by Academicians Anatoly KLIMOV, Vladimir NAGORNEV (Russian Academy of Medical Sciences), and Alexander DENISENKO, Dr. Sc. (Med.), RAMS Research Institute of Experimental Medicine, St. Petersburg
More than 15,000,000 people die of cardiovascular diseases every year all over the world, with nearly half of the lethal cases (7,200,000) being due to myocardial infarction that sails ever more frequently persons of the middle and even young age. This killer disease (as well as stroke and ischemia of inferior limbs) is caused by atherosclerosis by and large. Affecting the entire organism, it is responsible for pathologies in the trunk arteries of the heart, brain, kidneys and other organs. Identification of factors contributing to this disease enables fresh approaches to its diagnosis and treatment.
Atherosclerosis develops slowly, over decades. One of its main inducing factors is the high concentration of cholesterol in blood plasma where it is present as part of lipid-protein complexes, the lipoproteins.
These occur in several classes differing in dimensions, density, in the quantitative relations of lipids and proteins, and in other characteristics. Compared with other analogs, low-density lipoproteins (LDL) are the most cholesterol-rich (up to 45 wt. percent) and atherogenic (i.e. provoking atherosclerosis) particles. LDL account for about 70 percent of blood plasma cholesterol. These lipoproteins carry cholesterol from blood to organs and tissues, and their increased concentration in blood heightens the risk of pathology. Individuals with a homozygous (i.e. inherited from both parents) hypercholesteremia* develop a high level of LDL in blood and in the absence of medical treatment die of the aftereffects of atherosclerosis at an early age (10 to 20 years).
The initial stages of atherosclerotic lesions (fatty spots and streaks) are characterized by the intra- and extracellular accumulation of lipids in the arterial inner layer (intima). Its cells, which contain in their vacuoles a large amount of cholesterol and its esters with fatty acids, are mostly mononuclear phagocytes (macrophages). When observed with a microscope, vacuoles look like foam bubbles, and that is why constituent cells are called foam cells.
MODIFICATION OF LIPOPROTEINS AND ATHEROGENESIS
In their now classical experiments the American scientists Drs. Michael S. Brown and Joseph L. Goldstein (Nobel Prize, 1985) have demonstrated that cells capture low-density
* Hypercholesteremia (hypercholesterolemia) - increased concentration of cholesterol in blood. -Ed.
For their study of immunological mechanisms of atherosclerosis, and innovative methods of its diagnosis and treatment Drs. A. KLIMOV, V. NAGORNEV and A. DENISENKO were awarded a State Prize of the Russian Federation (science and technology), 2003.
lipoproteins by specific receptors recognizing a corresponding protein component. Since their activity is regulated by intracellular cholesterol via feedback, its excessive accumulation cannot be realized with the participation of LDL receptors. There is direct evidence for that. When native (not modified) human blood plasma LDL were incubated with mouse peritoneal macrophages (the procedure used in model experiments), there was no active uptake of lipoproteins by cells having a very small number of the LDL receptors. And yet patients with a history of homozygous family hypercholesteremia and rabbits having the same pathology, despite the total absence of such functionally active receptors, are rather quick in developing heavy atherosclerotic lesions of blood vessels with characteristic foam cells. Therefore the LDL receptors cannot be implicated in the accumulation of lipids by macrophages, though their deficiency causes hypercholesteremia and atherosclerosis.
But if macrophages cannot consume such large amounts of lipoproteins in this fashion, how do they manage to do it after all?
Ilya Mechnikov, an eminent Russian biologist and pathologist (Nobel Prize, 1908), described macrophages as "scavengers" because of their ability to take up and digest substances foreign to the organism. Proceeding from the same assumption, Drs. Brown and Goldstein (USA) have postulated that certain changes in the LDL structure should intensify the uptake and digestion of such substances by macrophages which come to be transformed to foam cells. As confirmed by experiments, modified low-density lipoproteins (mLDL) do exist, and they are taken up by a scavenger receptor that does not recognize and bind native LDL (essential difference!), which are not atherogenic per se, and its activity is not regulated by intracellular cholesterol.
The following dependence holds: the more of the LDL is present in blood, the higher the probability of structural changes in such lipoproteins (and of consequent changes in their physicochemical and biological characteristics), and the larger the proportion of modified lipoproteins circulating in the organism. In the 1980s Dr. Daniel Steinberg (USA) demonstrated the leading role of lipid peroxidation in this process. Although the initial stages of such peroxidation may take place in blood, the most radical changes occur in the arterial wall when the scavenger receptors of macrophage begin recognizing the modified particles as foreign substances. This is a signal triggering the mobilization and migration of blood cells, the monocytes and lymphocytes, into the vascular wall. In the arterial intima, where mLDL come to be accumulated, monocytes are converted to macrophages and, thereafter, into foam cells.
The further buildup of lipoproteins in the intima aggravates the atherosclerotic scenario which changes its subsequent course essentially. At this
Foam cell. V - lipid vacuoles; CC - cholesterol crystals.
stage macrophages are no longer able to cope with the overabundance of lipoproteins and perish because of the vast buildup of cholesterol and its esters. Ал emergency like that activates the reserve defenses of the organism-namely cells of other types, mostly the smooth muscle and lymph cells (lymphocytes). The process of such cumulative action can be described as aseptic inflammation with the characteristic aggregation of inflammatory cells, necrotic foci, connective tissue capsules and the like. It gives rise to atheromas (tissue degeneration and decay) which can cause vascular attenuation, aneurism and even ruptures. Should the endothelial monolayer be impaired thereby ("plaque rupture"), there follow such aftereffects as the deposition of fibrin as well as a parietal thrombus (the blood clot formed thereby may come off) and embolism; the atheroma gives way to thrombosis. Blood-vessel occlusion (clottage) may lead to disruption of the blood supply of tissue and cause myocardial infarction or insult (stroke).
Smooth-muscle cells getting into the focus of lesion start proliferating (growing apace) and synthesizing connective tissue proteins (collagen, elastin) so as to surround (isolate) the cholesterol mass by a tight fibrous capsule. However, this protective adjustment of the organism poses the danger of the constriction (and even total occlusion) of the vascular lumen and thus can much deteriorate the blood supply to the ambient tissues. Fibrous plaques, the chief morphological manifestation of atherosclerotic lesions, are formed as a result. Pathologies of different types and at different stage of progression may occur in different parts of a patient's cardiovascular system. Fibrous excrescence and vascular tissue necrosis may concur in the same place to generate new atheromas and new pathology.
Thus we have assumed that the modification of lipoproteins, being one of the first and obligatory events in atherosclerosis pathogeny, likewise induces the production of autoantibodies to them. That is to say, converts them to autoantigens, a process in which the immune system is implicated*.
Now what concerns the peculiar characteristics of atherosclerotic lesions attending immunopathologies, too. First, they are characterized by a focal pattern - that is lipoproteins are deposited in definite foci, or sites, of arteries, and that's where atherosclerotic plaques are formed. Second, as shown by experiments in animals, hypercholesterolemia develops in waves (wavy pattern) as seen in the formation of "layered" plaques with alternate layers of lipids, foam cells and fibrous tissue. And last, such lesions are remarkable for the monocytic and lymphocytic infiltration of arteries; besides, cell populations arising during atherosclerotic lesions and immune inflammatory diseases are alike.
Incubation of mouse peritoneal macrophages with an immune LDL-Ab complex for 48 h. Left, cells dead through necrosis; right-those in apoptosis.
* See: V. Chereshnev, B. Yushkov, "Immune System and Hemopoiesis", Science in Russia, No. 1. 2005. - Ed.
These peculiarities invite the idea: atherosclerosis is a disease in which an autoimmune factor could be playing a very important role.
Our theory of atherosclerosis pathogeny provides for a definite sequence of events: (1) modification of lipoproteins which develop autoantigen properties; (2) production of antibodies (Ab) against mLDL; (3) formation of mLDL-Ab autoimmune complexes which come to be attached to the arterial wall surface, with the endothelial integument damaged thereby, thus facilitating the invasion of the complexes and free lipoproteins (LDL above all) into the arterial intima; (4) uncontrolled uptake by arterial wall macrophages of the mLDL-Ab complex and transformation of these cells into foam ones; (5) progress of the focal lesion of the arterial intima, destruction of cells and fibers, and elimination of decomposition products as the lymph flows out to regional lymph nodes; (6) formation of antibodies to the structural components of vessel wall, their fixation on modified vascular structures followed by further progression of the pathological process. We began our studies by inducing atherosclerosis in rabbits.
WHAT DID EXPERIMENTS SHOW?
First we studied the formative dynamics and circulation periods of the autoimmune complex mLDL-Ab in experimentally induced hypercholesteremia. In test animals antibodies to LDL appeared already 7 days upon the introduction of atherogenic feed; their titer reached a maximum in the second week, whereupon it declined to naught by the sixth or seventh week. The mLDL-Ab complex was identified and isolated as early as the third week of the experiment when the number of free antibodies started shrinking; however, it could not be detected in the fifth week. Yet it came up again with an increase in arterial lesions to attain a maximum by the twelfth week of the experiment.
Simultaneously we investigated the dynamics of protein deposition (LDL, gamma-globulin and fibrin) in the aorta wall. We found that the diminished concentration of the immune mLDL-Ab complex in blood (fifth week) concurred with its appearance in the vascular wall. Thereupon destructive changes followed on the affected sites of the aorta: swelling and serous-fibrinous edema of the intima; impaired permeability of the endothelial barrier; fragmentation of elastic fibers; formation of foam cells and their destruction.
Our next step was to find out if it was possible to slow down the progression of atherosclerotic lesions through medication. For this purpose we used an immunodepressant, cyclophosphamide (cytoxan). Administered to rabbits on a cholesterol diet, this preparation prevented mLDL-Ab complexation; atherosclerosis of the aorta did not develop or else it was mild (mean lesion area amounted to 5.0±3.4 percent, while the figure for the control group was as high as 52.3±24.6 percent). Therefore these experiments showed the signifi-
Adhesion of monocytes to the endothelium (left) and their migration into the intima (right).
cant role of immimological mechanisms in the pathogeny of experimentally induced atherosclerosis.
An important proof of the autoimmune nature of the disease is that it could be provoked in a recipient inoculated with serum antibodies or lymphoid cells isolated from a sensitized* organism. We tried exactly that by experimenting on mice. Put on an atherogenic diet (that contained 4 percent of cholesterol) for three months, these mice registered a nearly 70 percent increase of cholesterol in their blood. Atherogenic changes were also detected in blood plasma as shown by a decrease in the concentration of high-density lipoproteins (HDL) inhibiting atherosclerosis and a simultaneous increase in the LDL concentration. Our experimental mice thus developed moderate atherogenic hyperlipemia (hyperlipidemia).
To transfer the thus induced LDL autosensitization to healthy animals on an ordinary diet, we injected intraperitoneally, spleen cells obtained from hyperlipemia - affected mice (each dose containing 107 cells). Autosensitization to LDL was detected three days thereafter and persisted at least up until the 14th day of the experiment. Cholesterol concentration in plasma increased by more than 40 percent. Both the donors and the recipients exhibited analogous changes of the lipoprotein spectrum: the concentration of LDL increased twofold, while that of HDL was down. The transfer of spleen cells from mice on a regular diet, who had no sensitization to lipoproteins, or liver cells from mice, who developed hyperlipemia and autosensitization to lipoproteins, had no effect on lipoprotein concentration in blood, nor did it affect immunological indexes either. Therefore the above changes cannot be explained by the presence of large doses of cholesterol in the spleen cell culture, for liver cells of hyperlipemia-affected animals had 3 to 4 times as much of it as the spleen cells did. That is the transfer of LP sensitization led to changes in the lipoprotein spectrum and concentration in blood-changes that could be evaluated as "autoimmune hyperlemia".
IMMUNE COMPLEXES IN BLOOD AND ARTERIES
For all the cogency of data obtained from experimental animals, it was important to look into the role of immune factors for the development of atherosclerosis in man. The first question to come up was whether mLDL-Ab complexes are present in the blood of individuals-those suffering from heart ischemia and not. To elucidate this point it was essential to devise methods of their isolation and quantitative assay.
The mLDL-Ab complex was found to be present not only in persons with clinical manifestations of atherosclerosis but also in those not affected by this pathology. True, mLDL-Ab complex concentration is rather low: in healthy individuals it averages 32 ng/ml (in gamma-globulin equivalent), while in the ischemic heart disease patients it is nearly tenfold as high (292 ng/ml). But even in heart ischemia cases only 0.27 percent of plasmatic LDL happens to be implicated in the formation of autoimmune mLDL-Ab complexes. This could be due to the vigorous elimination of the complexes from the blood flow because of their active capture by cells of the reticuloendothelial system.**
The concentration of the mLDL-Ab complex in the atherosclerotic intima of the human aorta was found to be much higher than in plasma. On the average 5.3 percent of LDL in the intima are associated with antibodies (against 0.27 percent in plasma). Which means that mLDL-Ab complexes are formed in the aorta wall. The antibody-producing cells (B-lymphocytes) detected in the zone of the coronary artery's atherosclerotic patch (plaque) are indirect evidence for that.
Over the past two decades antibodies to modified (oxidized) LDL have been found time and again in patients afflicted with coronary atherosclerosis. The increased presence of such antibodies could even portend myocardial infarction. On the other hand, deposits of gamma-globulin and components of the complement*** system have been spotted in atherosclerotic lesions of the human aorta and coronary arteries. Even if the presence or increased concentration of antibodies to mLDL and immune mLDL-Ab complexes in blood could be regarded as a marker, the data on the accumulation of the immune complex components in the foci of atherosclerotic arterial lesions attest to the active involvement of the immune complexes in atherogenesis.
The very notion of atherogenicity implies the grave danger of lipoproteins or immune complexes: circulating in blood, they provoke atherosclerosis. We have assessed but two of its essential indicators: its ability to induce cholesterol accumulation in macrophages (a process giving rise to foam cells) and its cytotoxicity and ability to promote the penetration of free lipoproteins into the intima.
ACCUMULATION OF CHOLESTEROL IN MACROPHAGES
The physiological meaning of any immune complex amounts to neutralization of an antigen's adverse effect and its elimination from the organism. In our case this is the suppression of mLDL atherogenicity and the subsequent capture of the complex by cells of the reticuloendothelial system (morphologically atherogenicity was assessed by the intensity of the conversion of macrophages to foam cells).
Our experiments have shown that incubation of native LDL (isolated from the blood of healthy individuals) in the presence of macrophages pro-
* With reference to sensitization (sensibilization), or enhanced sensitivity of organisms, their cells and tissues to a particular substance. - Ed.
** Reticuloendothelial (macrophagal) system- a totality of protective cells in the organism of mammals, man including. These cells capture and digest bacteria and foreign or toxic particles. -Ed.
*** Complement-a set of immune proteins contained in the fresh blood serum of animals and man. It contributes to the bacteriocidal effect of blood. - Ed.
duces but insignificant amounts of cholesterol esters and, morphologically, ends in the appearance of solitary lipid vacuoles. Yet LP-Ab artificial complexes formed in vitro produce far greater amounts of cholesterol esters (sixty times as much as in the incubation of cells with free LDL) under similar conditions, while macrophages come to be virtually stuffed with vacuoles containing the selfsame esters: in fact they transformed into foam cells.
We found it interesting to compare the atherogenicity of naturally modified lipoproteins with that of immune mLDL-Ab complexes isolated from the human aorta. It was aortic mLDL that had the most stimulating effect for the accumulation of cholesterol esters in macrophages. Although the mLDL-Ab complex is less atherogenic, its effect is quite sufficient for foam cell formation. The persistent atherogenicity of autoimmune mLDL-Ab complexes isolated from the aorta (and blood plasma as well) may be due to macrophages capturing them with the aid of a different type of receptors (Fc) that recognize and bind antibodies of these complexes. As it is with scavenger receptors, the intracellular cholesterol does not inhibit their activity.
Consequently, complexation of mLDL with autoantibodies does not result in the loss of mLDL atherogenicity which only declines somewhat. This is why so many attempts at various laboratories to induce resistance to atherosclerosis through immunization of test animals with modified LDL have had moderate effect.
And last, yet another feature of immune mLDL-Ab complexes. As we found, an increase in the concentration of antibodies in a synthesized analog activates its capture by macrophages, i.e. its atherogenicity intensifies, and so does its elimination (removal) from the blood flow of animals upon its intravenous injection. It is probable therefore that under natural conditions, too, the most atherogenic form among autoimmune mLDL-Ab complexes should be captured by cells of the reticuloendothelial system, macrophages including first of all.
CYTOTOXICITY OF IMMUNE COMPLEXES
Overall, cytotoxicity is characteristic of autoimmune complexes of any nature. It is particularly high in the mLDL-Ab complex (synthesized in vitro or isolated from blood plasma or aortic wall). A significant portion of cells perished when this complex was incubated with macrophages; this occurred both through necrosis (nonspecific form of death caused by grave injuries) and through apoptosis (programmed death). Among other things, we studied the impact of the LDL-Ab complex obtained in vitro on the endothelium of the rabbit carotid artery during its perfusion* in situ. And we found that a 45-minute circulation of the immune complex in
Perfusion of the myocardium (by single photon emission computer tomography, SPECT) before heparin cryoplasmaprecipitation (top) and after.
* Perfusion-here, injection or penetration of a liquid (e.g. blood) into the blood vessels of a particular organ, body part or the entire organism. - Ed.
the perfusing artery can cause appreciable damages to the plasmatic membranes of the carotid artery's endothelial cells and facilitate the LDL entry into the intima.
Lately a new approach has been adopted to the problem of atherosclerosis pathogeny - this malady is viewed as a sluggish immune inflammation. Drs. Goran Hannson (Sweden) and Peter Libby (USA) have made a tangible contribution to this idea. We, too, are devoting much attention to this aspect of the problem (Dr. V. Nagornev et al.).
Facts show certain similarity between the two processes - atherosclerotic lesions and those caused by inflammation during various immunopathological conditions. Both are accompanied by the formation of a neoantigen, by the focal accumulation of nongranular leukocytes (monocytes-macrophages and lymphocytes) migrating to the arterial wall from the blood flow, by the focal growth of connective tissue, and so forth. Besides, immune inflammation factors have been detected in atherosclerotic lesions - cytokins, growth factors, adhesive molecules, immunoglobulins, complement, and so on.
NEW METHODS OF TREATMENT
The proof of any theory, no matter how intriguing, is in practice. Accordingly, we have been paying much attention to finding fresh approaches to the treatment of myocardial ischemia and other manifestations of atherosclerosis by means of immunocorrective therapy. One is the use of the immunomodulator T-activin isolated from bovine thymus gland and containing a mix of short-chained proteins. This preparation is used for immunodeficiency conditions, particularly when the T-system of immunity is damaged. It stimulates the generation of certain cytokins, rehabilitates the activity of T-killers, activates the performance of stem hemopoietic cells and normalizes a number of other immunity indexes.
We used T-activin for cases with the unstable angina when the presence of autoimmune mLDL-Ab complexes in blood was combined with the enhanced sensitization of lymphocytes to LDL. Such sensitization disappeared in all the patients after two weeks of treatment, with the number of T-suppressor cells restored back to normal. Simultaneously, veloergometry indicators improved, and so did the patients' general state. Angina attacks occurred less often, and the need of nitrate medication diminished. The effect of treatment kept on for two months. Immunocorrective therapy works if supplemented with other remedies for ischemic heart disease.
Another method used in treating atherosclerosis and its complications involves extracorporal removal of autoimmune complexes from blood plasma by means of heparin cryoplasmaprecipitation (i.e. precipitation in cold). Briefly, it consists in the following. A relatively large amount of blood is taken from the patient. After centrifugation the blood cells are returned to the patient, while the plasma is allowed to stand at low temperature (down to -30°C) for 24 h. A dense precipitate containing predominantly fibrinogen, mLDL and immune complexes is formed thereby. The plasma is decanted and, upon being heated to body temperature, is given back to the patient. In all, 4 - 6 procedures with the interval of 1 to 2 days are needed for complete plasma exchange. Neither large volumes of plasma nor such valuable components as high-density lipoproteins (HDL) possessing antiatherogenic properties are lost in the process. A single course of treatment brought down the concentration of mLDL-Ab complexes in blood plasma (sixfold on the average); the amount of lipid peroxidation products dropped twofold, while resistance of lipoproteins to such oxidation increased by 80 percent. Plasma atherogenicity went down fourfold at a moderate decrease of cholesterol level (!), and more than half of the ischemic patients (51 percent) improved their condition. The number of angina episodes was down, too, and so was the amount of nitrate drugs taken.
Heparin cryoplasmaprecipitation improved reliably the veloergometry and electrocardiography records. The results thus obtained show the high therapeutic efficiency of the procedure. This is also confirmed by single photon emission computer tomography (SPECT) showing an increase of myocardial perfusion both in quiescence and at physical load.
As it was mentioned, not only autoimmune complexes but some of the mLDL and fibrinogen (factor I) is precipitated by heparin at low temperature. This has a favorable effect on the further course of the ischemic heart disease. The optimal longstanding results have been achieved in the reduction of plasma mLDL-Ab complex concentration.
T-activin and heparin-mediated plasmaprecipitation show a favorable effect already in the first weeks of therapy when regression of atherosclerotic lesions is absolutely out of the question. This effect must be due to a decrease in the content of immune complexes in blood which damage the vascular endothelium.
Thus, the autoimmune theory of atherosclerosis pathogeny allows to explain certain characteristic phenomena of the disease. Lately it has expanded into a broader view of atherosclerosis as chronic immune inflammation. Accordingly, new methods of treatment have been elaborated and introduced into clinical practice so as to prevent formation of modified lipoproteins and antibodies to them or to promote their elimination from blood.
Опубликовано 27 сентября 2018 года
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