesions (the beginning of "plaque" or "cholesterol deposits") was derived from LDL cholesterol, and that most of the cells containing lipid droplets (foam cells) arose from circulating monocytes (large general white blood cells) that entered the artery wall and became tissue macrophages (specialized white blood cells that engulf foreign material). The pioneering work of Drs. Michael S. Brown and Joseph L. Goldstein had shown that most of the cellular uptake of LDL occurred by way of a specific receptor on cell membranes, and that that receptor was missing in familial hypercholesterolemia.  Cells have various receptors to capture specific components transported in the blood and then carry them into the cell. Familial hypercholesterolemia is an inherited disease in which the LDL receptor is defective so they develop very high levels of blood cholesterol. These persons usually die of heart disease at a very early age.
Even though patients with familial hypercholesterolemia lack LDL receptors, they show enormous accumulation of LDL cholesterol in foam cells. Since some of these patients express absolutely no LDL receptors, it was necessary to conclude that the LDL must get in by some other pathway. Brown and Goldstein showed that chemical treatment of LDL with acetic anhydride converted the LDL to a form taken up more rapidly by macrophages, but there is no generation of acetyl LDL in vivo as far as anybody knows. 
The "eureka" experiment was done by Dr. Tore Henriksen and Dr. Eileen Mahoney in my laboratory in 1980. These findings were published in 1981.  Dr. Henriksen had done studies in Oslo showing that incubation of endothelial cells in culture with high concentrations of LDL led to cell death. He came to La Jolla to study this phenomenon further. I suggested that, in addition to trying to find out what the LDL did to the cells, he should concurrently ask what the cells were doing to the LDL. It turned out they were doing a lot!
The LDL reisolated from the cell culture medium after a 24-hour incubation with endothelial cells was markedly altered in its physical properties. More importantly, this physically modified (altered) LDL also showed one crucial change in biological properties--it was now taken up very avidly by monocyte/macrophages in culture. This is opposed to native (normal) LDL, which is not taken up very rapidly at all.
Passwater: That explains why LDL gets into macrophages to produce foam cells, but now the big question became what modifies the native LDL. How did you figure that out?
Steinberg: It took us more than six months to figure out what exactly was happening during the incubation that induced the alteration in the LDL. The "eureka" experiment there was done by Dr. Urs Steinbrecher who found that this change did not take place if we changed the medium in which the cells were grown. Only culture media that included some minimum concentration of metal ions was effective, and addition of antioxidants completely prevented the changes in the LDL. 
Passwater: So that's how the oxidative modification hypothesis got its start. Your group deduced that metal ions in the culture media modified native LDL via oxidizing the LDL. However, this is still a long way from actual body conditions. What happened next?
Steinberg: There quickly followed a number of other relevant findings, including the fact that oxidized LDL was chemoattractant for blood monocytes and could help recruit them into a developing lesion.  Also, it was soon determined that oxidized LDL inhibited the motility of tissue macrophages, which would tend to trap such cells in the artery wall once they got there.  Today the list of ways in which oxidized LDL behaves differently from native LDL has grown and we know a great deal more about the mechanisms involved. 
Passwater: You specifically said that the oxidized-LDL attracts monocytes and inhibits the resultant macrophages so as to trap them in artery walls. Why not veins?
Steinberg: Monocytes penetrate into vessels throughout the circulatory system at some rate, but they never accumulate in veins. Atherosclerosis simply does not develop in veins. But, if you surgically move a vein into the arterial system (as in a coronary bypass operation, for example), so that it is exposed to the high pressure of the arterial system, the vein will develop atherosclerosis. This process then is in fact quite similar to the process in arteries, including the migration of monocytes into the vessel wall and the accumulation of cholesterol, etc., etc..
Passwater: Is it accurate to say that only oxidized-LDL starts the plaque process?
Steinberg: No, it seems to me very likely that other modified forms of LDL are involved in plaque formation. What we know so far is that the use of antioxidants can decrease the rate of progression of lesions by 50-80%. That would speak to a major involvement of oxidation, but other things can also lead to foam cell formation. Studies by Dr. John C. Khoo in my laboratory have shown that aggregation of LDL with itself markedly increases the rate of uptake by macrophages.  The uptake in that case occurs by way of the native LDL receptor, not the acetyl LDL receptor or oxidized LDL receptor.
Studies by Drs. J. S. Frank and A. M. Fogelman at UCLA have demonstrated the generation LDL aggregates in the subendothelial space.  Aggregation does not depend upon prior oxidative modification. So here is a quite distinct mechanism by which LDL uptake into the macrophages can be accelerated and can perhaps initiate the fatty streak lesion.
Studies by Dr. Joseph L. Witztum and others in our laboratory have shown that minor modifications in the structure of LDL can render it immunogenic. Autoantibodies against oxidized LDL have been demonstrated in rabbits and in humans as well. Therefore, a complex of a modified LDL particle and an antibody against it can be taken up into macrophages by way of a completely different receptor, the receptor for immunoglobulins (the FC receptor).
So, there are at least two or three alternative modifications of LDL that could account for foam cell formation. These have not yet been studied in vivo as intensively as oxidative modification, and so we are not in a position to say with any confidence how important they may be.
Passwater: How does the body handle oxidized-LDL differently from normal LDL?
Steinberg: Whereas native LDL is recognized and taken into cells by way of the Brown-Goldstein receptor, oxidized LDL is recognized by the so-called scavenger receptors--the acetyl LDL receptor and a still incompletely characterized oxidized LDL receptor. The liver is very rich in receptors of the latter kinds. Consequently, when oxidized LDL is injected intravenously, it disappears from the blood at an enormous rate. Fifty percent of what you inject disappears in less than 5 minutes!
Of course, LDL that has only been oxidized minimally will not disappear so fast, and so, there may be a very, very small amount of oxidized LDL in the blood. Most of the oxidation that counts, however, probably occurs in the artery wall itself. Using appropriate antibodies that react specifically with oxidized LDL, we have been able to demonstrate its presence in arterial lesions (but not in normal artery).
Passwater: Can antioxidant nutrients reduce oxidation of LDL?
Steinberg: In the very early studies by Dr. Urs Steinbrecher in our laboratory, we showed that addition of vitamin E could completely prevent oxidation of LDL induced by incubation with cells in culture. 
Vitamin E is transported mainly in lipoproteins and presumably acts as an antioxidant defense, LDL actually contains a number of other antioxidant compounds, including beta-carotene, ubiquinol (coenzyme Q-10) and lycopene (a carotenoid found in tomatoes). When LDL is subjected to oxidative conditions, these antioxidants act as the first line of defense, and are themselves oxidized before the other component parts of the LDL molecule begin to undergo oxidative damage.
Dr. Hermann Esterbauer in Graz, Austria, was the first to show that when LDL is oxidized in the presence of copper, the first thing that happens is that the LDL content of vitamin E, beta-carotene, lycopene, ubiquinol, etc. drop sharply.  Only when they are all but used up do you begin to see oxidation of the fatty acids and of the cholesterol of the LDL. Vitamin C can also protect LDL, but it does it indirectly.
Vitamin C is soluble in water, but not in organic solvents or in lipids such as those found in LDL. So it can't act within the LDL particle. However, it can reduce oxidized vitamin E so that the molecule of vitamin E can act once again as a protective agent. In this indirect way, vitamin C "cycles" the vitamin E within the LDL particle.
The higher the vitamin E content of an LDL particle, the more it will be able to resist oxidative damage. However, the antioxidant content of the LDL is not the only factor determining its susceptibility. There is some evidence that the smaller LDL particles are more readily oxidized than the larger ones, and there may be other still undiscovered factors that play a role. However, it is well established now that adding supplements of vitamin E to the diet can increase the antioxidant content of the LDL and thus protect it, partially at least, from oxidative modification. [18-20]
The same is true for certain synthetic antioxidants. Probucol, butylated hydroxytoluene and di-phenyl-phenylenediamine also take up residence within the LDL, inhibit its oxidation and inhibit the progression of atherosclerosis. Supplements of natural antioxidants have so far been reported in only one study. Verlangieri and Bush fed monkeys supplemental vitamin E and found some inhibition of the progression of atherosclerosis. 
Passwater: Will you be involved in designing the prospective studies, and what are the chances of having these studies funded?
Steinberg: Dr. Witztum and I have joined hands with Dr. David Blankenhorn and his group at the University of Southern California, and Dr. B. Greg Brown and his group at the University of Washington in Seattle, to propose a three-center clinical test of the oxidative modification hypothesis. Our application has been submitted to the National Institutes of Health, and we are awaiting their verdict as to whether it can be funded or not. I will not be involved in the designing of other prospective studies unless invited to do so as a consultant. The NIH has just recently issued a request for proposals to test the antioxidant hypothesis as part of the Women's Health Initiative. So, the NIH is committed to exploring this new hypothesis intensively, and I think we can expect to see results in about four to six years from now.
Passwater: Dr. Steinberg, thank you for explaining your tremendously important research to us.
1. Antioxidants in the prevention of human atherosclerosis. Steinberg, D. and Workshop Participants Circulation 85:2338-2344 (1992)
2. 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 slowing the progression of atherosclerosis in the WHHL rabbit. Carew, T. E.; Schwenke, D. C. and Steinberg, D. Proc. Natl. Acad. Sci. 84:7725-9 (1987)
3. Probucol prevents the progression of atherosclerosis in Watanabe hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Kita, T.; Nagano, Y.; Yokode, M.; et al. Proc. Natl. Acad. Sci. 84:5928-31 (1987)
4. Probucol attenuates the development of aortic atherosclerosis in cholesterol-fed rabbits. Daugherty, A.; Zweifel, B. S. and Schonfeld, G. Br. J. Pharmacol. 98:612-8 (1989)
5. The antioxidant N,N'diphenyl-phenylenediamine prevents atherosclerosis in cholesterol-fed rabbits. Sparrow, C.; Doebber, T; Olszewski, J.; et al. Metabolism, p121 (abstract) (1992)
6. The antioxidant butylated hydroxytoluene protects against atherosclerosis. Bjorkhem, I.; Henriksson-Freyschuss, A.; Breuer, O.; et al. Arterioscler. Thromb. 11:15-22 (1991)
7. Effects of d-alpha-tocopherol supplementation on experimentally induced primate atherosclerosis. Verlangieri, Anthony J. and Bush, M. J. J. Amer. Coll. Nutr. 11:131-8 (1992)
(Also see: Reversing atherosclerosis: An interview with Dr. Anthony Verlangieri. Passwater, Richard A. Whole Foods p27-30 (August 1992)
8. A receptor-mediated pathway for cholesterol homeostasis. Brown, M. S. and Goldstein J. L. Science 232:34-47 (1986)
9. Binding site on macrophages that mediates uptake and degradation of acetylated low-density lipoprotein, producing massive cholesterol deposition. Goldstein, J. L.; Ho, Y. K.; Basu, S. K. and Brown, M. S. Proc. Natl. Acad. Sci. 76:333-7 (1979)
10. Enhanced macrophage degradation of low density lipoprotein previously incubated with cultured endothelial cells: Recognition by receptors for acetylated low density lipoproteins. Henriksen, T.; Mahoney, E. M. and Steinberg, D. Proc. Natl. Acad. Sci. 78:6499-6503 (1981)
11. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Steinbrecher, U. P.; Parthasarathy, S.; Leake, D. S.; Witztum, J. L. and Steinberg, D. Proc. Natl. Acad. Sci. 81:3883-7 (1984)
12. Endothelial cell-derived chemotactic activity for mouse peritoneal macrophages and the effects of modified forms of low density lipoprotein. Quinn, M. T.; Parthasarathy, S. and Steinberg, D. Proc. Natl. Acad. Sci. 82:5949-5953 (1985)
13. Oxidatively modified low density lipoproteins: A potential role in recruitment and retention of monocyte/macrophages during atherogenesis. Quinn, M. T.; Parthasarathy, S.; Fong, L. G. and Steinberg, D. Proc. Natl. Acad. Sci. 84:2995-8 (1987)
14. Beyond cholesterol: Modifications of low density lipoprotein that increase its atherogenicity. Steinberg, D.; Parthasarathy, S.; Carew, T. E.; Khoo, J. C. and Witztum, J. L. New Engl. J. Med. 320:915-24 (1989)
15. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Khoo, J. C.; Miller, E.; McLoughlin, P. and Steinberg, D. Arteriosclerosis 8:348-58 (1988)
16. Ultrastructure of the intima in WHHL and cholesterol-fed rabbit aortas prepared by ultra-rapid freezing and freeze-etching. Frank, J. S. and Fogelman, A. M. J. Lipid Res. 30:967-78 (1989)
17. Vitamin E and other lipophilic antioxidants protect LDL against oxidation. Esterbauer, H.; Rotheneder, M.; Striegl, G.; Waeg, G.; Ashy, A.; Sattler, W. and Jurgens, G. Fat Sci. Technol. 91:316-24 (1989)
18. Effect of oral supplementation with d-alpha-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. Dieber-Rotheneder, M.; Puhl, H.; Waeg, G.; Striegl, G. and Esterbauer, H. J. Lipid Res. 32:1325-32 (1991)
19. Effect of dietary supplementation with alpha-tocopherol on the oxidative modification of low density lipoprotein. Jialal, I. and Grundy, S. M. J. Lipid Res. 33:899-906 (1992)
20. Effect of dietary antioxidant combinations in humans: Protection of LDL by vitamin E but not by beta-carotene. Reaven, P. D.; Khouw, A.; Beltz, W. F.; Parasarathy, S. and Witztum, J. L. Arterioscler. Thromb. 13: in press (1993)
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