|The Discovery that Changed the Direction of Heart Disease Research |
|Interview with © Dr. Daniel Steinberg|
as Interviewed By© Richard A. Passwater PhD
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
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
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
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
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
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
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
Passwater: Dr. Steinberg, thank you for explaining your tremendously
important research to us.