Arteriosclerosis and Its Origins
Some of the earliest knowledge about arteriosclerosis came from dissections of the human body that were carried out by the anatomists of Renaissance Italy. Leonardo da Vinci, the famous Italian artist, engineer and scientist, is generally credited with the first of these dissections in the 15th century. Some years later, Andreas Vesalius, a member of a prominent Belgian medical family and a founder of modern human anatomy, described abnormalities that he found in the aorta and artery branches during his dissections of deceased human subjects in Venice and Padua in the 16th century. Even in this early period, the abnormalities of the arteries, later called arteriosclerosis, were related to social standing. In dissections performed in 18th century Holland, those persons with the most severe abnormalities were found to be from the wealthy classes of Dutch merchants or professionals.
In the 19th century two of the founders of modern medicine, pathologists Karl Rokitansky and Rudolf Virchow, began the systematic study of changes in the tissues of deceased persons. In 1842 Rokitansky published his comprehensive and influential handbook of the changes that he had observed in the tissues of people who died of many different diseases. In a later edition of that handbook he suggested that the elements of the blood, including blood clots and blood serum, formed layers on the internal lining of the arteries. 1 With time, these layers incorporated blood elements into a tough, hardened, calcified artery wall that characterizes the striking changes of arteriosclerosis, according to Rokitansky.
In the mid- and late-19th century, Rudolf Virchow, the famous German pathologist who introduced microscopy of tissues to the study of pathology, offered his own version of the origin of arteriosclerosis. 2 He pointed out that in this disease the wall of the artery first undergoes a form of degeneration in which mucoid substances become deposited in the tissue. Then fatty substances from the blood enter the artery wall and become deposited, forming atheromas—raised swollen areas on the internal lining surface (intima) of arteries with arteriosclerosis. The inside of atheromas contains a porridge-like substance. The word atheroma is derived from the Greek words for porridge (athere) and swelling (-oma). The word atherosclerosis refers to the advanced form of the human disease characterized by multiple atheromas. Virchow also pointed out that arteriosclerosis, hardening of the artery wall, is caused by fibrous tissue and deposits of calcium salts. The word arteriosclerosis comes from the word artery and the Greek word for hardened (sclere).
In the 19th century the most common cause of death was infectious disease, especially pneumonia and tuberculosis. Virchow suspected that a type of infection or inflammation might contribute to arteriosclerosis. As evidence for this idea, he pointed out that cells associated with infections (leukocytes) are sometimes found in arteries with arteriosclerosis. He also likened the swellings of the artery wall, atheromas, to tumors of the blood vessels. He found that the cells of the arterial wall are stimulated in arteriosclerosis, thereby increasing in number and narrowing the channel within the artery.
Diet and Arteriosclerosis
In 1908, after learning the methods of tissue examination that were introduced by Rokitansky and Virchow, M.A. Ignatovsky, a young teacher in a military medical school in St. Petersburg, Russia, decided to apply these methods to investigating the cause of arteriosclerosis. Like the Dutch anatomists of the 18th century, he had observed that his patients with severe arteriosclerosis tended to be from the wealthy class, making their diet suspect. Doctors in England had also suspected the diet of the wealthy class, which contained a larger amount of meat, butter, eggs and milk than the diet of poorer classes. Ignatovsky decided to feed this type of high animal protein diet to rabbits to determine whether he could observe changes in the arteries similar to those of his patients with arteriosclerosis. The rabbit normally eats a diet without any animal protein, obtaining its protein entirely from grains and vegetables.
After feeding the animal protein diet to rabbits for several months, Ignatovsky found that the animals had developed hardening and plaques of the arteries and aorta. He demonstrated that these changes were very similar to the arterial plaques of patients with arteriosclerosis. Because of these findings, Ignatovsky suggested that a diet with abundant meat and dairy products could cause arteriosclerosis in his patients. In his publications he meticulously drew by hand beautiful illustrations showing the changes he had found in the rabbit arteries through direct examination and with his microscope. He published his findings in several prominent French, German and Russian medical journals. 3 4 Infection could not explain the arterial changes, since no infectious agents were given to or found in his rabbits. Therefore, he suggested that it was the high protein diet that was responsible for the hardening of the rabbit arteries.
Several years later, at another medical school in St. Petersburg, two young doctors, Nikolai Anitschkov and S. Chalatov, became interested in Ignatovsky's findings. Previously, they had little success in producing arteriosclerosis in animals by injecting bacteria or by other methods. Although Ignatovsky had attributed his results to the protein in the meat and dairy products he fed the rabbits, the plaques in the rabbit arteries contained fats and cholesterol crystals. The German pathologist Ludwig Aschoff had also found cholesterol in human atheromas. The chemists of the late 19th century had isolated cholesterol in a pure form and determined its chemical structure. An-itsckow believed that the cholesterol in the meat and dairy products of the Ignatovsky diet could have produced the arteriosclerotic plaques in the rabbit arteries. Therefore, Anitschkov and Chalatov tried giving cholesterol to rabbits either mixed dry with rabbit chow or dissolved in vegetable oil.
After feeding rabbits this diet for several months, they found that the arteries of the animals developed the changes of arteriosclerosis similar to what Ignatovsky had observed. They found a great deal of fat and some cholesterol crystals in the artery walls. The liver and other organs also contained abundant fat and cholesterol, a condition known as cholesterolosis. They believed that Ig-natovsky's results could have been produced by the cholesterol in the meat and eggs in his experimental diet and not by the animal protein. Their paper was published in a German medical journal in 1913. 5
All of these studies, carried out in rival medical schools in pre-revolutionar> St. Petersburg, clearly established that experimental arteriosclerosis could reliably be produced in animals by dietary manipulation. For the first time, the cause of arteriosclerosis could be related to a nutritional origin, replacing the somewhat vague concepts of causation by infection or inflammation that were inherited from the previous century. Of course, the results of the studies implied that arteriosclerosis in humans is also induced by nutritional modifications or abnormalities. It was not clear, however, from these early experiments, which factor or factors in the diet were responsible for the experimental disease in rabbits or for human arteriosclerosis and heart disease.
Protein Intoxication and Arteriosclerosis
In 1922 Dr. Harry Newburgh, a full-time professor of clinical medicine and clinical research at the University of Michigan School of Medicine, began to devote a major portion of his efforts to his passion for clinical research on nutrition and the physiology of human disease.
Newburgh painstakingly reviewed the provocative results published by Ignatovsky, Chalatow and Anitschow and other investigators of arteriosclerosis during the previous decade. He was intrigued that the disease arteriosclerosis could be induced in rabbits by manipulating their diet. He repeated the earlier Russian experiments and confirmed their results. He anticipated that a systematic investigation of the effect of meat protein would clarify the picture. He and his associates prepared lean beef muscle that was dried, powdered and extracted with solvents to remove all traces of fat and cholesterol. They fed increasing doses of the powdered meat protein to rabbits, along with white flour and bran. Newburgh found that the higher the protein content of the diet, the sooner and more severe were the arteriosclerotic changes in the rabbits' arteries. 6 Further experiments were needed to prove that some constituent of meat protein was producing these striking changes.
Newburgh reasoned that if meat protein in the diet produces arteriosclerosis in rabbits, he should be able to produce similar changes in the arteries by intravenously injecting one by one, the individual amino acid components of protein into dogs or rabbits to discover which amino acid is the most damaging to the arteries. After these experiments were completed, he concluded that the arteries were not damaged by any single amino acid.
However, Newburgh discovered that cystine, the first amino acid to be identified in 1810 by William Wallaston, is toxic and very damaging to the kidneys when it is injected intravenously. (The name cystine is taken from the Greek word for bladder— kystis —because it was first isolated from human urinary bladder stones.) Cystine contains sulfur and is chemically related to homocystine, which does not occur in proteins. Several other amino acids in protein (lysine, histidine, tyrosine and tryptophan) also cause kidney damage when injected intravenously. 7
Subsequent events explain why none of the amino acids of protein injected singly into animals were found to cause arteriosclerosis. The amino acid methionine, discovered in 1922 by Herman Mueller, and the amino acid homocysteine, discovered in 1932 by Vincent DuVigneaud, were not among the amino acids that Newburgh tested. At the time of his experiments, methionine was not known to be an essentia] component of proteins and homocysteine was completely unknown.
Newburgh's experiments were a "near miss" in medical science. If he had been able to give methionine or homocysteine by intravenous injection, the damaging effect of homocysteine on arteries would have been discovered in 1925! Nevertheless, Newburgh's discoveries were put to good use in treating patients with kidney failure and diabetes. The "Newburgh-Marsh low-protein diet" became the standard treatment for kidney failure until the introduction of artificial kidney dialysis and kidney transplantation over a generation later. Newburgh also introduced a low-carbohydrate, high-fat diet for treatment of severe diabetes in the era before the discovery of insulin.
Vitamins and Arteriosclerosis
The first indication that a vitamin deficiency could be blamed for causing arteriosclerosis was the series of experiments with vitamin B6 deficiency in monkeys, as described in Chapter 1. In these experiments James Rinehart and his associates formulated a synthetic diet that contained no vitamin B6. After periods of vitamin B6 deficiency lasting 6 to 18 months, the monkeys' arteries were found to contain arteriosclerotic plaques characterized by loss of elasticity, hardening and narrowing of the lumen from fibrous tissue and deposits of mucoid substances containing tissue fluids. 8 Only with prolonged intermittent or partial deficiency of vitamin B6 were deposits of fats found in some of these plaques. When the synthetic diet contained added vitamin B6, no changes were observed in the arteries. Deficiencies of other vitamins did not cause arteriosclerotic plaques in monkeys.
Rinehart, like Virchow before him in the 19th century, suggested that mucoid degeneration is the earliest change in arteries with arteriosclerosis. He believed that fats from the blood plasma, including lipoproteins and cholesterol, could enter the artery wall and become deposited in association with the mucoid substance. In a meticulous study of the coronary arteries of persons who had died of arteriosclerosis and heart disease that was published in 1963, this sequence of events was generally confirmed in human subjects. 9
Rinehart's results were independently reproduced and confirmed in dogs and monkeys at the Merck Institute. 10 Subsequent experiments in Japan also confirmed them, and the scientists there went on to show that the plaques induced by vitamin B6 deficiency regressed following intensive periods of vitamin B6 treatment. 11 The experiments with animals suggested that a widespread deficiency of vitamin B6 in the human population could be a factor in causing arteriosclerosis. Rinehart's early results suggested that the amount of vitamin B6 in human blood samples was sub-optimal. Furthermore, the amount of vitamin B6 that was necessary to prevent weight loss in vitamin B6-deficient monkeys (50 meg per kilogram of body weight) was considerably greater than that consumed by humans (20 meg per kilogram of body weight) according to nutritional surveys.
Why was this significant discovery of the relation between vitamin B6 and arteriosclerosis neglected in the 1950s and 1960s? Although Rinehart related his findings to the importance of vitamin B6 in the breakdown of proteins, there was no theory which could explain why a vitamin deficiency produces changes in artery walls. Furthermore, vita-min-B6 deficient monkeys and dogs had normal blood cholesterol levels and only a small amount of fat and cholesterol was found in a few of the plaques of the arteries. When James Rinehart died in 1955, no investigator was able to continue his pioneering work.
When scientists a$ the Harvard School of Public Health took up the question of vitamin B6 and arteriosclerosis, 12 they made their animals so deficient in vitamin B6 that the animals lost weight, became severely anemic and died within 4 to 9 months. Rinehart had subjected his monkeys to intermittent periods of complete vitamin B6 deficiency or prolonged periods of partial vitamin B6 deficiency, allowing the animals to survive for periods as long as 23 months. Arteriosclerotic plaques require prolonged periods of intermittent or partial vitamin B6 deficiency of 6 to 12 months to allow the change in arterial cells and arterial elasticity and hardening to develop. Finally, vitamin B6-deficient animals develop plaques that are easily seen only by microscopy. The Harvard scientists used a method of staining with fat-soluble dyes to search for fatty plaques in the lining. Rinehart had found no fatty plaques there and neither did the Harvard scientists.
The seemingly minor differences between Rinehart's experiments and the Harvard experiments, however, led to a drastically different interpretation of their significance. The Harvard scientists concluded that vitamin B6 deficiency produces no arteriosclerosis in monkeys, even though their animals died of deficiency before the changes could have developed and the method they used to study the arteries would not have detected the significant changes Rinehart had found. Furthermore, the Harvard scientists could not accept the finding that arteriosclerotic plaques were developing in the monkeys without the apparent involvement of cholesterol and fats. The result of this contradictory interpretation of Rinehart's discovery was that other scientists lost interest and felt that this area of investigation was not promising.
Cholesterol, Fat and Arteriosclerosis
Since the early years of the 20th century, the prevailing theory about arteriosclerosis has been that cholesterol and fats in the diet are responsible for the buildup of cholesterol in the plasma and for the deposition of cholesterol in arteriosclerotic plaques.
Prompted by the demonstration of cholesterol crystals and fatty deposits in arteriosclerotic plaques by Ludwig Aschoff and by the finding of similar plaques in rabbits that were fed cholesterol by Nikolai Anitschkov, many medical scientists incriminated dietary cholesterol and fats in the causation of arteriosclerosis. Nutritional surveys of susceptible populations showed a general correlation between the fat content of the diet and the incidence of arteriosclerosis. 13 However, other surveys showed that consumption of dietary sugar, white flour and protein from foods of animal origin also correlated with the incidence of arteriosclerosis. Finally, a more detailed examination of dietary composition revealed a correlation between consumption of unsaturated oils of plant foods or fish with protection against arteriosclerosis.
The populations of Asian countries are less susceptible to arteriosclerosis than populations of western countries, suggesting that racial factors may be of importance in causing the disease. Early studies, however, showed that Asian populations consuming a Western diet, such as Americans of Japanese ancestry in Hawaii and California, developed increased cholesterol in the blood and increased risk of arteriosclerosis, compared with their relatives living in Asia. Indonesian stewards aboard Dutch ships were found to develop increased blood cholesterol and increased risk of arteriosclerosis after eating the Dutch diet. 13 These studies suggest that dietary factors are more important than racial factors in the cause of arteriosclerosis.
Another line of evidence supporting the correlation between elevated blood cholesterol and susceptibility to arteriosclerosis is derived from the study of genetic diseases affecting blood lipoproteins. The clearest example of such a disease is familial hypercholesterolemia, a rare condition occurring in one person per million. In this disease all cells of the body are unable to process and internalize LDL normally. As a result, the level of LDL and cholesterol in the blood becomes extremely elevated, leading to rapidly progressive and sometimes fatal arteriosclerosis.
Another example of a genetic disease affecting blood lipoproteins is dense LDL hypertriglyceridemia, in which small dense aggregated LDL particles are correlated with a high risk of arteriosclerosis. In yet another example, the condition affecting a group of individuals with familial dyslipidemic hypertension is believed to be of genetic origin. These individuals have small dense LDL particles, hypertension, diabetes mellitus and reduced HDL levels, greatly increasing their risk of arteriosclerosis.
In addition to familial diseases of cholesterol and lipoprotein processing and diets containing abundant fats, sugars and animal protein, other major factors contributing to human arteriosclerosis have been identified. These factors include advanced age, male gender, postmenopausal status, smoking and other toxins, hypertension, diabetes, hypothyroidism and lack of exercise. The influence of these major risk factors for arteriosclerosis on levels of blood cholesterol, LDL and HDL has been intensively studied for many years. The general results of these studies have shown that there is a correlation between these major risk factors and the elevation of LDL and total cholesterol and the decrease of HDL in the blood.
A problem with the risk factor approach is that a majority of individuals with arteriosclerosis have no abnormality of total blood cholesterol or lipoprotein fractions. Practicing physicians know that many of their patients with severe or fatal arteriosclerosis, including myocardial infarction, kidney failure or gangrene of the toes, have normal cholesterol and lipoprotein levels. Furthermore, no comprehensive theory has been developed which satisfactorily explains how arteriosclerosis risk factors affect cholesterol levels or how elevated LDL and decreased HDL levels initiate formation of arteriosclerotic plaques.
The development of a reliable and versatile clinical blood test for measurement of cholesterol in plasma (the Lieberman-Burchard test) greatly aided the study and understanding of the importance of cholesterol in human arteriosclerosis. The development of ultracentrifugation, electrophoresis and chromatography to determine blood lipoprotein fractions enabled medical scientists to explore and evaluate the importance of LDL, HDL and other lipoprotein fractions in the cause of arteriosclerosis.
In studying the formation of cholesterol in the body, scientists discovered that most of the cholesterol in the blood is formed by biochemical processes in the liver. Cholesterol is an important major constituent of the membranes of all cells of the body. Furthermore, cholesterol is needed for the production of the sex hormones estrogen and androgen in the ovary and testis. Cholesterol is also needed for production of stress and mineral hormones of the adrenal gland. Finally, cholesterol is excreted in the bile in ihe form of bile salts that are made from cholic acid, the degradation product of cholesterol, and two amino acid derivatives, glycine and taurine. The amount of cholesterol that is formed in the liver is carefully controlled and adjusted according to the needs of the different organs of the body. If the amount of cholesterol is increased in the diet, a healthy, well-functioning liver makes less cholesterol for the needs of the body. If the amount of cholesterol in the diet is decreased, the liver makes more cholesterol. In this way the body regulates very precisely how much cholesterol is produced for its needs.
Oxidation, Cholesterol and Arteriosclerosis
If cholesterol is produced within the body to make cell membranes, steroid hormones and bile salts, how could an excess of dietary cholesterol induce arteriosclerosis? In the 1950s the tantalizing clues that were uncovered by Ignatovsky, Newburgh and Rinehart to incriminate dietary protein receded into the background of medical thinking and were largely ignored because no theory could explain how protein intoxication or vitamin deficiency could produce arteriosclerosis. On the other hand, the cholesterol/ fat approach became the favorite of the medical/pharmaceutical establishment because of the gradual development of knowledge in this field. Yet the cholesterol/fat hypothesis also needed a comprehensive theory to explain how cholesterol, a normal chemical constituent of the body, could, when overeaten in the diet, induce a generalized disease of the arteries by causing an increased level of LDL and a decreased level of HDL in the plasma. Two related questions are (1) how does a diet high in saturated animal fats promote arteriosclerosis and (2) how does a diet high in unsaturated plant or fish oils protect against the disease?
In trying to understand how cholesterol and lipoproteins of plasma could cause arteriosclerotic plaques, medical scientists considered the nature of these plaques as they occur in human disease and in experimental animals. In the early stages of human arteriosclerosis, a mucoid change occurs in the artery wall, and soon thereafter fatty substances from the plasma enter the artery wall, forming "lipid streaks." These early changes are seen as flat areas on the lining of arteries containing fats as demonstrated by fat-soluble stains. Wandering cells of the blood called monocytes enter the lining of the artery and take up lipoproteins, transforming them into "foam cells" that contain abundant fat droplets. As the disease progresses, however, changes in the artery wall occur to produce loss of elasticity and hardening caused by fibrous tissue and deposits of calcium salts. In later stages of human arteriosclerosis the calcified plaques of fibrous tissue give the walls of most affected arteries a tough, brittle, hardened consistency that is very difficult to cut either with scissors or with a scalpel blade. Only in the aorta and some large arteries do the excessive deposits of fats and proteins cause soft atheromas to become prominent. The idea that arteries in human arteriosclerosis are narrowed only by greasy fatty deposits is simply not true for most arteries. The main changes, as pointed out by Rokitansky, Virchow and other scientists in the 19th century, involve increased fibrous tissue and calcium deposits, making the artery wall hardened, brittle and tough, hence the name arteriosclerosis.
One of the problems with the cholesterol-feeding experiments of Anitschkow and similar experiments by other scientists is that the arterial plaques in the animals contained too much fat and cholesterol in comparison with the plaques found in the human disease. Another problem with the cholesterol/fat experiments is that the LDL fraction ot lipoprotein is largely innocuous when injected directly into arteries or other tissues, failing to damage artery walls. Moreover, added LDL is not readily taken up by cell cultures of macrophages to form the foam cells that are known to be key players in the formation of arteriosclerotic plaques. Some further change seems to occur that makes the LDL of individuals with arteriosclerosis injurious to the lining surface of arteries. This change in LDL leads to the formation of foam cells, fatty streaks and subsequent transformation of these early abnormalities into advanced plaques that are brittle, tough and hardened by fibrous tissues and calcium deposits.
Early attempts to identify which component of LDL is injurious to artery walls led to the testing of many chemical relatives of cholesterol. A group of cholesterol compounds that contain extra oxygen atoms was found to be highly toxic when tested in cell cultures and in fragments of aorta maintained in culture. 14 Moreover, several of these oxidized cholesterol compounds, called oxycholes-terols, produced injury to artery walls, deposition of fat and arteriosclerotic plaques when fed to rabbits. One of the most toxic and injurious of these oxycholesterols is cholestane triol, which contains three added oxygen atoms per cholesterol. Oxycholesterol was also found to greatly reduce cholesterol formation by cultured cells because of its toxic effects on the enzymes of these cells.
In a series of studies of oxycholesterols, medical scientists at Albany Medical College showed that highly purified cholesterol, chemically freed of all traces of oxycholesterols and protected from the oxygen of air, does not produce arteriosclerosis when administered to rabbits or monkeys. 15 The method that these scientists used to purify cholesterol was developed in the 1950s by Professor Louis Fieser, a prominent chemist at Harvard when I was a student in his organic chemistry course. He selected several of his students (including me) to help him develop this method. We added bromine to cholesterol to produce di-bromocholesterol, which was highly purified by repeated crystallization. When the bromine was removed by acid, extremely pure cholesterol was then protected from the oxygen of air by storage under nitrogen gas in the deep freezer.
These experiments with oxycholesterols showed not only that these chemical derivatives are highly effective in producing arteriosclerosis in animals, but they also showed that highly purified cholesterol, freed of all traces of oxycholesterols and protected from the oxygen of air, does not injure the arteries of animals. This important discovery means that the experiments of Anitschkow and other scientists who had fed cholesterol to animals have to be reinterpreted. Unless these scientists took precautions to prevent the exposure of the cholesterol that they used in their experiments to the oxygen of air, their experiments could be interpreted as showing that oxysterol contaminants (other than cholesterol itself) were producing arteriosclerosis in their experimental animals. In addition, these oxycholesterols have been discovered in foods in association with fats of animal origin, particularly those foods which have been heated in the presence of air. Finally, these oxycholesterols have been discovered in the LDL fraction of human blood plasma and in the arteriosclerotic plaques of human arteries.
Modification of LDL by Arterial Cells
Following the discovery of the toxic effects of oxycholesterols, scientists studied the process by which LDL reacts with oxygen in the body. Experiments with cells cultured from arteries (endothelial cells, smooth muscle cells) and from blood (macrophages/monocytes) showed that these cells are able to add oxygen to LDL particles. 16 These living cells use oxygen in a way that causes LDL to become dense and to contain oxycholesterols, oxidized fats and oxidized proteins. In experiments using solutions without exposure to living cells, this modified LDL is formed by free radical oxygen damage in LDL samples that are incubated with copper or iron salts. Several compounds containing sulfur, including homocysteine, have been found to hasten this modification of LDL when solutions are exposed to the oxygen of air.
The experiments in which LDL is exposed to living cells in culture or exposed to metal salts in solution show that oxygen causes LDL to change into a form that is readily taken up by macrophanges to form foam cells in cultures. There is doubt, however, that this process occurs in this way in individuals with arteriosclerosis. Modified LDL does not occur in plasma, and antioxidant substances in plasma prevent formation of modified LDL. It is much more likely that LDL is taken up by arterial cells and that oxygen is added to LDL within these cells during the early stages of formation of arteriosclerotic plaques. Other experiments with animals have shown that there is a generalized disturbance in the way that all tissues of the body handle oxygen in arteriosclerosis.
The studies of Drs. Michael Brown and Joseph Goldstein in the 1970s showed that cells cultured from individuals with familial hypercholesterolemia lack a cell membrane receptor that is responsible for internalization and processing of LDL. 17 As the result of this failure to process LDL normally, extremely high levels of cholesterol and LDL build up in the blood of these individuals. The victims of this disease frequently develop heart disease and arteriosclerosis in their teens and 20s. These studies show that the receptor for LDL on cell membranes is an important factor in controlling how cells process and utilize the cholesterol and other consitituents of LDL.
The process by which cells take up LDL has been studied intensively by medical scientists because of the importance of foam cells in arteriosclerotic plaques. Wandering cells of the blood (monocytes) ordinarily take up very little LDL, as shown by experiments with cultures of these cells. When the LDL is first modified by artificial chemical reactions (such as acetylation or carbamylation), however, the cells eagerly take up modified LDL by means of a membrane protein called a "scavenger receptor." As the result of this process, the cells take up abundant modified LDL and store cholesterol within their cytoplasm to form "foam cells." In the progression of arteriosclerotic plaques, foam cells release cholesterol and fats into early plaques.
In advanced plaques, cholesterol crystals and fatty deposits are formed by the continued release of cholesterol and fats from foam cells. The artificial chemical modification of LDL is very unlikely to be of importance in human arteriosclerosis, however, since this chemical process does not occur in living cells and tissues.
While these studies of the interaction of LDL with cells and its modification by reaction with oxygen have illuminated important aspects of the formation of arteriosclerotic plaques, there remain many unanswered questions about the relation of these processes to the underlying causes of arteriosclerosis in the general population. What chemical modification of LDL occurs in human arteriosclerosis that leads to its internalization and formation of foam cells in early plaques? How do dietary fats and cholesterol affect the process of modification of LDL by the cells of developing arteriosclerotic plaques? Is there any relation between changes in LDL processing or LDL modification and the effects of protein intoxication on artery walls as studied by Ignatovsky, Newburgh and Rine-hart? How can the process of LDL internalization and modification by reaction with oxygen within arterial cells be altered by dietary changes, drug therapy or other measures for the prevention or treatment of arteriosclerosis? Current scientific investigation of the cholesterol/fat hypothesis is focused on answering these questions.
Blood Clotting and Arteriosclerosis
An increased tendency to form blood clots within arteries, a process called thrombosis, is characteristic of human arteriosclerosis. During the progression of plaques, the blood clots that form in areas of damage to the arterial lining contribute to the narrowing of the artery lumen. Small blood clots that adhere to the surface of plaques gradually become incorporated into the plaque, increasing the thickness of the plaque over a period of weeks and months. In advanced arteriosclerosis, therefore, incorporated areas of blood clots, deposits of cholesterol and fats, fibrosis and deposits of calcium salts form complicated arteriosclerotic plaques. If this process is gradual and progressive, the function of vital organs is affected over a period of months or years. For example, when the arteries to the legs are gradually and progressively narrowed by these advanced complicated plaques, the amount of blood flow in the toes becomes progressively compromised and greatly diminished. The result of this process is that the tissues of the toes and feet gradually die, a condition known as gangrene. This painful condition is commonly treated by surgical grafting of artificial arteries in order to restore blood flow to the toes and feet. If grafting is unsuccessful, or if the arteriosclerosis is too far advanced for surgical therapy, amputation of the toes, foot or leg may become necessary because of pain and life-threatening gangrene. A similar progression of advanced arteriosclerotic plaques commonly affects the arteries leading to the brain, heart and kidneys, causing gradual loss of function of these vital organs.
A more dramatic and sudden effect of arteriosclerosis occurs when a large blood clot forms in an artery that is already severely narrowed by plaques. In the coronary artery a thrombosis of this type deprives a portion of heart muscle of blood flow, causing death of part of the heart. This condition results in acute heart attack, a complication known as acute myocardial infarction. Careful studies have shown, however, that complete coronary occlusion by thrombosis is the end result of a complex series of changes in the wall of the artery including degeneration of plaque contents, rupture of arterial wall tissue and bleeding into the affected plaque. 9 When the carotid artery to the brain is affected by sudden and complete occlusion by thrombosis at the site of advanced arteriosclerotic plaques, a stroke is caused by death of brain tissue.
The propensity for individuals with arteriosclerosis to develop blood clots which gradually or suddenly reduce blood flow in vital arteries has led to the use of anticoagulant drugs to control this complication. Aspirin is a drug that decreases the reactivity of blood platelets, the cell-like components of blood that are required for blood clotting. Population studies have suggested that small daily doses of aspirin may prevent the occurrence of thrombosis in individuals susceptible to coronary heart disease or stroke. Another approach is to use drugs, such as tissue plasminogen activator [TPA] or streptokinase, that activate plasminogen, a normal blood enzyme that helps to dissolve blood clots. These drugs help to limit damage to the heart or brain when given promptly after formation of an occlusive thrombus in a coronary or carotid artery. By helping to dissolve the blood clot that is occluding the artery, the blood flow is reestablished, reducing the size of the damaged area in the heart or brain.
The injury to arterial cells and tissues in the early stages of arteriosclerosis triggers complex cellular and molecular interactions, known as the response-to-injury hypothesis. 18 The injury of the lining cells of arteries triggers a reaction by blood-clotting factors and platelets that leads to the formation of fibrin, the principal component of blood clots. This reaction causes the platelets and arterial cells to release protein growth factors that stimulate growth of the muscle cells of artery walls. This injury also causes white blood cells to adhere to the site of injury, forming more foam cells and releasing more growth factors and other cell-signalling molecules called cytokines. The result of these complex interactions is increased growth of the muscle cells of the artery wall, production of fibrous tissue and ground substance by these cells and the deposition of fatty substances, including cholesterol, from LDL within the site of arterial injury.
Lipoprotein(a) is a genetically determined component of lipoprotein that has been found to be correlated with susceptibility to arteriosclerosis in several studies. 19 Because of its close chemical relation to the enzyme plasminogen, which normally controls blood clotting, lipoprotein(a) is widely assumed to be a factor in promoting blood clotting. Thus, because of its chemical composition and structure, this substance is likely to be involved both in the deposition of fats in arteriosclerotic plaques and in limiting the ability of the body to dissolve blood clots. The therapeutic action of streptokinase or tissue plasminogen activator in heart attack or stroke depends on activation of the plasminogen of blood plasma to dissolve blood clots. However, some doubt has been cast upon the importance of lipoprotein(a) in arteriosclerosis by failure to find an association between elevated lipoprotein^) and risk of myocardial infarction. 20
Shortcomings of the Cholesterol/Fat Approach
During its 80-year reign, the cholesterol/fat explanation of the cause of arteriosclerosis became, despite its many shortcomings, the favorite of the medical-pharmaceutical establishment. A reason for its wide acceptance is that it offers a general explanation for the correlation between elevated levels of LDL and decreased HDL and susceptibility to arteriosclerosis. The approach also offers a general explanation of the correlation between composition of the fatty constituents of the diet and susceptibility to the disease. It does not, however, address adequately the other correlations between susceptibility to arteriosclerosis and the consumption of animal protein and highly processed foodstuffs, including sugars and white flour. The approach also explains the experimental induction of elevated LDL levels and deposition of fat in arteriosclerotic plaques in experimental animals. Insufficient attention, however, has been paid to the key role of oxycholesterols in the experimental disease.
Some of the shortcomings of the cholesterol/fat approach are of major significance. Perhaps its most important failure is the lack of explanation for the rapid escalation of incidence of arteriosclerosis, heart disease and stroke during the mid-20th century in America and its subsequent dramatic decline beginning in the mid-1960s. Detailed studies of the composition of the American diet failed to reveal a correlation between cholesterol and fat content and the major changes produced by arteriosclerosis. In general, the fat and cholesterol content of the American diet has changed very little during recent decades which saw a two- to three-fold decline in the incidence of heart disease, stroke and other manifestations of arteriosclerosis. Moreover, there were no significant changes in blood levels of LDL and cholesterol during this period. Other factors, including changes in medical therapy and lifestyle factors such as smoking and exercise, cannot explain why the incidence of arteriosclerotic disease has declined.
Another related shortcoming of the cholesterol/fat approach is its failure to demonstrate a correlation between the cholesterol and fat composition of the diet and the level of LDL in susceptible populations as exemplified by the 50-year-old Framingham Heart Study. Experiments with animals also fail to demonstrate a correlation between dietary cholesterol and the level of cholesterol in the LDL of plasma. The feeding experiments in which cholesterol was added to the diet of animals were compromised by failure to consider the potent effects of the oxycholesterol contaminants of the added cholesterol.
In accordance with its position as the leading cause of mortality in America, arteriosclerotic heart disease and stroke have been the subject of intensive clinical investigation over the past four decades. Starting with the Heart-Diet Pilot and the Coronary Drug Project in the 1960s and 1970s, continuing with the Multiple Risk Factor Intervention Trial, the Coronary Primary Prevention Trial, and other studies of the 1980s, a tremendously expensive and detailed effort was made to evaluate the efficacy of cholesterol-lowering drugs, hormones, vitamins and diets in the prevention of coronary heart disease. During this long period of clinical trials, low-cholesterol/fat diets; a series of drugs such as cholestyramine, clofibrate, gemfibrizol, colestipol and lo-vastatin; hormones such as estrogens and dextrothyroxine; and the vitamin niacin have been evaluated for their ability to lower serum cholesterol and LDL levels and to raise the HDL level. While modest reductions in cholesterol or LDL were observed in some studies, the overall reductions in coronary heart disease and mortality have been slight or negligible in most studies. More recently, aggressive anti-cholesterol and anti-LDL therapy has been claimed to provide some evidence of modest regression of plaques, as shown by angiographic X-ray studies of coronary arteries. 21 Despite the decades-long effort by thousands of medical investigators, however, coronary heart disease stubbornly remains the leading cause of death in America, and aggressive diet and drug therapy have shown only marginal to modest efficacy in combating the disease.
At a practical level, physicians know that the majority of their patients with coronary heart disease, stroke and other forms of arteriosclerotic disease have no evidence of elevated cholesterol or LDL levels. In a study of 194 consecutive autopsy studies of mostly male veterans, for example, I found that only 8 percent of cases with severe arteriosclerosis had total cholesterol levels greater than 250 mg/dL, and the mean blood cholesterol level in the group with the severest disease was 186.7 mg/dL. 22 This study did confirm that cholesterol levels are positively correlated with severity of arteriosclerosis in patients with minimal, moderate or severe disease. In this study two-thirds of the patients with severe arteriosclerosis had no evidence of elevated blood cholesterol, diabetes or hypertension.
It is significant that the 80-year history of the cholesterol/ fat approach has yet to provide a coherent and comprehensive scientific theory which explains in detail how cholesterol, a normal constituent of the body, or excess dietary fat in the diet of susceptible populations produces arteriosclerotic plaques. The current explanations of the modification of LDL 16 and the response-to-injury hypothesis 18 do not offer a comprehensive theory that links these pathogenic processes to known causative factors in the disease.
Recent epidemiological surveys have suggested that diets or supplements containing abundant vitamin E are of benefit in reducing the risk of coronary heart disease in both women and men. 23 Vitamin E is a potent fat-soluble antioxidant vitamin that may act by modifying the reaction of LDL with oxygen. Recent studies, however, have shown that another antioxidant vitamin precursor, beta-carotene, lacks a preventive effect on coronary heart disease, as predicted by the LDL modification hypothesis. Similarly, the findings that premenopausal women and postmenopausal women taking estrogens are protected against arteriosclerotic heart disease are not readily explained by the modified LDL hypothesis. No adequate epidemiological test of the response-to-injury hypothesis has yet been devised, so this explanation of the genesis of arteriosclerosis remains primarily a detailed description of cellular and molecular events in the progression of arteriosclerotic plaques.
Nutritional Deficiency and Arteriosclerosis
The entire history of the cholesterol/fat hypothesis explaining the cause of arteriosclerosis is based on the unproven assumption that the disease is produced by overconsumption of the normal dietary constituents cholesterol and fat. Only in the case of the oxycholesterols is there compelling evidence to suggest that a trace or contaminant constituent associated with fat and cholesterol in the diet is actually injurious and capable of initiating arteriosclerotic plaques. Overconsumption of cholesterol and fats in the diet, however, may be linked to concomitant underconsumption of nutritional constituents, in particular, the nonfat, water-soluble and indigestible fiber components of the diet.
The idea that vitamin deficiencies (vitamins B6, B12 and folic acid) could be responsible for or participate in the cause of the most common disease in America has not until recently been taken seriously by the medical community. Yet the overconsumption of fats, sugars and highly processed foods that are depleted of these vitamins is just the circumstance which could lead to widespread nutritional deficiencies of the water-soluble vitamins that are easily destroyed or depleted by food processing. In this way the concept of underconsumption of vital nutrients that are lost or destroyed in food processing, preservation or preparation is diametrically opposed to the assumption that overconsumption of a major dietary constituent could be the underlying cause of arteriosclerosis.
The cholesterol/fat approach has become so widely embraced for so long by the nutritional and food industry establishments that a different concept of the cause of arteriosclerosis has been difficult for many to accept. Because of the ingrained nature of conventional thinking, any approach based on other considerations, discoveries and theories that differ from conventional wisdom has been considered unthinkable and unacceptable.
The National Cholesterol Education Program of 1987 was founded by a consensus development conference on cholesterol and heart disease sponsored by the National Heart, Lung and Blood Institute of the National Institutes of Health in Bethesda in 1984. The focus of the recommendations of the "consensus conference" was to recognize that blood cholesterol, when elevated, increases the risk of arteriosclerotic heart disease. The National Cholesterol Education Program further went on to declare that dietary or drug therapy which succeeds in lowering blood cholesterol will reduce the burden of arteriosclerosis and heart disease in America. Nowhere in the proceedings of the "consensus conference" or in the National Cholesterol Education Program was there any consideration of the possibility that the underlying cause of the disease could be related to nutritional deficiencies. The idea that the food industry might cause the nation's number-one killer disease by creating a food supply that is seriously deficient in B vitamins has been considered preposterous by many.
The vitamin deficiency approach to the prevention of arteriosclerosis has until recently been regarded by the medical community as old-fashioned, ineffective, outmoded and without sufficient foundation in scientific evidence. The idea that expensive drugs or drastic diets to control elevated blood cholesterol could be ineffective in preventing vascular disease is generally considered unthinkable by the pharmaceutical-medical complex. The proposed comprehensive program of drug therapy to prevent elevated blood cholesterol levels in as many as 50 million individuals is potentially enormously profitable to the pharmaceutical industry. Moreover, recent efforts have been directed to another enormously profitable measure, using aggressive drug and dietary therapy in children in an effort to combat elevated blood cholesterol levels in childhood. The unproven assumption is that this strategy could prevent arteriosclerotic disease, especially coronary heart disease, in adulthood. In contrast, the vitamin deficiency concept of the cause of arteriosclerosis promises to be extremely effective without yielding excessive profits to the pharmaceutical companies that manufacture unpatentable, unprofitable and inexpensive vitamin additives to the food supply. This strategy will not allow the pharmaceutical industry to reap the bonanza of profits entailed in the anticholesterol drug market.
The risk factor approach has made important advances in understanding the relative importance of diet, family history, gender, smoking, lack of exercise, aging, obesity, diabetes, hypertension, elevated blood cholesterol and LDL and kidney failure in the causation of arteriosclerosis. However, this approach has not yet produced a coherent scientific theory that explains how these factors lead to and cause the arteriosclerotic plaques and blood-clotting abnormalities that produce the human disease. New thinking and new strategies, provided by the discovery of the connection between homocysteine and vascular disease as described in the chapter which follows, are needed to develop a scientifically coherent and persuasive theory of the underlying cause of arteriosclerosis in susceptible populations. 24
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