Understanding Atherosclerosis by Stevan

The Accumulation of Fat in Injured Tissue Cells is Key to Understanding the Degeneration of the Heart, the Kidneys, and the Disease, Atherosclerosis (A Review)

Non-common abrevations: OCD â&#x20AC;&#x201C; Oncosis Cell Death or the other cell death, historically called Necrosis. Acknowledgments: Frank B. Johnson, M.D., Chief of Histochemistry Department, Armed Forces Institute of Pathology, Washington, D.C.; Assistance with references: Jackie L. Rogers, MLS, SLIS

The Accumulation of Fat in Injured Tissue Cells is Key to Understanding the Degeneration of the Heart, the Kidneys, and the Disease, Atherosclerosis (A Review) Introduction This review of medical research literature regarding the Accumulation of Fat in Injured Tissue Cells is broad in its scope of search, but limited to relevant issues in its presentation. It does not deal with the chemistry of fats. Although the terms fats and lipids are used interchangeably, all fats are lipids but not all lipids are fats. In the early medical literature, all lipids were called fats. There is an important thread of knowledge running through the literature on atherosclerosis and diseases of the myocardium which involves a physiological process common to tissue injury, which results in injury to the organs of the body with high energy requirements for metabolic life. This is the observation of the accumulation of fat in injured tissue cells. This concept is valuable because it points to the cause of susceptibility of tissues to injury involving a deficiency in oxygen necessary to support energy metabolism. Prominent in the understanding process is the knowledge of and definition of the cell death process, oncosis, â&#x20AC;&#x153;the other cell death,â&#x20AC;? hereafter referred to alternately as OCD. Two main issues supporting these investigations are: 1) that lipids accumulate in tissue when there is a deficit of oxygen to support the normal metabolism of the tissue; and 2) that this accumulation is reversible as the offending metabolic conditions are alleviated. Although this injury process seems to be very general in many organ tissues such as heart, kidney, and liver and even in bone; the heart and the blood vessels are emphasized here because, in those organs, the case was strongly researched and reported by investigators. This literature search has an objective to find a unity concept in all the occasions of oxygen deficiency which lead to injury and are characterized by fat accumulation in the injured tissue cells. Also a valuable concept would be the idea that the injury and subsequent degeneration of the vital organs were due to causes of deficiency which could be remedied. The outstanding message would be to remedy first all causes of oxygen deficiency to the cell. The second, more difficult message would be to protect a person from toxins such as bacterial toxins, viral disease, immunological insult, or other known conditions which compromise the energy generation process of the cell involving the use of oxygen. One of the problems which deterred researchers was the difficulty in characterizing fats and lipids with stains. This contributed to the relative obscurity of the studies of fats in tissues. The difficulty of preparation necessary for the microscopic examination for fats in organ tissues contributed to the neglect. Stains of oil red 0 and the Sudan fat stains only stained the triglyceride lipids. Therefore, special stains were necessary for phospholipids, and no practical stain was available to characterize cholesterol in tissues. The fat was often related to vacuoles (sites in tissue) where fat apparently resided before 2

the usual slide preparation process common to practice in general tissue pathology removed it. Osmium salts stain lipid in electron micrographs as black osmium residues in the tissue preparations. Cells progressing to necrosis leave cellular debris which contains some fat. Acute and rapid cell death results in no residual tissue fat. The cellular debris resulting from cell death by this process goes through a standard cleanup by phagocytes; then the tissue is repaired in the usual manner. The phagocytic activity is a part of the inflammatory process. It is demonstrated thus far that injured tissue cells under a slow chronic process will accumulate fat. The cells accumulating fat are usually being injured gradually, while acute injury resulting in rapid death does not result in tissue accumulation of fat. Understanding the physiological process involved in the degeneration of the vital organs and atherosclerosis provides a basis for early intervention to protect the health of individuals and slow the aging process. Early History Historically, Rudolf Virchow in 1858 observed the accumulation of fat in injured tissues (1). He subsequently studied the degeneration of the arterial wall, which now is recognized as the disease atherosclerosis. He also established the microscopic study of diseased tissue cells leading to the medical science of tissue pathology. He was the first pathologist. In 1932 Hans Ulrich studied the accumulation of fat in the vital organs of guinea pigs (2). He placed guinea pigs in a deficient oxygen atmosphere and over time studied the effect of hypoxia on the tissues of the heart, liver and kidneys. The results demonstrated the accumulation of fat in the tissues of these organs. The source of the accumulated fat was from the fat storage areas of the animals. An interesting side issue was the protection of one guinea pig from the characteristic accumulation process. That animal was found to be pregnant during the study. In 1958-59 while on a United States Air Force research assignment at the Armed Forces Institute of Pathology, Washington, D.C. (AFIP), the author conducted a research study using rats to confirm the findings of Hans Ulrich. The study was set up using four white rats. The selected rats included two lean rats and two fat rats. These specifications were defined by the AFIP which provided the animals. The rats did not look fat in appearance. The fat rats were only slightly larger than the lean rats. The rats were placed in an oxygen deficient environment employing a chamber with adjustments for altitude above sea level. The oxygen deficient altitude of 20,000 feet was selected. The animals were exposed to the deficient oxygen for 16 hours. During that time period, the animals were observed in order to check their activity at the end of a period of eight hours. The result of the observation at eight hours was dramatic in nature. The lean rats were moving about the chamber with normal activity, while the fat rats were stationary at the air intake port and struggling for breath. The fat ratsâ&#x20AC;&#x2122; breathing motion evidenced a dramatic struggle to increase their air intake, confirming that the fat rats were requiring more oxygen than the lean rats. This observation supports the idea that a limited diet is a healthy diet; and also defines the health problems of the obese as a problem of oxygen utilization. 3

The slides for microscopic examination were prepared from the liver, heart and kidney tissues of all four animals. The tissue slides for microscopic examination were stained for fat with Oil Red O. The results of microscopic examination showed a slight fat accumulation in the tissues of the lean rats and an intense accumulation of fat in the liver, heart and kidney tissues of the fat rats. This research experience motivated this further review of the medical literature. As early as 1944, Researchers reviewed the literature on the subject of accumulation of fat in tissues as an indicator of cell injury (3) (4). They recognized that the injury was caused by an inadequate supply of oxygen to the affected tissues. They recognized that the injury was also caused by a decrease in the phosporylation production of ATP. This medical literature search resulted in a number of issues which have broad support. The reporting of these issues serves to result in the understanding of the degenerative process in compromising the normal function of the vital organs. A summary of the issues and resulting processes sets the stage for the understanding of the benefits from this effort. Issues and Processes In order to set the stage for understanding the review presentation, the issues and processes must be understood. The issues and the resulting processes are: 1) The accumulation of fat in injured tissues was the central idea pursued during the literature search. 2) The cause of cell injury is an oxygen deficiency which causes an interruption in the production of ATP and ultimately the cutoff of production of ATP. The injury results in a unique process of cell death which must be contrasted and not confused with apoptosis. 3) There is evidence that the prominence of apoptosis, (programmed cell death in current medical research) may add confusion as to the type of cell death emphasized. The oncosis cell death or the other cell death (OCD) process is not apoptosis, which is programmed cell death. 4) Important to the understanding of the injury and repair process is the discovery that the so-called fibrocyte which functions in the tissue repair process is, in the case of vital organ tissues, a multi-functional cell which develops from the main functional cell of the organ under study. 5) The repair process most often results in a fibrosis of the injured site, not a replacement of the original functional tissue of the organ. This is especially true in the heart. 6) The cause of the tissue injury is primarily any situation which limits the oxygen available to cells of the tissues. 7) The organs which have a high energy requirement are those most affected by oxygen deficient injury, and the heart has the highest demand for oxygen. 8) Anatomical distance from the source of oxygen is a factor. 9) Exposure to carbon monoxide has a role in the tissue injury. The carbon monoxide (CO) combines with hemoglobin and thereby limits the distribution of oxygen. The CO also poisons the enzymes in the cytochrome system; thereby affecting the phosphorylation system producing ATP. 4

Among the important issues is the necessity to understand that the support of cell metabolism with nutrition and oxygen is only available from the circulatory blood in arteries and capillaries as distributors to the tissue cells. It is not evident that blood flowing through arteries and close to the artery wall contributes oxygen adequately to adjacent artery wall tissue. This is also true of the blood in the chambers of the heart. There is overwhelming evidence that the blood in the chambers of the heart does not nourish and provide adequate oxygen to the myocardium. Sub-Clinical Death of Cells The value of this review of the medical research literature has resulted in the discovery of a sub-clinical process of slow death of the functional cells of the vital organs, heart, kidneys, as well as the arterial wall disease, atherosclerosis. There is currently some recognition of a subclinical injury to the heart muscle which is known as “silent ischemia”. An understanding of this slow but eventually destructive process is based on an understanding of a tissue cell death process which exists as another cell death process and is not to be confused with apoptosis, which is programmed cell death. This other cell death (OCD) is a cell death resulting from cell injury and ends in necrosis. The cell injury is caused by a wide variety of recognized diseases, but the subclinical diseases caused by chronic and sometimes slow cell injury manifest themselves only after the condition has progressed to a level of clinical recognition, or at autopsy. Examples are the diseases of degeneration or weakness of the heart muscles, degeneration of the heart valves, and the various degenerative diseases of the kidneys. The accumulation of lipids in cells separates the two fundamental cell death processes. The process of programmed cell death is characterized by a shrinking of the cell followed by removal of the dead debris of the cells by phagocytes. This process of apoptosis serves to eliminate cells in a very effective manner to fulfill the purpose of a programmed cell death process. The other cell death process, which is not well-defined in the literature, is a degenerative process which is caused by the respiratory compromise of the tissue cells of the various organs such as heart, kidneys, liver and even the arteries. It is this poorly understood process which is central to the understanding of the degeneration process discovered by this effort. The death process or “other cell death process” (OCD) has been named oncosis because the first step involves swelling of the cell. However, the word oncosis could easily be confused with its close association with cancer. A more straightforward name would be “cloudy swelling”, which is the first cellular effect recognized by pathologists. The process as observed in the cell historically has been characterized by the light microscope as 1) cloudy swelling; 2) accumulation of fat; and 3) necrosis. It is important to understand this degenerative process of the cell because it is the key to understanding the degenerative diseases of the vital organs, and even the disease process of atherosclerosis. The entire book “When Cells Die” is devoted to the explanation of and the significance of apoptosis (5). However in chapter two, Benjamin F. Trump and Irene K. 5

Berezesky contrast in detail the differences between Oncosis, “the other cell death”, OCD and apoptosis. These authors present an excellent contrast between the two processes of cell death, but in doing so, they introduce labels to the steps in the injury and death process which may confuse rather than enlighten. The first of these is the choice of the pre-lethal phase of the OCD as oncosis which is derived from the Greek word, “oncos”, meaning swelling. The first evidence of the OCD is the swelling of the injured cell. In contrast, the process of apoptosis begins with a shrinking of the cell. It is unfortunate that oncosis as a term could be confused with the study of oncology which is historically involved with tumors and cancer. Therefore, this review will introduce and define the other cell death, OCD, as opposed to apoptosis, programmed cell death. The cell death process emphasized here is OCD which is characterized as: Step 1: Cloudy swelling. Step 2: lipid accumulation, and Step 3: necrosis. Further detailed explanation of the different characteristics of apoptosis is beyond the scope of this review. It is emphasized that the term necrosis must remain in its classical definition as the end point of cell death due to injury. In the literature, generally necrosis is used as the identity of the other cell death (OCD). The cause of the degeneration is a disabling of the oxygen related respiration process of the cell. Therefore, the degeneration is most prevalent and readily observed in the tissues of the body requiring high energy, such as the heart and kidneys. The second issue is, What is the cause of the onset of this degeneration process? The experimental data shows that hypoxia is the reference test condition. However, as a result of research from 1950 through 1990 and beyond, there are many agents and conditions contributing to the attacks on cellular respiration of the vital organs. They somewhat parallel the enumerated causes of atherosclerosis, among which are: bacterial toxins, viral effects and anemia, as well as the hypoxia caused by inadequate delivery of oxygenated blood. Organ health is dependent on the adequate vascular support for blood delivery, and susceptibility to injury is a function of the inadequacy of the oxygen transport of the arterial blood delivery system. The anatomical variability and the degree of adequacy of the arterial system could result in an inadequate oxygen delivery to specific areas of organ tissue, and thereby contribute to the degenerative process. This would provide a completely new concept of susceptibility. This new concept is valuable today because the remedy is on the horizon in the form of new blood vessels provided by the use of cytokines for angiogenesis. The main issue is to connect the research in this area to a unified cause for the cellular degeneration seen in the tissue of the heart, arteries and kidneys. The early investigation of hypoxia damage to tissues was conducted by Ulrich, but the degenerative process was recognized by Virchow even earlier. As stated previously, Rudolph Virchow, the first pathologist, reported the accumulation of fats in injured tissue cells. Although his report on fat accumulation in injured cells led to an understanding of the connection of fat accumulation in the artery wall to the disease atherosclerosis, it was to cause controversy through the years. Some investigators attempted to explain the process as not accumulation, but rather a revealing of the fat from a non-stainable state to a stainable state where the presence of fat in tissue was demonstrated. 6

Greater Understanding of the Degenerative Process The understanding of the degenerative process from chronic attacks on the vital organs was given a great step forward by Schlesinger and Reiner at Harvard Medical School (6). In 1955, Schlesinger and Reiner studied the small infarcts of the heart muscle and discovered very small foci of dead cells they called myocytosis. They attributed these infarct-like lesions to a â&#x20AC;&#x153;metabolic imbalanceâ&#x20AC;?, which, when defined, resulted from a deficiency of oxygen available to the cells. This deficiency resulted in a lowering of ATP production and subsequent cut off of ATP production. They linked the myocytosis to conditions of anemia and other conditions resulting in ischemia. Their greatest contribution to the understanding of the degenerative process was the stepwise explanation of a cell death sequence which has been substantiated by research on the other organs as the other cell death process or (OCD). It begins with swelling of the injured cell, the accumulation of fat as a second step, and finally results in necrosis. Their chart of the death sequence is reproduced from their research below as a quotation. In order to become manifest in routine microscopic sections. The metabolic imbalance will have to act with a great enough intensity and for a long enough time. The combination of these two factors may result in a degree of metabolic imbalance so slight as to cause only reversible lesions of the muscle fibers such as cloudy swelling, hydrops, or fatty infiltration. On the other hand, the degree may be so severe as to cause the irreversible necrosis of an infarct. These relationships are indicated in Table III, with the implication that the graded anatomical responses listed are not restricted to myocardial ischemia due to coronary atherosclerosis but may result from other disturbances of metabolism. Table III Schema of Regressive Cardiac Responses

Lesion

Reversibility of process End Result

Mild Cloudy swelling Hydrops Fatty degeneration

Degree of metabolic imbalance Moderate Severe I. Focal 1) active I.Miliary infarct Myocy- 2)inactive with stromal Tolosis 3)healing necrosis. II. Miliary infarct without stromal necrosis

II.Massive infarcTion

Reversible

Irreversible

Restitution to normal

Fibrous scar

Such a concept would explain, for instance, the occurrence of any of these lesions, including infarct necrosis in the absence of significant coronary atherosclerosisâ&#x20AC;Ś It is necessary to emphasize the importance of central characteristics of the other cell death (OCD) which are paramount to the understanding of the degenerative processes applied to the vital organs in future presentations. Historically, the research into the accumulation of fat in injured cells was the staining of triglycerides and later the recognition of the accompanying accumulation of the other lipids, phospholipids, and lipoproteins along with the deposits of cholesterol crystals. The necrosis process is associated with a strong inflammatory reaction which, in its end point, may involve deposits of calcium salts. Historically, the recognition of the lipid was limited to triglyceride stained with the Sudan stains and oil red O. Later in the 1950s and 1960s, the cholesterol in tissue was recognized as anisotropic deposits which were recognized by birefringence and also appeared in urinary sediments observed microscopically as a crystalline Maltese cross. It was not until the 1970s that researchers used monoclonal antibodies to identify the presence of the lipoproteins in the tissue with the use of immunological techniques to identify these lipids in tissue lesions (7). The fixation with Osmium salts, which were used extensively for electron microscope studies, detected the lipids as a black precipitate of Osmium. The necrosis process may result in tissue calcification. Another interesting fact is that the repair process which follows cell injury and death is a function of the main operational cell of the organ. These cells convert into a synthesis role which produces the fibrous tissues of repair. The idea that tissues could be nourished by the adjacent sources such as the lumen of an artery or the endocardial area of the heart is defined. There is very limited metabolic support other than the normal vascular distribution system. Two areas free of the destructive deficiency are, in the case of the artery, the tissue is not damaged in an area equivalent to the intima plus a few muscle layers in the media, and, in the case of the heart, the muscle area two millimeters adjacent to the endocardium. This indicates very limited nourishment by diffusion from adjacent flowing blood. The centers of the degenerative lesions are situated beyond the ends of the pure coronary circulation. The nourishment of the tissues of the heart and the artery wall, other than the normal vascularization, is the vasa vasorum and limited Tran endothelial diffusion. The degree of metabolic imbalance may be so slight as to cause only reversible damage of cloudy swelling or fatty accumulation, or it may be so severe and long-lasting as to cause irreversible necrosis of an infarct. The greatest contribution of Schlesinger and Reiner is their detailed description of the cell death process involved. This cell death process is the basis for understanding the degenerative process weakening the vital organs and resulting in the disease atherosclerosis. A key element to identify this death process is the accumulation of fat 8

in the tissue cells in step two of the process. Another important element is the type of necrosis which generates residue particles which support a subsequent inflammation process. This inflammation process is a well recognized factor in the disease process. The contribution of Schlesinger and Reiner is significant because they identified a degenerative condition of the heart muscle which is sub-clinical in nature as to not be identified as a heart attack with appreciable infarct, but a chronic condition which, if it becomes extensive enough, could weaken the heart muscle. This condition may be recognized as â&#x20AC;&#x153;silent ischemiaâ&#x20AC;?. The lesions of myocytosis were observed in the heart muscle of cats subjected to an oxygen-deficient environment. The next important issue is the fibrotic cell (which is recognized as the source of tissue fibrosis) versus the mesachondrial multi-function cell (8). It becomes established that the organ tissues did not have to depend on a fibrocyte to do repairs. There is a unity of agreement that the major functioning cell of the organ changes from its major role in the vital process of the organ into a role as synthetic cell to produce the chemical constituents of the fibrosis process. In the heart, it is the myocyte and in the arterial vessel wall, it is the smooth muscle cells of media origin. To summarize, the oncosis cell death process involves the following: initially, cell swelling (cloudy swelling), then the accumulation of fat (lipids), then cell degeneration which then leads to necrosis, which characteristically involves cell debris which supports an inflammatory process in the tissues. This sets the foundation for using this OCD to understand the degeneration of the vital organs. This cell death process is not to be confused with the apoptosis process which is programmed cell death. The scope of the study of the vital organs includes the heart, kidneys and a very specialized organ â&#x20AC;&#x201C; the arterial blood vessels. The arterial blood vessels must be viewed as a specialized tissue requiring metabolic support as an organ (9). Characteristic of all of these vital tissues is the high degree of the requirement for oxygen. The heart is the organ with the largest requirement for oxygen. At this juncture, some discussion is necessary concerning the physiological metabolic reserves actually available to these vital organs. The heart, as an example, does not have a situation of oxygen reserve. Contrary to the understanding propagated by early physiologists, that the human body has substantial reserves, the true picture is one of living on the edge. Under the most favorable circumstances of health, the truth about oxygen use in an organ with peak oxygen requirements has little margin on the adequacy of oxygen supply. It is noted by physiologists that when the blood being pumped to the heart through the coronary arteries leaves the heart, the oxygen content of the blood is practically depleted (10). Therefore, any circumstance or disease causing a deficiency of the oxygen content of a volume of blood will have a detrimental effect on the function of the heart. It can be expected that the other organs such as the kidneys and the artery wall as high energy tissues will be affected similarly. As the review progresses, this fact will become apparent for each organ under study. There will be a number of conditions which influence the amount of oxygen available to the organs but there is an anatomical variation to be considered. The degree of vascularity or adequacy of vascularity is emerging as a predisposing factor in the 9

degeneration of the vital organs. Although the arteries and arterioles of the heart, for the most part, could be considered normal in their distribution, some variation including insufficiency might be expected. It is known, for instance, that the vascularization of the apex of the heart can be deficient and that deficiency has been measured. Correction has been successful in early attempts to increase vascularization by punching holes into the wall of the heart and thereby increasing angiogenesis. In this review it will be noted that the degenerative processes and injury to tissues occur more intensely as the tissues are a greater distance from the heart which is the source of oxygenated blood. There are other additional reports of degeneration of the heart which are examples of injury and subsequent OCD. Research reports on other degenerating conditions of the heart are reported. The injury issues were demonstrated by Shnitka and Nachlas in their experimental production of heart muscle infarcts in forty-two dogs (11). The experimental infarcts were produced by tying off a branch of the coronary arteries. An hourly check on the progress of infarction was conducted. Microscopic histochemical observations were conducted on an hourly, daily and weekly basis. The results followed the characteristics of heart muscle tissue cell injury. Cloudy swelling was noted; accumulation of fat was noted; Necrosis of the heart muscle was noted; and during the injury process the loss of ATP production was noted. The accumulation was presumed to be the result of letting the usual fat into the cell, but the lower intracellular utilization might have produced the accumulation. There was evidence that some injured muscle tissues survived and were able to reverse the trend toward death and necrosis. Purkinje fibers located beneath the endocardium remained viable and retained normal glycogen stores and respiratory enzymes. These experiments with the production of injury and infarcts in the heart muscle of dogs supports the injury and repair process characteristics of other organs. Degeneration Related to Aging The presentation of investigative studies to highlight the cause of degeneration in the heart and kidneys results in very distinct characteristics of the OCD process. With aging, the aortic and mitral valves experience degrees of degeneration. This process of degeneration fits the OCD process with emphasis on the calcification of the valves. The degeneration of the valves includes accumulation of fat. The areas of greatest accumulation of fat are also the areas of greatest calcification. The degenerative changes were similar to the changes in arteriosclerosis but having distinctive features. Sell and Skully studied 100 specimens of the heart mitrial valve and 100 specimens of the aortic valve (12). The mitrial valve specimens were from subjects 1 day to 96 years in age while the aortic specimens were from subjects 5 days to 89 years in age. The investigators studied the various characteristics of the heart valves in relationship to age. The report describes the valve tissues of youth as fibrosis free healthy tissue cell structure. By the fifth decade, the valve tissue exhibited cells with fewer nuclear and an increase in fibrous tissue. Along the strands of fibrous tissue were increasing deposits of fat, and with increasing age the fibrous tissue increased. The accumulation of fat increased and deposits of calcium were noted. In the specimens from 10

the oldest subjects, the fibrous tissue was greatest and the maximum occurrences of lipid deposits were associated with the most calcified areas of the valves. These characteristics of degeneration were seen in both the aortic and mitral valves. These characteristics of degeneration of the valves with age represents an example of the OCD process which becomes progressively more degenerative as the healthy young tissue is replaced by fibrosis. Robert E. Olson researched in detail the role of energy metabolism in the development of congestive heart failure. (13). The results of his study showed how the deficiency of energy metabolism was one of the root causes of the decline in the effectiveness of the heart in sustaining the high energy requirement for active life. In some cases, there was a direct causal relationship between a disease-linked diminished production of ATP and the development of congestive heart failure. He studied the myocardial metabolism in congestive heart failure, and explained in detail every biochemical process in energy production. His report does not include the definitive characteristic of OCD. There is no report of fat accumulation in cells and no reports of necrosis or calcification of tissues; however, a very direct connection is made concerning the energy production and utilization in the myocardium. Two diseases are listed as resulting in interference with energy production and as causes of heart failure; anemia and beri beri. Anemia causes a lack of oxygen for oxidative phosphorylation, while beri beri results in a lack of a coenzyme necessary for the oxidation of key metabolites at the substrate level. The heart fails when the generation of free energy in the process of contraction of the myocardium is insufficient for the circulatory load imposed. When the generation of ATP is insufficient, the OCD conditions are met. The kidney diseases connected to the OCD process were studied by Flume and Ashworth (14). A number of degenerative diseases of the kidneys have a lipid accumulation as a result of the degenerative process. Some of these kidney diseases are characterized by tubular lesions and were studied by electron microscope techniques. The diseases studied were acute glomerulonephritis, chronic glomerulonephritis, nephritic syndrome, and diabetic nephropathy. The techniques for preparation of the specimens included the use of osmium tetroxide in the fixing of the tissue specimen. This is a common tissue fixing method used to fix tissues for electron microscopic examination. When osmium salts are used, the lipid in the tissue reduces the osmium salts to deposit osmium metal residues. When the black osmium metallic residues are observed, this indicates the presence of fats in the tissue. The most prominent observation reported from studies of injured and degenerating tubular cells of the kidney was the ever-present deposits of osmium indicating lipid accumulation in these tissues. Tissue specimens from 122 patients were studied. Both the proximal convoluted tubules PCT and the distal convoluted tubules DCT were studied. In the case of acute glomerulonephritis, renal injury by drugs and in reverse progressive renal disease, there were found changes that were sub lethal in both the PCR and the DCT. These changes observed by light microscope were characterized by cloudy swelling or hydropic degeneration. The electron microscope studies showed mitochondrial swelling in conjunction with the first observation of osmiophilic deposits, which are interpreted as fat deposits. As the disease became more severe, resulting in acute tubular injury, lipid 11

droplets were found in the basal portion of cells of the PCT and the DCT. In the more acute disease processes, tubular atrophy and tubular epithelial cell degeneration were observed. Both conditions were accompanied by evidence of the accumulation of fat. In these cases of tubular cell injury and death, the investigators reported that the injury was probably caused by failure of the ATP energy generation process. In acutely injured cells as well as tubular atrophy, the accumulation of lipid was characteristic. Reasoning from the underlying renal disease, it was surmised that tubular cell injury in most of the cases studied was due to ischemia resulting from obliterative or obstructive glomerula lesions. Although difficult to detect, abundant cholesterol deposits accompanied the accumulation of lipid in the disease, lipoid nephrosis. The Atherosclerosis Connection The disease Atherosclerosis is explained by the processes associated with the “other cell death” OCD. The understanding of the “injury and repair” process which is the defining process of atherosclerosis lesion development is based on the physiology of the artery wall in response to injury. The physiology involves a unique multifunction of the Smooth Muscle Cell (SMC). The SMC which resides in the media has a primary role in muscular function of the artery wall as the muscle tissue. The muscle functions of expansion and contraction are vital functions of the artery wall. The SMC takes on a multifunction to produce the chemical substances to form the repair tissue of fibrosis. The production of fibrous tissue becomes the major factor in the repair process. This role was recognized and supported by the reports of Wissler and other investigators. Russell Ross expanded on this important role as a fibrous production multifunctional SMC (15). Upon stimulation to do so, the media residing SMC migrates into the intima space and changes to a multifunctional synthetic cell. This migration is accompanied by a proliferation of multifunctional synthetic cells which thickens the intima and requires enhanced support from the nutritional and oxygen mediated ATP production. It is the thickening of the intima and the increased demand for metabolic activity supporting SMC proliferation which result in inadequacies of cellular life support. These inadequacies result in the injury leading to the formation of the atherosclerotic lesion. A closer look at the specific requirements for cell proliferation reveals the need for accelerated availability of the various lipids involved in cell growth. A major requirement is the need for cholesterol and phospholipids which are necessary to build cell walls and cell organelles. The thickened intima receives inadequate amounts of nutrition and oxygen, causing it to become more difficult for the cells to sustain the production of ATP; then the cells begin to suffer injury and death. The first factor supporting the development of atherosclerotic lesions is the thickening of the intima. At birth, the intima is thin and contains very few cells. As the decades of life pass, the intima thickens as cells move into the space behind the endothelium. Normally, (in the absence of early risk factors for the development of the atherosclerotic disease, such as cigarette smoking, diabetes, or anemia), the intima does not thicken enough to bring on dangerous atherosclerosis until the fourth decade of life. The investigators report and support varying views as to the life functions which attribute to the development of the atherosclerotic lesion; the fibrous capped plaque. The fibrous plaque is the mature form of the lesion and is the cause of the life threatening phase of the 12

disease which the investigators recognize as a basis on which to build an understanding of the disease. The major issues are: 1) the role of the multifunctional smooth muscle cell to produce the fibrous tissue important to the repair process. 2) the idea that the development of the plaque in the intima space is caused by a failure in the function of the endothelial cells lining the lumen of the artery as a barrier to the infiltration of lipid into the intima space to form the characteristic lipid center of the mature plaque. This idea is supported by Ross and refuted by Campbell; 3) the idea that the fatty streak which is composed of fatfilled cells occupying the intima space is the precursor of the mature plaque development. This idea is supported by Ross but refuted by Velican; 4) the idea that there is a group of necrotic cells developing in the intima space, which results in a foci of necrosis in the intima, which is the precursor to the plaque development. This idea is supported by Velican (16); 5) the fact is, that as the intima thickens with age, and the thickening is speeded up by the risk factors for atherosclerosis, the conditions for cell injury are enhanced. The healthy life process of the cells in the thickened intima is diminished by the lack of adequate nourishment and oxygen support. Thereby the SMC of the intima succumbs to the other cell death (OCD) process and contributes to the characteristic fat and cellular debris in the lesion center of the fibrous plaque. Russell Ross dedicated his studies to understanding the disease process of Atherosclerosis. The listed issues will be supported or refuted by the evidence reported by the references selected. Two of the issues listed, however, are not supported by the facts. These two issues are: 1) the idea that atherosclerosis is caused by the dysfunction of the endothelial cells, (refuted by Campbell and Campbell); and 2) the idea that the fatfilled cell of the fatty streak is a predecessor to the fibrous plaque (refuted by the work of Velican). Review Objective The purpose of this review is to support the concept that the other cell death (OCD) is the key to understanding the degenerative processes that result in the decrease in healthy function of the vital organs, and including the cause for the disease process of atherosclerosis. In the case of atherosclerosis, the main investigators are Russell Ross, the Velicans, and the Campbells. Ross recognized atherosclerosis as a healing response to injury of the artery wall. It became the â&#x20AC;&#x153;injury and repairâ&#x20AC;? theory of atherosclerosis (17) (18). He worked to understand the process of SMC migration from the media to the intima and described the proliferation of the synthetic capabilities of the SMC in the intima to produce fibrous tissue to preserve the structural integrity of the artery wall. This process became recognized as a repair process that evolved to prevent a lesion which would have ended in perforation of the artery wall, resulting in loss of the integrity of the arterial system and subsequent bleeding. It will be noted that the degenerative processes and injury to tissues occur more intensely as the tissues are a greater distance from the heart, which is the source of oxygenated blood. A major example is the fact that the atherosclerotic lesions develop to a larger degree in the abdomen, and to a lesser degree in the thoracic area (19) (20). 13

The anatomy of the arterial blood vessel wall was defined by Russell Ross. The blood vessel wall must be recognized as a living organ with substantial oxygen requirements to support healthy tissue. The arterial wall surrounds the lumen of the vessel which is located in the center of the artery and serves as a conduit for the distribution of blood to the tissues. The innermost layer of the artery wall is a layer of endothelial cells behind which is a space with few cells, and this space is bordered on the outside by an internal elastic layer. The space behind the endothelial cells is the intima; next to it resides the muscular part of the artery called the Tunica media; and outside the media resides the Tunica Adventitia. Between the media and the adventitia is the external elastic layer. There are blood vessels in the adventitia called Vasa Vasorum. The Vasa Vasorum provides the supply of blood to the arterial wall. However, the effective reach of the Vasa Vasorum is limited to the media while the vessel wall is normal. The Vasa Vasorum may move into the intima as the intima thickens with age and as the atherosclerotic lesions develop. The research literature, in a number of instances, reports that the metabolic nourishment of tissues (as in the case of the heart), is not served by the blood flowing inside the heart, except for a thin layer of tissue just inside of the endocardium, and that distance is only 0.5mm to 1mm thick. Likewise, the artery wall does not depend on oxygen or nourishment from the blood flowing in the lumen of the vessel. The understanding of the degeneration of the vital organs is based on a deficiency in the process providing energy for the metabolism of the organ tissue cells, and the heart is the most vulnerable organ. The human heart has the greatest demand for high energy. Based on the research of Schlesinger and Reiner, the fact of the development of small foci of dead cells (necrosis) illustrates the vulnerability of the heart muscle to significant chronic damage caused by the deficiency in the high energy needs required. This may be an excellent insight to a subclinical condition recognized by cardiologists as silent ischemia. The Artery Wall Viewed as a Living Organ The artery wall, when considered as a living organ, needs to be presented in its functional parts. The lumen is the center of the artery and is a conduit to distribute blood which delivers nutrients and oxygen. The layer next to the lumen is a layer of endothelial cells which protects the outer wall from the physical dynamics of the moving blood in the lumen. The endothelial cells play a vital role, which is to prevent the initiation of any of the coagulation mechanisms in the circulating blood. There is a space behind the endothelial layer of cells which, in the early stages of life, contains very few cells. This area is known as the intima. As the decades of life pass, the intima becomes thickened when cells move in the space behind the endothelial layer. The next layer is the media, which consists of smooth muscle cells (SMC). These media cells are the main body of the artery wall and function to resist the pulsing of the moving blood and expand and contract the wall of the vessel as directed to fulfill the muscular functions. The next layer is the outermost layer. The outer layer, or the adventitia, contains vital blood vessels called the Vasa Vasorum. These blood vessels provide oxygen and nutritional support to the cells of the media. In the early years of life, the Vasa Vasorum resides only adjacent to the media and adventitia. As the intima thickness and the abnormal lesions of atherosclerosis develop, the Vasa Vasorum eventually moves into the intima. The Vasa 14

Vasorum and the fully developed atherosclerotic lesions (the fibrous plaque) do not develop until the fourth decade of human life. The physiology of the artery wall is functional in the injury and repair process. The process of atherosclerosis was recognized as a disease lesion arising out of the organismâ&#x20AC;&#x2122;s attempts to repair injury to the arterial wall. The repair process was initiated by the movement of smooth muscle cells from the media to the subendothelial space known as the intima. Over the years of research, the role of a fibrocyte cell was thought to be responsibility for producing the fibrosis tissue which resulted in the repair. However, as the years of research progressed, the fact became evident that the traditional fibrocyte was not available or active in the repair of injury to the vital organs. It became apparent that the major functioning cell of the particular organ became a multifunctioning synthetic cell, and thereby provided for the substance necessary for the production of the fibrous tissue of repair. For the heart myocardium it was the myocyte, and for the artery wall it was the smooth muscle cell located in the media. Wisslerâ&#x20AC;&#x2122;s research reported and enlarged on the understanding of these facts. The Contribution of Russell Ross Dr. Ross was the greatest contributor to the understanding of the disease, atherosclerosis. His research group investigated many approaches to the understanding of the disease process. Acceptance of the injury and repair concept of the disease was supported and explained. His group used immunological specific techniques to identify the tissue cells present during lesion development. This research confirmed that the most active cell in both the repair process and subsequent accumulation of lipids within the cell was the smooth muscle cell which migrated from the media to the subendothelial intima. This process was responsible for the thickening of the intima over time. The migration of the SMC and its proliferation as a multifunctional synthetic cell was the basis for the fibrosis process of repair and subsequently contributed to the formation of the fibrous cap on the mature atheroma, the fibrous plaque. This fibrous plaque as it matured became the most dangerous lesion of atherosclerosis. The mature lesion protruded into the lumen of the artery, grew its own blood vessels (Vasa Vasorum), which contributed to the dangerous rupture which released blood to form a clot in the lumen of the artery. The result of the rupture of the mature fibrous plaque produced a clot in the vessel and produced the blockage causing the heart attack or stroke. The thickened intima predisposes an injury to poorly oxygenated tissues in the intima, thereby supporting the inflammation and necrosis of these tissues. This is the initiating process leading to development of the young lesion which over time produces the mature fibrous plaque. Aside from the classical lesions, Ross investigated the unique lesions of the superficial femoral arteries (21). Ross reported that the arteries developed a fibrous tissue blockage without the classical atheroma development with the characteristic foundation of a lipid filled gruel center. Although there was some of the characteristic lipid accumulation, the major blockage was caused by the proliferation of the fibrous tissue. In this case, growth of fibrous tissue accumulated very little lipid. 15

The Ross research included, as a major part, the explanation of the proliferation of the SMC in the intima. He saw this process of injury repair as a process he wanted to control. If controlled successfully, this could result in atherosclerosis prevention. He also believed that the lipid accumulation entering the intima and subsequently accumulating in the SMC was caused by a failure of the endothelial layer of the artery wall. The research of the Campbells showed that the cells in the intima still accumulated fat even after the repair process was complete and the endothelial layer restored to the healthy state. This fact worked to refute the failure of the proper function of the endothelium theory of plaque development. Ross believed that the process of atherosclerosis was an out-of-control overrun of the beneficial repair process (22) (23). This is true of other diseases; and the arthritic diseases of bone joints are one example. The work of Lent Johnson reported lipid accumulation in cells during growth which resulted in arthritic disease (24). In the heart muscle, the repair phenomenon results in repair followed by fibrosis with little lipid residual. However, the evidence of lipid involvement in the process is the residual lipofuscin which is accumulating in small quantities in the heart muscle (25). The Contribution of C. Velican and Doina Velican The Velican report of the finding of 400 selected cases aged 1-40 years establishes the severity of the plaque development in each age group (26). The groups were: children 610 years old, juveniles 11-15 years old, adolescents 16-20 years old, young adults 21 to 30 years old, and mature adults from 31 to 40 years old. The coronary atherosclerotic plaques observed in this 1-40 year old category remained uncomplicated by thrombosis, hemorrhage or ulceration, which leaves these aspects of the complicated fibrous plaque to the age group beyond 40 years of age. The Velican report also found that the mature fibrous plaque in their material was not covered by a thick sclerotic fibrous cap, nor did their observed sclerotic fibrous plaques involve the inclusion of remnants of fibrin. These observations are significant, since they indicate that the clinically dangerous lesions causing the bleeding thrombosis and other manifestation of heart attack and stroke are not characteristic of early fibrous plaques up to 40 years old. This is also the message of the Pday study group. The most valuable work by the Velican researchers is the study of early necrosis and its role as the initial event which leads to the development of the atherosclerotic plaque or mature athroma. This is important because the initial occurrence of a necrosis lesion in its development follows the steps of the OCD. This cell death begins with a degenerative foci of tissue cell swelling. Step two is the accumulation of fat, ending in dissecting necrosis, with its stage of tissue remnants and debris supporting an inflammatory reaction and its most advanced form supporting the calcification of degenerative sites in tissue. Early plaques found in adolescents and young people were located at sites of pads and cushions. With later age groups of young adults and mature adults the site of plaque development shifted almost exclusively to the thickened intima. The Velican research group did amazing work. They investigated autopsy specimens from persons aged one year to age forty and some months before birth. Their most 16

amazing work was the investigation of the development of the coronary arteries in the human fetus. These investigations covered the period some months before birth. The fetus investigation followed the fetal development of the coronary arteries, revealing early problems in the development of these arteries. The investigations of the coronary arteries from age one to forty years established the role of the thickened intima and the role of early necrosis in the development of the fibrous cap atheroma, which is the mature form and the most characteristic lesion of atherosclerosis. The study of the fetal development of the coronary arteries centers on the development of the anterior descending branch of the coronary arteries. This coronary artery branch is selected for study because it is known as the most dangerous of the coronary arteries to develop the degree of atherosclerosis which threatens life. This particular branch is known as the “sudden death artery,” or the “widow maker.” (27). The Velican researchers reviewed a wide range of research articles and learned interesting facts about the embryonic development of the coronary arteries which address the issues concerning developmental problems leading to susceptibility of the coronary arteries in both child and adult arteries to develop the lesions of atherosclerosis. A broad spectrum of atherosclerosis research has reached the conclusion that the thickening of the intima to include protective pads which occur at the branches of arteries generally are the prevalent locations for the development of mature lesions known as fibrous plaque. The Velican investigations followed the development of these pads and other aspects of intima thickening in the embryonic development. The results show that the intima thickening and pad development were most severe in the anterior descending branch. These developments were contrasted with other arteries in the same fetus. The findings were that the injurious developments in the anterior descending branch of the coronary arteries were unique in that this troublesome development was not occurring in the basilar and renal arteries of the same fetus. The investigators then looked at the implications of these harmful early developments in the specimens after birth and early childhood. The results proved that harmful fetal developments in the anterior descending branch led to susceptibility to early manifestation of atherosclerosis, with such manifestations starting with an early form of cellular necrosis in the thickened areas. This was seen in the children as especially affecting the anterior descending branch of the coronary arteries. These degenerative changes did not occur in the basilar and renal arteries of the same children. The unique susceptibility of the anterior descending branch to atherosclerotic involvement and lethal thrombosis was attributable to a complex of physical, mechanical and hemodynamic factors, including more vigorous vibration than other coronary branches; greater bending and knuckling during myocardial contraction than other epicardial vessels, and an increased rate of flow-off which resulted from the rapid tapering associated with rapid runoff from the numerous branches. The maximum stress falls upon this vessel just where it becomes attached to the myocardial surface by the first main septal branch. When taken with other reported observations, there is a definite connection that these developmental conditions of hemodynamic stressors (compression, stretching vibration and lifting) represent an important factor in atherogenesis. 17

This investigative report by the Velicans fits in with the supposition that intimal thickening and early signs of cellular necrosis are a key to understanding the underlying conditions which contribute to the development of the atherosclerotic plaque, the fibrous plaque. It is important while reporting the results of investigations by the Velicans to report and set aside the role of the fatty streak in the development of the atherosclerotic plaque. Some investigators have implicated the fatty streak as a precursor to the fibrous plaque. The fatty streak is a ubiquitous lesion which forms early in the arteries of children and young adults. It may be a round speck or a streak. It consists of a group of cells which are filled with fat of the triglyceride variety. The fatty cells of the fatty streak are not characteristically accumulating dots of fat in the cytoplasm of the cell, as is characteristic of the smooth muscle cells of the developing athenoma; but the streak is filled with cells engorged with fat which occupy the major space within the cell. The Velican investigation reported that there was never seen any evidence of a transitioning of these fatty streaks into the fibrous plaque of atherosclerosis. Their other evidence was the presence of the fatty streak developing decades before the fibrous plaques and after the development of fibrous plaques, which was never seen in transition into the development of the fibrous plaque. The fatty streaks are numerous in areas where fibrous plaques never develop (28). Campbell & Campbell contributed to the understanding of endothelial injury and thickening intima (29). Researchers Campbell & Campbell have reviewed the atherosclerotic disease process extensively. The topics of interest from their review are: endothelial injury; phenotypic changes in the role of SMC; and diffuse intima thickening. The extensive studies of atherosclerosis development have implicated endothelial injury to the inner lining of the artery wall as a player in the development of plaque characteristic of the disease. The process studied implies that the endothelium is damaged, and subsequent processes let lipids diffuse into the subintima area or that phagocyte cells pick up lipids from the circulating blood and transport the lipids to the intima area. This becomes an explanation for lipids occurring in SMC resident in the subintima area, which subsequently are responsible for the lipid cholesterol crystal deposits in the center of the mature atheroma, the fibrous plaque. The evidence reported by the Campbells does not support these ideas about plaque development. Beginning with an investigation and report on the experimental damage to the endothelial layer, it was noted that when the endothelial layer was damaged, the damage had to exceed an area which was not repaired (replaced) for seven days before the sequence of events moved forward to remedy the damage. If the endothelium did not replace itself within seven days, the SMC migrated from the media into the damaged area and produced a so-called neointima to replace the normal endothelium. This neointima remained the repair until the endothelial cells of the normal intima were replaced. Other evidence that the damaged endothelium theory of atherosclerotic development did not hold true was the fact that the cells resident in the thickened intima continued to accumulate lipid after the replacement of the normal endothelium. The role of the phenotypic change of the SMC from the media moving to the intima was investigated and reported. The SMC which is in the media has a muscle contraction 18

role; yet, when it receives a stimulative message, it moves from the media to the intima and becomes a synthetic cell which produces the chemicals necessary to produce fibrous tissue. This tissue produced by the cells residing in the intima does repairs and subsequently accumulates fat, and then some cells die and become the cellular and lipid debris characteristic gruel of the center of the mature atheroma called the fibrous-capped plaque. This Campbell and Campbell review emphasizes the role of “diffuse intimal thickening” in the disease atherosclerosis. Intimal thickening precedes the development of the fibrous plaque which is the most advanced form of the atherosclerotic lesion. The question arises, then, Why does the intima thicken? The answer to this question is investigated and elaborated upon in the extensive work of the investigators, Velican and Velican. The Campbell researchers reviewed the issue of the migrating SMC from the contractile media into the intima. The migration is accompanied by a change in the roles of the SMC from contractile to a cell with a new phenotypic role as a synthetic cell which manufactures the chemicals necessary and subsequently produces the fibrosis tissue which becomes the fibrous tissue of the cap on the fibrous plaque. The SMC of the media, although proliferative, do not accumulate fat. While the SMC synthetic cells are both more proliferative in the subendothelial site and accumulate fat, these synthetic cells in their role to produce fibrous tissue interact with the lipids which are necessary for production of new cell component parts (cell walls and mitochondria) and they also subsequently accumulate fat. Some of these cells may become senescent, having exhausted their proliferative capacity. Upon the death of these cells, the debris resulting may become a part of the characteristic cholesterol containing debris found in the center of the fibrous plaque. There are three characteristic features of atherosclerosis according to the research of Russell Ross: 1) focal proliferation of SMC in the thickening intima; 2) synthesis of large amounts of connective tissue, particularly collagen and glycoaminoglycols; and 3) deposition of intra cellular and extra cellular lipid. The “synthetic state” smooth muscle cells of the thickened intima explain why diffuse thickened intima are sites of predilections for atherosclerosis. The Carbon Monoxide Connection P. Astrup is a medical researcher with world recognition as an expert in the measurement of blood gases. Astrup had some ideas about the effects of carbon monoxide on the development of the lesions of atherosclerosis (30). His ideas were based upon his knowledge of the carbon monoxide content of a cigarette smoker’s hemoglobin. He knew a smoker could have 10-12% of hemoglobin combined to carbon monoxide. He then studied the effect on rabbits exposed to carbon monoxide while being fed a cholesterol diet. The results showed an increase in the prevalence of the lesions of atherosclerosis. The deficiency of oxygen caused by the presence of carbon monoxide was the cause of the increased frequency of the lesions. He also thought, correctly, that the carbon monoxide might also poison the cytochrome system of phosphorylation, the energy process generating ATP. Subsequent research findings have proven the poisoning of the phosphorylation process to be true. Thomsen repeated the Astrup experiments by testing the effects of carbon monoxide on monkeys with results that support Astrup. (31). The connection of cigarette smoking to heart disease and atherosclerosis was reported by Astrup. Since the cigarette smoker has a 10-12% 19

carbon monoxide connected to the blood hemoglobin, this results in a measurable deficiency of the oxygen carrying capacity. This deficiency of oxygen to the heart, kidneys, and artery walls answers the question about the source of the systemic disease effects of cigarette smoking on the vital organs, especially the heart. Astrup also studied the effects of hyper oxygenation on the development of the lesions of atherosclerosis, and found a decreased rate of development of lesions when animals were exposed to an atmosphere of increased oxygen (32). Supporting evidence that the destructive OCD which weakens the vital organs is the result of a deficiency of the oxygen available to these organs is found in the study of wild animals and birds. To provide adequate oxygen to the artery wall, the Vasa Vasorum must develop an adequate number and reach into the artery wall. The fibrous plaque which is the mature lesion of atherosclerosis eventually obtains a Vasa Vasorum, but not until the atherosclerotic lesions have developed into the mature and dangerous fibrous plaque. Some wild animals and birds studied have poorly developed Vasa Vasorum associated with the extent of development of the lesions of atherosclerosis (33). Some birds with spontaneous atherosclerosis show inadequate development of the Vasa Vasorum, which seems to be the root cause of their extreme susceptibility to the disease. The study of the effects of low hemoglobin levels in women emphasizes the need for the maintenance of the oxygen support for the health needs of the vital organs (34). The report on the study of anemia in women was an extensive picture of demise of health expectations in keeping with low levels of hemoglobin, specifically the evidence connecting the poor medical outcomes of women with low hemoglobin values. It stated that each number of hemoglobin below 12 g% was an additional 20 percent negative against a favorable outcome of any medical problem suffered by the women. Furthermore, for each number below 12, there was an additional 20% burden on recovery from the medical problems experienced by the women studied. This report reflects the extreme importance of the maintenance of adequate oxygen to the vital organs. The concept of the function of the many physio-chemical processes of the cell as a pump contributes to the understanding of those processes (35). These pumps must depend on energy to operate and achieve their physiological functions. This means that the pumps are dependent on ATP. As a case in point, the function of HDL to remove lipoproteins such as LDL from the tissue cells and take it to the liver for outprocessing is dependent on adequate ATP levels in the cell. If there is a deficiency of ATP available in the cell, the blood level of HDL, which is seen as a healthy level and functions to reduce the atherosclerosis risk, becomes non-functional. This fact contributes to the understanding of the accumulation of fat in the injured cell when adequate lipids are delivered to the mitochondria of the cell to be used; but the low cell ATP levels prevent the physiological pumps from functioning properly, thereby allowing the injured cells to accumulate fat in the â&#x20AC;&#x153;other cell deathâ&#x20AC;? process.

Discussion This review of the medical literature reporting instances where tissue cell injury has resulted in an accumulation of fat in the injured tissue cells has revealed a degenerative process weakening the vital organs. The injury is slow and of a chronic nature. The 20

persistence of the injury process results in a destructive weakening of the organ, which, over time, is realized by the medical community as a disease with systemic effects, whereas the process itself remains subclinical in nature. The death process caused by injury to the cell is not well recognized and the effects are obscured by the popularity to recognize all cell death as apoptosis linked. Virchow and Ulrich recognized the disease process as being unique in its association with the accumulation of fat in cells. Schlesinger and Reiner defined small cell death foci in the heart muscle as caused by a unique cell death process which, in its second step, included the accumulation of fat in injured cells. They defined the cell death process in step by step detail as: 1) cloudy swelling; 2) accumulation of fat; and 3) necrosis. They reported in the final analysis that the cell death was caused by a deficiency of oxygen, which led to a decrease in the production of ATP, and subsequently the loss of ATP produced in the affected cells. They also reported that adequate oxygen and nutrition was not supplied to the myocardium from the blood circulating within the chambers of the heart. Also reported was the fact that the foci of dead myocardium cells occurred toward the end of the arterial distribution of blood. They reported that the lesions were reproduced in the hearts of cats experimentally exposed to an atmosphere deficient in oxygen. This other cell death process (OCD) was reported in other experiments with the myocardium, and a connection was made to the degeneration of the heart valves. Kidney diseases were also connected with the cell death process. This review, in its investigation process, centers on the disease characteristics of atherosclerosis. When the artery wall is recognized as a living organ, rather than just a tube, the development of the atherosclerosis connection progresses because there is a clear picture and there are multiple references for the development of the atherosclerosis connection. The process of atherosclerosis needs to be emphasized as a major finding. Without arguing the present approaches to handling the disease process, the facts revealed represent a new outlook. First, atherosclerosis begins to become dangerous usually only after the fourth decade of life, unless there are unusually intense risk factors such as familial lipid disease or cigarette smoking or conditions which limit the oxygen supply to the tissue cells. In the injury and repair process of atherosclerosis development, there occurs a pinpoint injury to the artery wall in the intima area. This injury activates the movement of SMC from the media to the intima site of injury; then the proliferation of the synthetic SMC in the intima demands more lipids to accommodate the building of the structural matrix for new cells. The new proliferation of SMC exceeds the demand for oxygen caused by the lack of adequate Vasa Vasorum development toward the intima area. The result is a dying off of SMC in the intima, leaving a deposit of lipids in the center of the fibrous capped lesions. An interesting side issue is why the development of the lesions is not concentric in the artery wall, but develop characteristically on only one side. An explanation could be that the Vasa Vasorum development is adequate to provide adequate oxygen to the side of the artery wall spared from lesion development. The lesion development is not from the endothelial side, but develops from inadequacy of oxygen to 21

support the proliferation of SMC in the intima; yet the proliferation of SMC functions to repair the injury to the wall of the artery. Although it is the protective repair process which serves to protect the integrity of the artery wall, an overreaction to the need for repair seems to be a part of the disease process. The fact that carbon monoxide enhanced the atherosclerosis disease process made a valuable connection to cigarette smoking. Even at this late date, the systemic effects of cigarette smoking on the heart are not understood, and detrimental effects on the kidneys are not recognized. There are many disease connections made to cigarette smoking when it is realized that oxygen is extremely necessary to prevent disease. An example is the connection of cigarette smoking with dental gum disease. Changes must be made in how hemoglobin blood concentrations are regarded in clinical practice. A medical dictionary even reports that a person can be healthy with a 9 g/% hemoglobin blood level, if the body has accommodated to that level. The truth, as reported by the study of anemia in women, is that any value lower than 12g % is a health hazard. As a matter for maximum health purposes the recommendations should read 1314 g% for women and 15-16 g % for men. As a matter for clinical practicality, every opportunity must be made to investigate every aspect of life which might hinder adequate oxygen for all tissues of the vital organs. Some areas for study would be; 1) the realization that estrogen replacement therapy supports angiogenesis; 2) reverse the idea that estrogen hormone replacement therapy does not protect the heart; 3) develop a program to encourage people to know their hemoglobin blood concentration number; and 4) educate the public as to the requirements for Vitamin B12 and iron to support the healthiest concentration of hemoglobin. As for the helpfulness of dealing with preventing the disease atherosclerosis, the enlightened concern for the sense of the disease and the process of the disease should take pressure away from the extreme attempts to control cholesterol blood levels in the public. There should be reason to return to more traditional diets of red meat and unlimited use of dairy products. Red meat supplies iron and co-enzymes necessary to the phosphorylation process of energy generation; these essentials are Q10 and carnitine. Future research could seek out for study methods to assure proper delivery of oxygen to the vital organs. The understanding of the role of cigarette smoking in the slow degeneration of the heart and kidney would increase the pressure for most people to quit smoking. As an application to improve clinical practice, the most valuable reference presented is the study of anemia in women. This is extremely valuable because it provides a practical opportunity to intervene, and results of intervention can provide more healthful medical outcomes for women. REFERENCES 1. Virchow R. Cellular pathology as based upon physiological and pathological histology. Lecture XVI (1858). Nutr Rev 1989 Jan;47(1):23-25. 2. Ulrich H. Organverfettngen bei sanerstoffmangel und hunger. Frankfurter Zeitschrit fur Pathologie 1938;52:81-98. (English translation available.) 22

3. Hueper WC. General reviews. Arteriosclerosis. Arch Pathol.1944;38:162-81. 4. Dixon KC. Fatty deposition: a disorder of the cell. Q J Exp Physiol Cogn Med Sci 1958 Apr;43(2):139-59. 5. Trump BF, Berezesky, IK. The Reactions of cells to lethal injury: oncosis and necrosisâ&#x20AC;&#x201D;The role of calcium. In: Lokshin RA, Zakeri Z, Tilly, JL, editors. When cells die: A comprehensive evaluation of apoptosis and programmed cell death. New York: Wiley-Liss; 1998. p.57-78. 6. Schlesinger MJ, Reiner L. Focal myocytolysis of the heart. Am J Pathol 1955 May-Jun; 31(3):443-459. 7. Shogo K, Boyd HC, Fligner C, Ross R, Gown AM. Human atherosclerosis. III. Immunocytochemical analysis of the cell composition of lesions of young adults. Am J Pathol 1992 Apr;140(4):907-14. 8. Wissler RW. The arterial medial cell, smooth muscle or multifunctional mesenchyme? J Atheroscler Res 1968 Mar-Apr;8(2):201-13. 9. Holman RL, McGill HC Jr, Strong JP, Geer JC, Guidry, MA. The Arterial wall as an organ. In: Pincus G, ed. Hormones and Atherosclerosis. New York: Academic Press; 1959. p. 123-129. 10. Guyton AC. Textbook of medical physiology. 7th ed. WB Saunders; c1986. Chapter 25. The coronary circulation and ischemic heart disease; p. 296-297. 11. Shnitka TK, Nachlas MM. Histochemical alterations in ischemic heart muscle and early myocardial infarction. Am J Pathol 1963 May;43(5):507-21. 12. Sell S, Scully RE. Aging changes in the aortic and mitral valves; histologic and histochemical studies with observations on the pathogenesis of calcific aortic stenosis and calcification of the mitral annulus. Am J Pathol 1965 Mar; 46(3):345-55. 13. Olson RE. Myocardial metabolism in congestive heart failure. J Chronic Dis 1959 May; 9(5):442-64. 14. Flume JB, Ashworth CT, James JA. An electron microscopic study of tubular lesions in human kidney biopsy specimens. Am J Patho 1963 Dec;43(6):1067-87. 15. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993 Apr 29;362(6423):801-9. 23

16. Velican C, Velican D. Coronary intimal necrosis occurring as an early stage of atherosclerotic involvement. Atherosclerosis 1981;39(4):479-96. 17. Ross R. George Lyman Duff Memorial Lecture. Atherosclerosis: a problem of the biology of arterial wall cells and their interactions with blood components. Arteriosclerosis 1981Sep-Oct;1(5):293-311. 18. Ross R. The arterial wall and atherosclerosis. Annu Rev Med 1979;30:1-15. 19. Masuda J, Ross R. Atherogenesis during low level hypercholesterolemia in the nonhuman primate. II. Fatty streak conversion to fibrous plaque. Atheriosclerosis 1990 Mar-Apr;10:178-87. 20. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Natural history of aortic and coronary atherosclerotic lesions in youth: findings from the PDAY Study. Artherioscler Thromb 1993 Sep;13(9):1291-8. 21. Ross R, Wight TN, Strandness E, Thiele B. Human atherosclerosis. American Journal of Pathology: I. Cell constitution and characteristics of advanced lesions of the superficial femoral artery 1984 Jan;114(1):79-91. 22. Ross R. Rous-Whipple award lecture. Atherosclerosis: a defense mechanism gone awry. Am J Pathol 1993 Oct; 143(4):987-1002. 23. Ross R. Cellular and molecular studies of atherogenesis. Atherosclerosis 1997 Jan (suppl);131: S3-4. 24. Johnson LC. Kinetics of osteoarthritis or degenerative arthrosis. Lab Invest 1959 Nov-Dec; 8:1223-41. 25. Roffe C. Ageing of the heart. Br J Biomed Sci 1998 Jun; 55(2):136-148. 26. Velican C, Velican D. The precursors of coronary atherosclerotic plaques in subjects up to 40 years old. Atherosclerosis 1980 Sep;37(1):33-46. 27. Velican C, Velican D. Intimal thickening in developing coronary arteries and its relevance to atherosclerotic involvement. Atherosclerosis 1975;23:345-55. 28. Velican C. A dissecting view on the role of the fatty streak in the pathogenesis of human atherosclerosis: culprit or bystander? Med Interne 1981 OctDec;19(4):321-337.

29. Campbell GR., Campbell, JH. Recent advances in molecular pathology. Smooth muscle phenotypic changes in arterial wall homeostasis: Implications for the pathogenesis of atherosclerosis. Exp Mol Pathol 1985;42:139-162. 30. Astrup P, Kjeldsen K, Wanstrup J. Enhancing influence of carbon monoxide on the development of atheromatosis in cholesterol-fed rabbits. J Atheroscler Res 1967 7:343-54. 31. Thomsen HK. Carbon monoxide-induced atherosclerosis in primates; an electronmicroscopic study on the coronary arteries of Macaca irus monkeys. Atherosclerosis 1974;20(2):233-40. 32. Stender S, Astrup P, Kjeldsen K. Hyperoxia-induced decrease in aortic accumulation of cholesterol in rabbits previously fed a cholesterol-enriched diet. Exp Mol Pathol 1976; 25(2):221-6. 33. Cowan DF. Abnormalities of vasa vasorum in exotic mammals and birds. Am J Pathol 1967 Oct;51(4):591-599. 34. Arant CB, Wessel TR, Olson MB, Merz CN, Sopko G, Rogers WJ, Sharaf BL, Reis SE, Smith KM, Johnson BD, Handberg E, Mankad S, Pepine CJ. Hemoglobin level is an independent predictor for adverse cardiovascular outcomes in women undergoing evaluation for chest pain. JACC 2004 June 2;43(11):2009-2010. 35. Owen JS, Mulcahy JV. ATP-binding cassette A1 protein and HDL homeostasis. Artherosclerosis supplements 3, 2002:13-22. About the Author: Marion H. Mixson Jr. Lt Col USAF retired (Air Force from 1950 to 1974 where he worked as Biomedical Laboratory Officer) Clinical Chemist Memorial Mission Hospital Asheville NC 1974-1982 as Laboratory Manager Masters Degree from Ohio State University in 1958 Member of the American Association of Clinical Chemistry since 1962, member subdivision group: Lipoproteins and vascular diseases. Member of the New York Academy of Science since 1992 privately published "Mixson's Family Guide to Cholesterol and Heart Disease" in 1988 deceased 9/4/2012

To the reader: If you would like to make a $10 donation to support the cost of this website. I will send you a copy of Marion Mixson’s 1988 42 page booklet “Mixson’s Family Guide to Cholesterol and Heart disease”. I also will send you the 3 page “Hemoglobin initiative.” Please send the $10 check with the address you want the booklet mailed to to: Stevan Mixson 11 Ballantree Drive Asheville, NC 28803 Please allow 2 to 3 weeks for delivery. Thanks! Stevan Mixson (son of Marion Hagood Mixson Jr.)