Majid Ali, M.D.
Redox regulation in the circulating blood is a dynamic, elaborately integrated complex of diverse energetic-molecular events that involve all plasma and cellular oxidant-antioxidant systems. Changes of redox dysregulation in circulating blood comprise cell erythrocyte membrane damage and cell lysis, zones of plasma congealing, activation of polymorphonuclear leucocytes and monocytes, transformation of monocytes into macrophages, and formation of microclots and microplaques. We designate this broad spectrum of changes as AA oxidopathy. Derangements of the clotting-unclotting equilibrium (CUE), involving both established and as yet unrecognized coagulation pathways, are designated oxidative coagulopathy. Spontaneity of oxidation in the circulating blood assures that oxidative coagulopathy, and fibrinolytic response triggered by it, occurs in health at all times. Oxidatively-triggered molecular responses to AA oxidpathy occurring in the endothelial cells, myocytes and fibroblasts that constitute atherogenesis are regarded as consequences of unrelenting AA oxidopathy.
Plasma cholesterol, a weak antioxidant, initially prevents AA oxidopathy—albeit inadequately—and, once oxidized, feeds the oxidative fires set off by a host of oxidative stressors discussed above. Chronic use of HMG Co-reductase inhibitor statin drugs provides minimal short-term clinical benefits and, as yet undefined, long-term chemical consequences of disruption of lipid metabolism and carcinogenicity. The short-term benefits of statin drugs, in our view, may be largely attributed to their ability to address the single issue of mycotoxicity in the pathogenesis of oxidative coagulopathy.
We propose that ischemic heart disease (IHD) is caused by “AA oxidopathy”—a state of accelerated oxidative molecular injury to blood corpuscles and plasma components. Although AA oxidopathy eventually results in the formation of microclots and microplaques, it begins with oxidative permutations of plasma lipid, sugar and protein molecules, and is not merely confined to oxidative activation of recognized coagulation pathways which we collectively designate as “oxidative coagulopathy”. AA oxidopathy comprises localized areas of blood cell damage and congealing of plasma in its early stages, fibrin clot and thread formation with platelet entrapment in the intermediate stages, and microclot and micro-plaque formation in late stages. Such changes can be directly observed in peripheral blood smears with high-resolution phase-contrast and dark-field microscopy. As observed microscopically, the early changes of oxidative coagulopathy are reversible and constitute what we designate as “clotting-unclotting equilibrium (CUE)”. The AA oxidopathy hypothesis seeks to establish oxidative “clotting-unclotting disequilibrium” and related molecular and cellular events occurring in the circulating blood as the primary pathogenetic elements of IHD, and not those taking place in the vessel wall. This hypothesis challenges two fundamental assumptions of the prevailing cholesterol, inflammatory, infectious, autoimmune and gene hypotheses: 1) that IHD is caused by initial tissue injury occurring in the arterial wall; and 2) that optimal therapeutic approaches must focus on lowering blood cholesterol levels and/or revascularization procedures such as angioplasty and coronary bypass surgery.
AA oxidopathy is caused by oxidant stressors of all types; however, our morphologic observations lead us to recognize the following four groups of oxidants as the principal factors in its pathogenesis: 1) chronic adrenergic hypervigilence associated with lifestyle stressors; 2) rapid glucose-insulin shifts and hyperinsulinemia; 3) mycotoxins, and to lesser degrees, other microbial
oxidants; and 4) ecologic oxidants. Native, unoxidized cholesterol, a weak antioxidant, plays no direct role in the pathogenesis of IHD which is an oxidative phenomenon. Clinical outcome data obtained with nondrug, nonsurgical therapies that arrest and reverse oxidative coagulopathy—without focus on blood cholesterol levels—validate the theoretical tenets of AA oxidopathy as the core pathogenetic mechanism of IHD.
Why should any hypothesis be put forth? One reason is to merge the apparently incongruous parts into a whole, and to facilitate comprehension of the whole. A more important reason is to seek a simpler concept that integrates new observations with established knowledge. In clinical medicine, perhaps the compelling justification for a new hypothesis is to provide the scientific basis and/or rationale for therapies that are more logical, safer and clinically more effective than those based on pre-existing theories. We believe the proposed AA oxidopathy hypothesis meets all of the above three criteria. We introduce the term oxidative coagulopathy for a state of accelerated oxidative intravascular clotting involving known coagulative pathways, and the second term AA oxidopathy for a much broader range of oxidative injury to all the components of circulating blood as well as to coronary arteries and myocardium that cause IHD. In the context of coronary artery disease, our high-resolution, phase-contrast observations are new and are not explained by the existing cholesterol, inflammatory, autoimmune and gene activation/mutation theories of pathogenesis of atherosclerosis. The concept of AA oxidopathy is simple and does integrate the new observations with established knowledge. And, finally, as we demonstrate later in this article, AA oxidopathy does provide a sound scientific basis for therapies that are more logical, safer and clinically more effective than those based on existing cholesterol, inflammatory, infectious, autoimmune and gene theories of atherosclerosis.
In introducing our hypothesis that AA oxidopathy is the core pathogenetic mechanism of IHD, we address the following five basic questions:
- Since blood is the medium of the circulatory system, should inquiry into the pathogenesis of IHD be directed to the primary events occurring in the circulating blood rather than to secondary changes in the arterial wall that result in atheroma formation?
- Since all the primary plasma and cell membrane events occurring in the circulating blood are oxidative in nature and since cholesterol is an antioxidant, is the prevailing focus on blood cholesterol as the centerpiece molecule under scrutiny in experimental and clinical research justified?
- Do the cholesterol, inflammatory, infectious, autoimmune and gene hypotheses adequately explain the pathogenetic mechanisms of the established risk factors of IHD?
- Does the proposed AA oxidopathy hypothesis completely explain the pathogenetic mechanisms of all of the established risk factors of IHD?
- How good are the long-term clinical outcomes of therapies for reversing IHD that are based on the cholesterol, inflammatory, infectious, autoimmune and gene mutation hypotheses? How good are the therapies based on the AA oxidopathy hypothesis?
In 1985, one of us (MA) published the hypothesis that the phenomenon of spontaneity of oxidation in nature is the core mechanism of molecular and cellular injury in all diseases1,2. Since the publication of that hypothesis, we have surveyed a host of natural oxidative phenomena and drawn support for the hypothesis3-8. The notion that a single mechanism can serve as the core pathogenetic mechanism for molecular injury in all disease process appears too simplistic to be valid. Yet, diligent search of the literature of oxidative phenomena in nature and biology fails to uncover any evidence to the contrary9. Specifically, we have investigated oxidative phenomena in peripheral blood in a variety of clinical settings9-11 and have described reversibility of oxidatively-induced erythrocyte membrane deformities12 in patients with chronic fatigue syndrome and platelet aggregation13 by ascorbic acid.
The core concepts of oxidative coagulopathy and AA oxidopathy also evolved from many of our previous morphologic findings of myocardial injury as well as clinical and electrophysiologic observations of heart function14-31. Specifically, we observed microscopic evidence of cardiac myocytolysis in cardiomyopathy14,15; myocardial fibrosis associated with iron deposits in hemodialysis patients16-18, with calcium19 and oxalate deposits20,21. Some parallel morphologic observations concerned vascular intimal proliferative changes and other vascular alterations in hemodialysis patients22-24. We also documented anatomic and enzymatic evidence of ischemic myocardial injury unaccompanied by occlusive coronary artery disease25,26. We have also discussed many clinical implications of the hypothesis of spontaneity of oxidation in integrative medicine in areas of clinical nutrition27,, fitness28, adrenergic hypervigilence29, and meditative self-regulatory methods for stress reduction9,30,31. In 1995, such experimental and clinical observations led us to define ischemic coronary artery disease as a specific example of disease processes that are initiated by oxidative injury29. In two companion articles in this issue of the Journal, together with our colleagues, we present clinical outcome data obtained in patients with advanced IHD and renal failure32-4.
We introduce the terms AA oxidopathy and oxidative coagulopathy in this article for sound reasons. Our morphologic observations of peripheral blood indicate to us that the molecular events involved in recognized intrinsic and extrinsic coagulative pathways are oxidative in nature, though the redox dynamics of such pathways have not been fully investigated. Limited published studies on this subject support our viewpoint35-40. Recently, a third coagulative cascade, Bradford-Allen Coagulation Pathway, has been proposed that involves activation of sialidase enzyme by reactive oxidative species41. It seems likely that additional oxidatively-triggered coagulative pathways will be discovered and characterized in the future. We introduce the term oxidative coagulopathy as an all-compassing term for all such oxidative coagulative events that result in “microclot” and “micro-plaque” formation within the circulating blood. We introduce the term AA oxidopathy for a broader spectrum of morphologic patterns of blood cell and plasma component damage that involves oxidative injury to lipids, sugars, protein moieties as well as diverse enzymatic pathways of intracellular, extracellular and interstitial compartments. The concepts of oxidative coagulopathy and AA oxidopathy evolved from our high-resolution, phase-contrast studies of smears of peripheral blood in patients with advanced coronary artery disease, congestive heart failure, cardiac arrhythmias, hyperglycemia in poorly controlled diabetics, in smokers before and after smoking three cigarettes in three minutes, and a host of other clinicopathologic entities associated with accelerated oxidative molecular injury.
Support for AA oxidopathy as the fundamental pathogenetic mechanism of IHD is drawn from many lines of evidence, and the oxidative nature of the involved processes is recognized as the common denominator. The clinical implications of AA oxidopathy are radically different from those of the prevailing cholesterol, inflammatory, infectious, autoimmune and gene theories. Specifically, it requires that therapeutic strategies be directed to issues of blood ecology (all oxidative phenomena of circulating blood) and not on aspects of vessel wall ecology (cellular events of the vascular wall).
A Need for A Unifying Concept of Atherogenesis
The need for ‛innovative research’ into the pathogenetic mechanisms of IHD was emphasized recently42. The relationship between flow-limiting stenosis and ischemic coronary disease is weak43. Reduction in the extent of atherosclerosis does not correlate with reduced mortality44-8. Treatment with lipid-lowering drugs does not reduce total and IHD mortality in women49-51. A growing body of evidence points to a significant pathogenetic role of chronic inflammation52-56. Among normal men, base-line serum levels of C-reactive protein, an acute-phase reactant, correlate well with future myocardial infarction and stroke, and the increased risk is independent of lipid-related and non-lipid-related risk factors of atherosclerosis57; intriguingly, the benefits of aspirin in risk reduction diminish significantly with decreasing serum levels of C-reactive protein. Other evidence points to the roles of leukocyte activation and an autoimmune process58-61. Several lines of evidence support the critical roles played by platelets, monocytes, macrophages, myocytes and fibroblasts that are unequivocally independent of blood levels lipids63-8. Recent investigations into the molecular basis of IHD have revealed activation of genes that encode for several mediator molecules such as platelet aggregation, vasoactive and chemotactic factors as well as cytokines and interleukins69-71.
The prevailing cholesterol theory of coronary artery disease ascribes the primary pathogenetic mechanism of coronary artery disease to a disturbance of cholesterol metabolism. This view holds up to careful scrutiny neither on theoretical grounds nor on the basis of known experimental and clinical observations. The clinical benefits of lipid-lowering drugs are limited72-78, and their toxicity, including carcinogenicity, is increasingly recognized79-88. The advocacy of some proponents of the cholesterol theory for use of such drugs for persons without raised blood cholesterol levels in disturbing89.
Below, we include a brief survey of pertinent literature as a frame of reference for presenting our AA oxidopathy hypothesis.
The AA oxidopathy hypothesis provides a rational explanation of atherogenic mechanisms of risk factors of IHD as well as for the coronary vasospastic events that cause clinical ischemic heart disease without coronary occlusive disease. The proposed hypothesis also calls for a radically different clinical approach to prevention and reversal of ischemic heart disease. Specifically, it requires an integrative approach that addresses all of the following principal categories of chronic oxidant stressors: 1) adrenergic hypervigilence; 2) glucose-insulin dysregulation; 3) fungal and bacterial stress proteins as well as other types of toxins; and 4) ecologic oxidants. The dominant prevailing approaches to ischemic coronary artery disease—such angioplasty, coronary bypass and multiple drug therapies that focus on calcium channel and adrenergic blockade—evidently do not address any of the causes of AA oxidopathy, and thus cannot be regarded as optmal therapies.
In 1842, T.W. Jones, a British physician asked the question: Why doesn’t the blood circulating in the vessels coagulate90? This question has intrigued blood coagulation researchers ever since. In 1845, Rudolph Virchow, the German physician and father of pathology, responded to the question raised by Jones by stating that under certain circumstances circulating blood does coagulate, and speculated what those pathologic states might be91. Almost simultaneously, A. Trousseau, a French physician, observed that circulating blood does coagulate in the vessels in certain conditions and reported clinical observations to support Virchow’s speculation92. Trousseau’s syndrome is the name still used when thrombophlebosis is associated with malignant diseases.
In 1893, Dastre first proposed the term “fibrinolyse” for his observations on the dissolution of blood clots93. However, his were not among the earliest observations on fibrinolysis. John Hunter, the eighteenth-century English surgeon, included his observation on clot dissolution in his famous treatise on blood94. In 1887, Green’s publication of his studies of the effect of sodium chloride on the dissolution of plasma clot also preceded those of Dastre95. In 1903, Delezenne and Pozerski reported activation of serum proteolytic activity by chloroform96, and four years later, Opie and Barker separated albumen from globulin and proteolytic activity was associated with the globulin fraction97. In 1933, rapid lysis of plasma clots by extracts of beta-hemolytic streptococci was noted by Tillet and Garner98, and in 1944 Kaplan demonstrated that the streptococcal factor was an activator for the proteolytic enzyme precursor in human plasma99.
Fibrinolysis is generally assumed to occur only as a part of the spectrum of pathologic coagulative disorders. Our morphologic observations challenge this assumption. We have repeatedly observed congealed plasma and microclots in healthy subjects without history or demonstrable evidence of any coagulative disorders. We have also observed, as illustrated in this article, that such congealing of plasma and microclot formation is easily reversed by addition of antioxidants, proving that such coagulopathy is oxidative in nature. Our microscopic findings show that clotting and “unclotting” within circulating blood occurs with high frequency in a variety of cardiovascular disorders as well as in otherwise healthy subjects with established risk factors of IHD. In states of accelerated oxidative molecular injury, the rate of oxidative coagulation exceeds that of fibrinolysis, and the various patterns of oxidative coagulopathy and AA oxidopathy are readily observed. Clinicopathologic entities that are associated with disseminated intravascular coagulation, in our view, represent more advanced stages of the same process. While disseminated intravascular clotting in many acute and chronic disorders has been thoroughly studied, the occurrence and extent of such phenomena in the insidious development of molecular and cellular lesions that lead to IHD, to our knowledge, has not been previously recognized.
SPONTANEITY OF OXIDATION IN NATURE
Oxygen: A Molecular Dr. Jekyll and Mr. Hyde
We include below brief comments about some fundamental aspects of the phenomenon of oxidation as a framework for our discussion of oxidative coagulopathy. Oxidation is a spontaneous process—it requires neither an expenditure of energy nor any outside cues. A flower wilts spontaneously; a wilted flower does not “unwilt” spontaneously. Fish rot spontaneously; rotten fish do not “unrot” spontaneously. Cut grass decomposes spontaneously; decomposed grass does not “undecompose” spontaneously. Thus, spontaneity of oxidation in nature is the natural phenomenon that provides the core mechanism of molecular injury in biology. Stated in another way, spontaneity of oxidation is nature’s grand scheme to assure that no oxygen-utilizing form of life remains immune to the immutable law of oxidative death. Oxidation plays a similar role in the decay of inanimate matter as well. Iron rusts spontaneously; rusted iron does not “unrust” spontaneously. Reduction, the other side of the redox equation of life, requires expenditure of energy.
What is the energetic basis of spontaneity of oxidation in nature? A simple analogy may be used to answer this question. A boy is playing with a ball attached to a string. He keeps the ball flying in an orbit around him by moving his extended arm in a circle above his head. In this circumstance, the kinetic energy of the ball seeks to move the ball away from the boy, but it is counterbalanced by the pull of the string on it so that the ball stays in a circular orbit. If the boy lets go of the string, the ball will (“spontaneously”) fly away. The same thing would happen if the boy were to spin the ball with a greater force than can be sustained by the string. The above analogy may be completed by imagining that the ball moves in elliptical orbits—the string has extreme elasticity and pulls the ball closer to the boy’s head by shrinking the string at one time and allows the ball to move far away from the boy by stretching at another time. (Physicists believe that atoms exist in a simultaneous particle-wave state determined by a particle-wave probability distribution.) A similar set of conditions governs the motion of electrons as they spin around the nucleus of an atom. Thus, spontaneity of oxidation (electron loss) is in reality a function of the kinetic energy of electrons which favors their outward movement, hence their loss. Thus, no external source of energy is required in oxidation.
Electrons within atoms and molecules do not orbit the nucleus of an atom in the sense that the earth orbits the sun. Rather, electrons occupy regions of space called orbitals which can hold no more than two electrons. A characteristic of electrons in a given orbital is that they demonstrate opposite spins. Within a molecule, two electrons sharing the same orbital exist in a bond called a covalent bond. A lone electron within an orbital is considered unpaired. This leads us to the definition of a free radical: any atomic or molecular species capable of an independent (“free”) existence that contains one or more unpaired electrons in one or more orbitals. Some common naturally occurring free radicals are included in the following table.
|Types of Radical*||Examples|
Lipid peroxyl lipid-O*
|Hydrogen-centered||Hydrogen atom H*|
|Delocalized electrons||Phenoxyl (delocalized into benzene ring) C6H5O*
Nitric oxide NO*
* Adopted from Halliwell100. Carbon- and sulfer-centered radicals usually react rapidly with oxygen.
Molecular Duality of Oxygen
Oxygen is a molecular Dr. Jekyll and Mr. Hyde. Oxygen ushers life in. Oxygen terminates life. We believe the comprehension of the molecular duality of oxygen is essential to understanding both oxidative coagulopathy and AA oxidopathy—and, hence, to an understanding of atherogenesis.
Diatomic oxygen in ambient air is considered a radical because it contains two unpaired electrons. This structural characteristic of oxygen, according to thermodynamics, should allow oxygen to cause immediate combustion of all organic molecules that come in contact with it. Why does that not happen? The explanation is that the two unpaired electrons of diatomic oxygen in two different orbitals have the same spin quantum number. If oxygen were to directly oxidize organic molecules, it would have to accept two electrons from a donor with spins that are opposite to its own two unpaired electrons so as to be properly accommodated into the vacant spaces in oxygen’s two orbitals containing unpaired electrons. This, of course, cannot be achieved by electrons in covalent bonds, which spin in opposite directions. Such spin restriction explains oxygen’s poor reactivity even though it is a powerful oxidizer.
More than 90% of the oxygen used in the human body is utilized by mitochondrial cytochrome oxidase, which transfers four electrons into an oxygen molecule to produce two molecules of water.
O2 + 4H+ + 4e– = 2H2O
Under ordinary circumstances, reduction of oxygen by cytochrome oxidases in the above reaction does not release reactive oxygen radicals. This is assured by transitional metal ions—such as iron, copper, vanadium and titanium, which are carried in the active sites of cytochrome oxidases. Such metal ions occur in variable states of oxidation, and changes in such states facilitate transfer of single electrons in an orderly fashion in which various partially reduced forms of oxygen are held bound to the metal ions. These ions also play essential roles in spontaneous oxidation (autoxidation) of several nonradical compounds including ascorbic acid; thiols such as cysteine, homocysteine and reduced glutathione; catecholamines such as epinephrine and norepinephrine; and a host of amines such as 3,4-dihydroxyphenylalanine (DOPA) and 6-hydroxydopamine.
Molecular oxygen has an interesting “love-hate” relationship with electrons. It avidly picks up free electrons in its vicinity, then just as avidly spins them out. In a vacuum, electrons travel at the speed of light. Even though the speed of an electron in tissues would be expected to be drastically reduced, the electron-oxygen transactions must still take place at amazingly fast speeds. During oxidative phosphorylation in the generation of ATP, molecular oxygen accepts an electron—is reduced—to become superoxide. Superoxide then loses it electrons spontaneously—is oxidized—in initiating the free radical chain reactions that result in the formation of peroxides, oxyacids, aldehydes and hydroxyl radicals. Such free radicals oxidize proteins of coagulation cascades, thus triggering oxidative coagulopathy, which further fans the fires of AA oxidopathy. However, our high-resolution microscopic observations described in this article lead us to conclude that accelerated oxidative stress on components of circulating blood is neither confined to oxidative injury of coagulation pathways nor, indeed, are the coagulative phenomena the initial events. We introduce the term AA oxidopathy to encompass a broad range of oxidative events that include: 1) peroxidation of plasma and cell membrane lipids; 2) oxidative permutations of plasma and cell membrane sugars and proteins; 3) accelerated autoxidation of nonenzymatic plasma antioxidants such as thiols and ascorbic; 4) inactivation or saturation of plasma enzymatic antioxidant mechanisms; 5) endothelial injury; and 6) later oxidative injury to subendothelial collagen and the muscularis of the arterial wall. Oxidation of LDL cholesterol—widely believed to be the critical event in atherogenesis—is, in our view, a relatively less significant event. We return to this essential issue later in this paper.
How Do Cells Autoregulate?
Teleologically, individual human cells may be expected to autoregulate, just as unicellular organisms regulate themselves. Indeed, human cells often regulate their own growth and other functions by affecting key transcription factors. One commonly used mechanism by which cells turn key proteins on and off is by adding or removing phosphate groups. A growing body of evidence shows that human cells may also use redox reactions in a parallel way. For example, hydrogen peroxide is involved in the signaling pathway of platelet-derived growth factor which stimulates proliferation of vessel wall myocytes 101. Other examples of autoregulation of cells by employing their oxidants include activation of nuclear factor Kb, which turns on genes for some mediators of inflammation, and inactivation of AP-1, which controls some genes involved with growth102. Nature seems to have yet simpler and more elegant ways to allow individuals cells to autoregulate. For example, it assigns important cell signalling functions to molecular oxygen by simply adding an electron to it. Recently, it has been shown that superoxides relay Ras protein’s oncogenic message in transformed fibroblasts103.
Abnormal coagulative phenomena within the circulating blood occurs in diverse pathologic entities such as eclampsia, anaphylaxis, localized and generalized Schwartzman reactions, hemorrhagic diathesis in clinical and experimental acute viral infections, bacterial endotoxic shock and others104-8. Some earlier investigators mistook such coagulative phenomena within the vascular lumina—fibrin threads and amorphous deposits as well as the classical thrombi—as postmortem events. However, this mistake was recognized by Ingerslev and Teilum who described fibrin thrombi in hepatic periportal sinusoids in the liver of women who survived eclampsia 109.
The occurrence and patterns of free radical injury to the myocardium, the conducting system of the heart and coronary arteries has been investigated extensively with ischemia-perfusion studies110-115. Specifically, free radical, particularly superoxide anion (O2._) and hydroxyl radical (OH*) are produced during and after ischemia and reperfusion, and cause oxidative functional and structural injury to the heart, including the loss of myocardial contractile function. Superoxide anion is a relatively weak oxidant and owes most of its destructive potential to its ability to generate hydrogen peroxide by reacting with molecular oxygen. Hydrogen peroxide, in turn, generates highly toxic OH* radicals in the presence of transition metals such as iron and copper.
Morphology of Atherosclerosis
By gross morphology, atherosclerosis is a simple process that progresses in three stages. In the first stage, a yellow-gray fatty streak appears on the inner surface of the vessel wall. Histologically, it comprises foamy, lipid-laden macrophages in the subendothelial space. The second stage is characterized by a fibrous plaque formation. The plaque is composed of a central necrotic, acellular area of fatty deposits covered by a “fibrous” cap which in reality is made up of proliferating myocytes and fibroblasts in a matrix of collagen. In the third stage, hemorrhage occurs within the central necrotic area resulting in a thrombus composed of fibrin threads with trapped platelets. The smooth fatty streak is often present in children and progresses to the two later stages with time.
The common atheromatous plaque is a raised area of white-gray-yellow discoloration on the inside wall of a vessel. As a plaque grows in size, it protrudes into the vascular lumen and begins to reduce the inner caliber of the vessel. Plaques may vary in size from 0.1 to 2 cm in diameter, but may grow to much larger sizes when they coalesce. In the aorta and larger arteries, plaques may extend for several centimeters. The luminal surfaces of plaques are usually irregular and indurated; small erosions covered with fibrin thrombi are commonly observed. On section, the center of large plaques often exudes viscous, yellow-gray-brown gumous material—hence, the name atheroma from the Greek word for gruel. Histologically, plaques are composed of necrotic tissue with cholesterol crystals and other lipid deposits; degeneration and necrosis of the collagen and the muscle in the vascular wall; smooth muscle proliferation; and fibrous scarring at the periphery of the plaque. In advanced stages, hemorrhage and dystrophic calcification frequently occur in necrotic tissues.
It is the presence of cholesterol crystals and other lipid deposits in the plaque that has misled generations of pathologists and cardiologist into thinking that cholesterol is the cause of atherosclerosis—just as a prior generation of pathologists made a similar error, mistaking deposits of dystrophic calcification in injured tissues as evidence for dysregulated calcium metabolism. In atherosclerosis, cholesterol deposits occur as a consequence of oxidative coagulopathy just as calcium deposits are often seen in organized hematomas. As we mention briefly earlier in the abstract of this paper, and discuss at length below, cholesterol is an antioxidant and cannot cause the lesions of coronary artery disease which are produced as a result of oxidative injury.
Morphologic Patterns of AA Oxidopathy
Below we describe our high-resolution, phase-contrast morphologic observations that comprise oxidative coagulopathy. The degree and extent of such coagulopathy, of course, varies over a broad range depending on the number and the nature of oxidative stressors. In the context of IHD, important oxidant stressors include hyperadrenegic state, smoking, hyperglycemia, excess of oxidized plasma lipids, obesity and cardiac arrhythmias. We have observed oxidative coagulopathy to generally progress in the following seven morphologic stages:
- Erythrocyte and Leukocyte Membrane Deformities
- Diaphanous Congealing of Plasma
- Platelet Aggregation and Membrane Damage
- Filamentous Coagulum (Fibrin Needles)
- Lumpy Coagulum
The patterns of oxidative coagulative injury described in this article were observed in extensive studies of blood morphology in a host of acute and chronic cardiovascular as well as non-cardiovascular disorders, including advanced IHD, unstable angina, congestive heart failure, cardiac arrhythmias, hypertensive crises, acute and chronic viral and bacterial infections, fungemia, acute and chronic atopic disorders, chemical sensitivity reactions, acute and chronic degenerative disorders and malignant diseases.
Erythrocyte and Leukocyte Membrane Damage
Erythrocytes when observed with an ordinary bright-light microscope appear as rigid biconcave disc-shaped corpuscles. When examined with high-resolution, phase-contrast microscope, these cells are seen as malleable round cells that readily change their shape to ovoid, triangular, dumb-bell or irregular outlines to squeeze past other erythrocyte in densely populated fields. Such cells assume their regular rounded contour as soon as they find open space. The earliest and most common abnormality we observed in oxidative coagulopathy was erythrocytic rouleaux formation (figure 1), erythrocyte lysis (figure 2), leucocytic clumping (figure 3) and platelet aggregation (figure 4). Notably, we often observed erythrocytic and leucocytic changes of coagulopathy independent of platelet clumping (figure 5). Frequently observed erythrocytic membrane abnormalities included wrinkling, tear drop deformity, sharp angulations and spike formations (figure 6). We established the oxidative nature of such abnormalities by demonstrating their reversibility with antioxidants such as taurine (figures 19 and 20), vitamin E (figures 17 and 18), vitamin A, and vitamin C reported previously12 but nor shown in this article. Parenthetically, we add that we have observed similar evidence of erythrocyte membrane injury in diverse clinical entities associated with accelerated molecular injury such as disabling chronic fatigue, fibromylagia and a host of severe nutritional, ecologic and autoimmune disorders.
Erythrocyte Homogenate, Free Iron and AA Oxidopathy
In deliberations of atherogenesis, oxidative injury to erythrocytes, the presence in the plasma of free hemoglobin leached from damaged red cells—and the presence of excess iron in the plasma as a result of those factors—are seldom, if ever, considered to play any roles. Our morphologic findings lead us to propose that oxidative erythrocyte injury plays an important role in the genesis of AA oxidopathy—and, hence, atherogenesis. We observed erythrocyte damage and lysis with high frequency in all clinical states accompanied by accelerated oxidative stress on the components of circulating blood.
Iron, like oxygen, is a molecular Dr. Jekyll and Mr. Hyde. It is needed for molecular transport (in hemoglobin for oxygen), for storage (in myoglobin), for energy functions (in cytochrome oxidase and other cytochromes), for respiration (in non-heme-iron proteins), and for antioxidant defenses (in catalase). In its Mr Hyde role, iron (in free form) is a potent oxidant and catalyzes the generation of many dangerous oxygen-derived radicals116-119. In health, the Mr. Hyde roles of iron are minimized by transferrin, an iron-binding protein which rigidly limits the availability of free iron. In normal plasma, only 20 to 30 percent of transferrin occurs in a saturated state.
Free hemoglobin has been considered a dangerous protein—a biological Fenton catalyst120,121. It rapidly quenches free radicals in an highly oxidizing environment and becomes oxidized, thus turning into a potent oxidant. It is readily degraded by H2O2 to release free iron which initiates and propagates several free radical reactions122,123. Hemoglobin also reacts with H2O2 to produce a protein-bound oxidizing species capable of causing lipid peroxidation 124. Free hemoglobin also avidly binds with nitric oxide radicals and induces vasospasm, triggering yet other oxidizing events125, which, in turn, feed the “oxidative fires” of AA oxidopathy.
Beyond ample evidence of destructive oxidizing capacity of erythrocyte-derived factors discussed above, there is also direct evidence that red blood cells play a role in atherogenesis. Sambrano and colleagues have shown that certain receptors on macrophages for oxidized LDL also bind to oxidatively-injured red cells prior to their internalization and lysis126. Lipid and carbohydrate moieties of erythrocyte membranes can be expected to play of host of roles in oxidative coagulopathy and AA oxidopathy just as they do in attachment, endocytosis, membrane fusion and viral hemagglutination in viral infections127-129.. As we discuss in Part-II of this article, a growing body of evidence points to the roles of strong inflammatory, infectious and autoimmune mechanisms in atherogenesis. It seems obvious to us that additional evidence for inflammtory and immunogenic roles of erythrocyte-derived factors in oxidative coagulopathy, AA oxidopathy, atherogenesis and IHD will be forthcoming as those areas are explored further in future.
Of considerable interest in this context is the matter of electrostatic interactions among oxidatively-damaged eryhtrocyte membranes and other oxidized elements in the circulating blood ecosystem. Phospholipids and lipid components of LDL inhibit infectivity and hemagglutination of rhabdoviruses, probably because of structural similarity between such compounds and the receptors for the viruses and cell membranes130. In the case of vesicular stomatitis virus, phosphatidylinositol, phosphatidylserine and GM3 ganglioside show inhibitory activity131. But what are the mechanisms of action of such lipid moieties? Some light on this question is shed by studies of Mastromarino and colleagues131 in which removal of negatively charged molecules from membrane lipids by enzyme treatment significantly reduces their inhibitory activity, suggesting that electrostatic interactions play important roles in viral-cell membrane dynamics. It is likely that similar electrostatic roles involving platelet, monocyte and other elements in circulating blood ecology will also be discovered in the future.