How Vitamin E Prevents Heart Disease: An interview with Dr. David Janero

by Richard A. Passwater, Ph.D.

In recent columns, I have presented the evidence that vitamin E and other antioxidant nutrients are protective against heart disease. People -- especially scientists -- have trouble understanding this fact until they know "how" vitamin E accomplishes this feat.

One scientist -- a cell biologist and biochemist -- has done much to establish the necessary cellular evidence elucidating the protective mechanism. Dr. David Janero is a member of the senior staff in the Cardiovascular- Atherosclerosis Research Department of CIBA-GEIGY Corporation, Pharmaceuticals Division. He conducted his Ph.D. work in cell biology at the Yale University School of Medicine and was a National Institutes of Health Postdoctoral Fellow in biological chemistry at the Johns Hopkins University School of Medicine. Since 1983, he has held various research and development positions in the pharmaceutical industry. Dr. Janero and his research team have contributed to an increased understanding of the mechanisms of cardiovascular disease and have helped define the potential of both novel therapeutics and natural products (particularly vitamin E) to satisfy associated medical needs.

Their research findings are published in over 90 scientific reports and have been presented at many professional meetings, most recently at the Federation of American Societies for Experimental Biology conference "Vitamins E and C and Free Radical Reactions." I have asked Dr. Janero to explain how vitamin E protects against heart disease. I have set the stage in the previous column with Dr. Lester Packer, who explained how vitamin E stops free radicals including the lipid peroxidation process. Now Dr. Janero will help me continue the story. We discuss many of the ways in which vitamin E works to prevent heart disease, and then we look at the specific role of how vitamin E prevents low-density lipoprotein from becoming oxidized. This is the common ground that now has all cardiovascular researchers excited.

Passwater: You study heart disease from a different perspective than "traditional" heart researchers -- pathologists, cardiologists, and epidemiologists. You are trained as a cell biologist and biochemist, yet clinical cardiologists are learning from your work. What does your cellular and biochemical perspective bring to heart disease research?

Janero: Modern cell biology uses concepts and technology from many disciplines, including physical sciences such as chemistry and physics, to help elucidate how the basic unit of life, the cell, functions in health and disease. The critical importance of cell biology to medicine stems from a principle elucidated over a century ago: all disease has a basis in abnormal cell function. Biochemistry provides ways of probing and explaining cell function in quantitative terms. Consequently, the combination of cell biology and biochemistry is a powerful experimental avenue for me as a researcher on the origins and mechanisms of cardiovascular disease. Cell biology and biochemistry provide essential information to the physician who must understand disease mechanisms in treating cardiac patients and optimize potential means of prevention/therapy. The link between modern medicine and modern therapeutics is the cell biology of disease.

Passwater: The timing seems good for new research approaches such as your laboratory's. Many theories on the development of atherosclerosis that once were thought to "hold water" are now considered full of holes. Would you give us a brief overview of the current theory of "spontaneous atherosclerosis?"

Janero: In the 1800's, anatomists recognized that atherosclerosis is manifest as lipid-rich deposits in the walls of blood vessels, particularly certain arteries (hence, the alternative name "arteriosclerosis"). Such fat deposits reduce the vessel opening, thereby limiting the nutritive blood supply to whatever tissue or organ is downstream. Despite considerable research, the details of how atherosclerotic vessel disease develops remain elusive. Early thinking favored the concept that disruption or loss of the thin layer of cells lining the artery (the endothelium) precipitated atherosclerosis. Although rapid vessel blockage does occur after physical endothelial dosage, this accelerated form of vessel disease is distinct from "spontaneous" atherosclerosis.

Routine, spontaneous atherosclerosis is an injury response by the artery wall which develops chronically (over decades in humans) in the presence of extrinsic "risk factors" (e.g., high blood pressure, smoking, diabetes, excesses of certain blood lipids) that increase the likelihood of the first overt sign of atherosclerosis, the lipid deposit in the vessel wall termed the "fatty streak" because it consists of frothy-looking, lipid-laden "foam cells."

Although arteries in the body change structurally with time, this age-related differentiation is not pathologic and does not itself lead to atherosclerosis.

Passwater: Tell us a little about the structure of arteries.

Janero: Arteries are a type of blood vessel which receive nutrient-rich blood from the heart and conduct it to the major body organs. In humans, relatively large artery diameters mean that about 20% of the total circulating blood volume flows within these vessels. Generally, the artery wall contains an endothelial lining, one cell-layer thick, in direct contact with the circulating blood. Considerable connective tissue and smooth muscle are below the endothelium to support the artery and at the same time give the artery wall a fair degree of flexibility.

Passwater: Why did early researchers believe that cholesterol would just "zap" from the blood into the artery wall?

Janero: The mechanism by which certain cells in the artery wall (particularly so-called monocyte-macrophage cells) internalize circulating fat to form lipid-rich foam cells has been detailed only within the last 20 years or so. The research sought to explain a contradiction: lipid-laden foam cells tend to develop when blood low-density lipoprotein (LDL) levels are high, yet circulating LDL "fed" to isolated macrophages is not taken up by these cells.

Research on the cell biology of this seeming anomaly demonstrated that unregulated internalization of damaged or "modified" LDL particles through specific "scavenger receptors" on the macrophage surface leads to foam cell formation. The most pathologically relevant modification of LDL seems to be oxidation (i.e., free radical-mediated peroxidation of LDL polyunsaturated fatty acids). Therefore, oxidative lipoprotein damage can be considered a driving force for early lesion (i.e., fatty streak) formation [1]. Passwater: Conversion of smooth muscle cells of the artery wall into proliferating cells also seems important to the progression of atherosclerosis. What causes this conversion? Free radicals?

Janero: Smooth muscle proliferation is likely induced by small-molecule "messengers" released from nonmuscle cells (such as endothelial cells) in the artery wall. Growth factors within smooth muscle cells may regulate their proliferative response to exogenous chemical signals. It remains to be determined whether oxidants directly modulate the proliferative response.

Passwater: Do atherosclerotic lipid deposits appear mostly in specific arteries and/or at particular locations within arteries?

Janero: Atherosclerosis has a predisposition for critical arterial beds comprised of medium-sized arteries, e.g., the coronary and carotid arteries supplying heart muscle and brain, respectively, with nutritive blood. Potentially important predisposing factors in arteries may be the rather high oxygen tension of the blood flowing through them and the considerable hydrostatic pressure generated. But there is significant variability both along the length of the artery and around its circumference regarding the pattern of lipid deposition within the arterial wall. This variation is believed to reflect both blood-flow dynamics and the chronic, cumulative nature of the lipid build-up itself.

Passwater: Why might vitamin E protect against atherosclerosis?

Janero: The idea that vitamin E may help prevent the initiation and/or progression of spontaneous atherosclerosis is suggested by five main lines of largely experimental evidence:

(a) Vitamin E influences responses of cells in the artery wall and inhibits lipoprotein oxidation in ways which would be anti-atherogenic if occurring in the organism.

(b) Long-term vitamin E deficiency in animals causes atherosclerotic-like vessel lesions which can be limited, if not reversed, by vitamin E administration.

(c) Dietary vitamin E supplementation suppresses atherosclerosis in at least some animal models of atherosclerotic vessel disease.

(d) Vitamin E supplementation may help normalize a pro-atherogenic blood lipid profile.

(e) Epidemiologists have inversely correlated blood vitamin E levels in humans with mortality from coronary heart disease.

Although this evidence as a whole strongly suggests that vitamin E has therapeutic potential against atherosclerosis, definitive proof of such a role in man is lacking at present. The details can be found in a recent review [2].

Passwater: How does vitamin E affect the function of the endothelial lining of arteries in ways that might help prevent atherosclerosis?

Janero: Data on this question come mainly from work on isolated endothelial cells in culture. Vitamin E is a critical antioxidant protector of endothelial-cell membranes against the consequences of oxidative stress, particularly lipid peroxidation. More limited data suggest that vitamin E can promote endothelial repair and even suppress harmful oxidant production within the endothelium itself which could lead to, for instance, oxidation of lipoproteins.

Passwater: Would you describe the monocyte-macrophage and explain how vitamin E affects the functioning of this cell as a potential antiatherosclerotic agent?

Janero: The monocyte is a type of white blood cell produced in the bone marrow. Monocytes circulate in the blood for a limited time (about 40 hours in humans), whereupon they may enter connective tissue throughout the body, increase in size and metabolic activity, and be recognized operationally as macrophages. Through production and release of various chemical mediators, macrophages can recruit more monocytes into connective tissue from the circulation. Macrophages actively take-up and kill disease-causing microorganisms, making them critical to the inflammatory response. Active macrophages in the connective tissue of the artery wall have an impressive capacity for oxidizing biomolecules and lipoproteins.

The relationship between vitamin E and the macrophage is complex. The vitamin E in macrophage membranes helps the cell itself withstand oxidative stress. Vitamin E could prevent lipoprotein oxidation (and, thus, uptake of oxidized lipoproteins by the macrophage to form foam cells) by limiting macrophage oxidant production. It is particularly interesting, and more than a little ironic, that oxidized lipoproteins themselves stimulate monocyte differentiation to macrophages.

Passwater: You have mentioned oxidized lipoproteins several times now. Do you feel that lipoprotein oxidation is an important target for vitamin E as an antiatherosclerotic agent?

Janero: Inhibition of lipoprotein oxidation is increasingly being recognized as one of the most conceptually attractive and therapeutically important means by which atherosclerosis might be prevented and possibly even reversed.

Passwater: Let's discuss oxidized lipoproteins in detail later. You haven't mentioned ischemia-reperfusion injury to the heart. Isn't this what brought your work to the attention of classical heart-disease researchers in the first place?

Janero: My own work in the cardiovascular area began with lipoprotein synthesis and metabolism. Over the ten years since, my laboratory has conducted research in several areas, some well outside the cardiovascular system, largely due to my good fortune of having talented, dedicated colleagues and collaborators. Discussions with Drs. Adrianne Bendich and Lawrence Machlin stimulated me to study vitamin E in the heart muscle cell [3].

A constant research focus throughout this time has been cardiac ischemia, a well-recognized clinical complication of coronary-artery atherosclerosis because of diminished blood supply to heart muscle. The reduction in nutritive blood flow does not allow the heart-muscle cell to maintain normal function and metabolism. Several means (especially "clot busters" such as streptokinase and tPA) are now being used in the clinic to reperfuse the ischemic heart. Although reperfusion is essential to prevent death of ischemic heart muscle, reperfusion itself appears capable of extending ischemic cardiac damage by killing heart muscle cells which would have been expected to remain viable.

Our research on ischemia-reperfusion injury has focused on several potential mechanisms and mediators, including membrane lipids, derangements in energy metabolism, and oxidative stress [4]. We try to understand what factors act as determinants of heart muscle injury and how one might intervene to prevent post-ischemic reperfusion damage. Our overall goal in this area of our research is to help the cardiologist restore maximal pump function to the post-ischemic heart. For this discussion, it's especially noteworthy that vitamin E may have direct beneficial effects against cardiac ischemia [5].

Passwater: Earlier, you mentioned oxidized lipoprotein as a major target for the protective effect of vitamin E against atherosclerosis. What are lipoproteins?

Janero: Lipoproteins represent nature's elegant engineering solution to the following problem: How can a rather large amount of lipid (fat) be continuously transported through the body in blood, which is water-based and in which fats are not readily soluble? Lipoproteins are microscopic complexes of various types of lipid (triglyceride, cholesterol, phospholipid) and protein ("apoprotein") which, as particles, are blood-soluble. Structurally, the more polar lipid (i.e., phospholipid) as well as large portions of water-soluble apoproteins form a "shell" which surrounds a "core" of nonpolar lipid (triglyceride, cholesterol ester) having negligible water solubility.

Lipoproteins are classified according to their density, which reflects their lipid composition and lipid-to-protein ratio. In turn, the specific lipid and protein contents of the six major types of lipoprotein particle reflect their different functions and their metabolism. For example, triglyceride-rich very low density-lipoprotein (VLDL) transports triglyceride from the liver to peripheral tissues which use triglyceride as fuel, whereas low-density lipoprotein (LDL) is the major carrier of blood cholesterol. Normal lipoprotein metabolism interconverts lipoprotein particles of varying densities.

Passwater: What holds lipoproteins together to give them a particle-like character?

Janero: Several factors define and stabilize the organization of lipoprotein lipids and proteins. One factor is lipoprotein synthesis itself, which is highly organized within cells (especially liver cells) much like an automobile assembly line [6]. Other factors have to do with the physical chemistry of lipid-protein interactions in water: apoproteins and lipids tend to undergo self-association reactions which result in a more stable, low-energy aggregate. The three-dimensional structure of apoproteins also exerts some control over lipoprotein stability. Recent molecular analyses of apoproteins indicate that they possess lipid-binding regions which also help keep the lipoprotein particle intact.

Passwater: How does LDL become oxidized? Is it a simple oxidation reaction?

Janero: "Oxidation" is a very general term encompassing a variety of degenerative changes to LDL molecular constituents, mainly as a consequence of the peroxidation of LDL lipid and the decomposition of the resultant fatty peroxides. [7] Lipid peroxidation, the introduction of molecular oxygen into a polyunsaturated fatty acid to form a fatty hydroperoxide, is chemically complex and not well understood. [8] As with oxidative damage in any living system, LDL oxidation can occur only when oxidant stress on the LDL particle exceeds the ability of endogenous LDL antioxidants to detoxify the stress. LDL can be oxidized by any of the three major cell types in the artery wall (endothelial cells, smooth-muscle cells, and monocyte-macrophages). Biochemical studies on LDL isolated from humans clearly show that oxidants (free radicals/hydrogen peroxide), in the presence of transition metals (e.g., iron, copper), can initiate the peroxidation of polyunsaturated fatty acids in LDL phospholipid, cholesterol ester, and triglyceride molecules. Endogenous LDL antioxidants (e.g., vitamin E, various carotenoids) are consumed during LDL oxidation. Key intermediates generated during lipid peroxidation are lipid free-radicals. Lipid radicals can react with other polyunsaturated fatty acids in LDL particles to set up a "chain reaction" and rapidly generate oxidatively-modified LDL. The decomposition products of fatty peroxides damage LDL apoproteins, disrupting the organization of the LDL particle itself. The end result of LDL oxidation is an LDL particle with striking physical and chemical abnormalities.

Passwater: Why is oxidized LDL more likely than native, unmodified LDL to form foam cells?

Janero: Decades ago, pathologists recognized that macrophages in the arterial wall helped form the lipid-rich atherosclerotic lesion called the "fatty streak." Studies in the 1970's produced the surprising result that macrophages do not take up native LDL rapidly enough to become lipid-loaded, even when exposed to much higher concentrations of LDL particles than in the body. The Nobel laureates Drs. Brown and Goldstein at the University of Texas Medical Center, Dallas, provided the following explanation in a now classic series of experiments which exemplify the power of modern cell biology: the macrophage has on its surface LDL receptors which not only bring native LDL into the macrophage, but simultaneously reduce its uptake as the cholesterol level within the macrophage increases. [9]

Oxidized-LDL is recognized not by this receptor for native LDL, but by so-called "scavenger" receptors also on the macrophage surface. A critical distinction between these LDL receptor types is that scavenger receptors for modified LDL are not sensitive to cholesterol levels within the macrophage. Thus, the scavenger receptor brings cholesterol-rich, oxidized-LDL into already cholesterol-loaded macrophages, whereas the receptor for native LDL would not. With continued uptake of oxidized-LDL, the macrophages become filled with lipid and trapped within the artery wall. Their now frothy appearance in the light microscope has given them the name "foam cells." Accumulations of dead foam cells constitute the earliest overt sign of atherosclerosis, the fatty streak.

Passwater: Although lipoproteins circulate in the blood, it seems that LDL oxidation would have to occur within the artery wall, below the endothelial lining, where macrophages reside. How can this be?

Janero: Circulating LDL particles interact with endothelial cells lining the arteries and, as they move into and out of the subendothelial space, with macrophages and smooth muscle cells of the middle layer of the artery wall. All three of these cell types have the capacity to oxidize LDL. It is likely, although not yet proven, that most of the LDL oxidation which supports atherosclerosis takes place in LDL-rich "microenvironments" in the middle layer of the artery wall. Both macrophages and smooth muscle cells in this layer can internalize oxidized-LDL to form foam cells. Oxidized-LDL in the artery wall may recruit monocytes into the developing atherosclerotic lesion, greatly increasing the possibility of foam cell formation.

Passwater: Is there evidence that LDL oxidation represents a pivotal process in atherosclerosis?

Janero: Most of the evidence indicating that oxidized-LDL is a critical source of lipid for foam cell formation comes from studies in animal models of coronary artery disease. In such models, oxidative LDL modification has been documented, and antioxidant therapy can inhibit the development of early atherosclerotic lesions. Oxidized-LDL and lipid peroxides are present in human atherosclerotic lesions and even in the circulation of coronary heart-disease patients. Administration of a cholesterol-lowering antioxidant drug, probucol, to hypercholesterolemic patients reduces the oxidizability of their LDL. So there are several lines of evidence associating oxidative LDL modification with early atherosclerosis. [10]

Passwater: How could vitamin E help prevent LDL oxidation and fatty streak development?

Janero: Vitamin E is the principal, if not sole, chain-breaking antioxidant in the circulation. As a lipoprotein constituent, it reacts with lipid free-radicals to neutralize them and suppress LDL lipid-peroxidation. Indeed, only when endogenous LDL-vitamin E is virtually depleted can peroxidation of LDL lipid occur at maximal rates. Vitamin E may also modify the physical properties of the LDL particle, making it more resistant to free-radical attack, and spare other antioxidants in the LDL particle. At least in principle, the vitamin E of endothelial calls, smooth muscle cells, and macrophages could inhibit their ability to oxidize LDL. Equivocal data exist suggesting that vitamin E can alter LDL lipid composition in a way which would decrease its content of oxidizable lipid.

Passwater: What blood level of vitamin E would offer optimal LDL protection?

Janero: This question remains to be answered. The mere presence of vitamin E in the LDL particle is not enough to suppress LDL oxidation, suggesting that the ratio of vitamin E to peroxidizable polyunsaturated fatty acid in the particle must be above some threshold for vitamin E to be a decisive factor in reducing LDL-oxidation. It is likely that risk-factor variation within the human population may prohibit definition of a general "optimal" blood vitamin E level for LDL antioxidant protection.

Passwater: What might increase the need for antioxidant protection of LDL?

Janero: Any condition which would predispose LDL to oxidative stress or increase its susceptibility to lipid peroxidation would argue for fortifying the antioxidant content of LDL. For instance, vitamin E supplementation inhibits oxidative LDL modification induced by a major risk factor for atherosclerosis, cigarette smoking, which imposes a major oxidant burden upon the smoker. Diets which increase the polyunsaturated fatty acid content of LDL would also potentiate the LDL antioxidant requirement, as might administration of drugs whose metabolism leads to free-radical production. In contrast, it is interesting to note that some commonly-prescribed cardiovascular drugs, such as calcium antagonists, posses antioxidant properties themselves which may help explain their efficacy in some models of atherosclerosis. [11]

Passwater: Clearly, we don't have all the answers regarding vitamin E's impact on atherosclerosis. How do you envision research in the area through the 1990's?

Janero: I am very optimistic about future research on vitamin E (and other nutrients) and cardiovascular disease. The idea that diseases such as spontaneous atherosclerosis which develop over a long period of time are more easily prevented than cured gives particular urgency to defining the therapeutic potential of dietary vitamin E supplementation. Mechanistic investigations on nutrients and atherosclerosis, aside from their value to preventive medicine, should also help improve our understanding of how atherosclerotic vessel disease develops. Research regarding vitamin E's ability to protect the ischemic heart should not be forgotten either in our age of reperfusion injury and increasing awareness of the "silent ischemic" heart-disease population [12].

Passwater: What supplements do you take?

Janero: Aside from my research, I try to maintain some status as a "radical fighter" by taking a general antioxidant mixture including vitamin E, beta-carotene, cysteine, glutathione, and selenium. (Total vitamin E, 400 IU daily.) I also take a multivitamin-mineral supplement.


1. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. Steinberg, D.L., et al. New England J. Med. 320:915-924 (1989).

2. Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis. Janero, D.R., Free Radical Biol. Med. 11:129-144 (1991).

3. Oxidative injury to myocardial membrane: direct modulation by endogenous alpha-tocopherol. Janero, D.R., et al. J. Mol. Cell. Cardiol. 21:1111-1124 (1989).

4. Hydrogen peroxide-induced oxidative stress to the mammalian heart-muscle cell (cardiomyocyte): lethal peroxidative membrane injury. Janero, D.R., et al. J. Cell. Physiol. 149:347-364 (1991).

5. Therapeutic potential of vitamin E against myocardial ischemic-reperfusion injury. Janero, D.R. Free Radical Biol. Med. 10:315-324 (1991).

6. Sequential assembly of very low density lipoprotein apolipoproteins, triacylglycerol, and phosphoglycerides by the intact liver cell. Janero, D.R. and Lane, M.D. J. Biol Chem. 258:14496-14504 (1983).

7. Free radical modification of low-density lipoprotein: mechanisms and biological consequences. Heinecke, J.W. Free Radical Biol. Med. 3:65-73 (1987).

8. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Janero, D.R. Free Radical Biol. Med. 9:515-540 (1990).

9. A receptor-mediated pathway for cholesterol homeostasis. Brown, M.S. and Goldstein, J.L. Science 232:34-47 (1986).

10. Therapeutic potential of vitamin E in the pathogenesis of spontaneous atherosclerosis. Janero, D.R. Free Radical Biol.Med. 11:129-144 (1991).

11. Protection of cardiac membrane phospholipid against oxidative injury by calcium antagonists. Janero, D.R., et al. Biochem. Pharmacol. 21:4197-4203 (1988).

12. Therapeutic potential of vitamin E against myocardial ischemic-reperfusion injury. Janero, D.R. Free Radical Biol. Med. 10:315-324 (1991).

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