VESSEL WALL METABOLISM I N S H R RATS IN RELATION TO ATHEROSCLEROSIS 0. Mrhovl, 1. Albrecht,* and D. Urbanovl Insritute f o r Clinical and Experimental Medicine and *Institute of Physiology Czechoslovak Academy of Science Prague-KrF, Budgjovickd. Czechoslovakia
The problem of atherosclerosis is undoubtedly one of the most pressing in modern medicine. Because the target organ of this disease, namely, the vessel wall, has a relatively intensive metabolism, the relationship of this metabolism to the initiation and development of atherosclerosis has been studied. As stressed by the results of studies performed over many years, the problem associated with the development and therapy of atherosclerosis cannot be tackled without taking into account the metabolic processes of the vascular wall. Whatever the pathogenesis of the disease, the participation of the vascular wall and, above all, its metabolism in the formation of t h e pathologic changes currently appear to be the most important factors. Our study of the problem yielded the finding that the changes in the activity of some enzymes of basic metabolic cycles accompany, or even precede, the pathologic changes in the vessel that lead to the development of atherosc1erosis.l A lowered activity of Krebs cycle enzymes and enhanced activity of some phosphomonoesterases were found to be characteristic for the processes that lead to the deposition of lipids, alterations of connective tissue reactions, and the formation of atherosclerotic lesions in the vessel wall. This finding was true both in experimental animals under artificial conditions, wherein the dominant factor was a lesion of the vessel wall, and in human vessels or vessel segments with a predilection for the development of atherosclerosis, for example, abdominal aorta as compared with ~ . ~ metabolic condithoracic aorta, pulmonary artery versus aorta, and so ~ n . These tions, which are deleterious for the resistance of the vessel to the disease, were found to evolve gradually during the animal’s Iifetime.‘J The study of these problems has so far been entirely confined to investigation of experimental lesions of the vessel wall in animals or, with human vessels, of material obtained by dissection. A considerable step forward was therefore made by breeding a strain of spontaneously hypertensive rats.2R.”
Clinical, morphologic, and experimental investigations point clearly to a close relationship between intravascular pressure and atherosclerosis.6-8 The mechanism of this association is still obscure. According to the filtration theory of atherosclerosis, the increased transarterial filtration pressure during hypertension facilitates the initiation of the diseaseg Other studies, both morphologic and experimental, indicate that the actual mechanism is considerably more complex.’” The above findings. particularly the intimate association of hypertension with atherosclerosis, induced us to focus our attention on the relationship of the vessel wall enzymes to atherogenesis in spontaneously hypertensive rats. MATERIALS AND METHODS This investigation was performed on S H R rats of the Okamoto-Aoki strain” maintained in Czechoslovakia since 1969. These rats exhibit a sudden increase in
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blood pressure at 40 days of age; in males, this increase proceeds until the 90th day, when it reaches the first plateau. In females, the progress of hypertension is slower but continues even after 90 days of age. The mean blood pressure (BP) value in adult SHR rats is 200 mm Hg. The cardiovascular pathology of SHR rats has been described in detail mainly by Japanese authors at Kyoto Univer~ity.'~-'~ However, relatively little attention has been focused on the aorta; we will therefore briefly mention our results with adult SHR rats. The aorta of hypertensive rats is generally larger, as is its inner diameter, as compared that with normal rats (FIGURES l a & Ib.) This phenomenon is due to the hypertrophy of the aorta wall (i.e., hypertrophy of smooth muscle cells of the media) combined with dilatation. The intima exhibits no pathologic features. The spaces between the elastic cells are considerably extended and are filled mostly by hypertrophied muscle cells. The amount of the connective tissue in the media of both normal and hypertensive rats is the same. The adventitia is formed, in both groups, by a thin layer of connective tissue with a small amount of circularly and irregularly arranged elastic fibers and some smooth muscle cells without hypertrophy. The rats were maintained under normal conditions on a standard diet. The activity of lactic dehydrogenase (LDH), malate dehydrogenase (MDH), acid phosphatase (ACP), alkaline phosphatase (AP), 5'-mononucleotidase (5'-Nu), adenylpyrophosphatase (APP), and carboxyl esterase (Carb. Est.) were determined in aortas by methods currently in use in our 1aborat0ry.l~The activity was measured in a I % aorta extract in physiologic saline (pH 7); LDH isoenzymes were determined in a 10% extract of the aorta in VeronaP-acetate buffer (pH 8.6). Fractionation of LDH was achieved by electrophoresis on W, paper, followed by a quantitative detection and evaluation as described previ0us1y.l~All results were related to the protein content of the extracts and were evaluated statistically by Student's t test. For clarity, the mean activity in the aorta of normotensive (N) rats was taken as 100%. In a parallel determination, the activities of ACP, MDH, and Carb. Est. were established in most samples by histochemical techniques.16 The activities of these enzymes were determined in the vessel walls of adult male and female rats suffering from spontaneous hypertension and were compared with activities found in normal rats of the same age maintained under identical conditions. Moreover, the activities of the enzymes were assayed in the aortas of SHR rats during their development, that is, at 23,40, 60, and 180 days of age. Each group studied comprised 10-15 animals of both sexes. The body weight and blood pressure of all animals were measured daily; the BP values were obtained indirectly by the pletismographic technique on the tails of conscious unanesthetized animals. A N D DISCUSSION RESULTS
The aortas of SHR male rats exhibited changes in the activities of all of the enzymes studied, as compared to those of normal animals. The activities of AP, ACP, and 5'-Nu were significantly increased, whereas those of MDH, LDH, and Carb. Est. were significantly lowered. The aortas of female SHR rats displayed the same changes in the activities of AP and LDH, which were not significantly altered 2 & 3). The activities of enzymes in the as compared to those of normal rats (FIGURES aortas of SHR rats are thus analogous to those found in aortas with increased susceptibility to the formation of atherosclerosis and those found in various artificially introduced vessel lesions in experimental animals in earlier experiment^.^.^ This result corresponds to our findings with respect to the lowered activities of Krebs cycle enzymes and to the increased activity of phosphomonoesterases (AP and ACP) under conditions favorable for deposition of lipids and initiation of atherosclerotic le-
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C A R L EST.
FIGURE2. The activity of acid phosphatase (ACP), alkaline phosphatase (AP), carboxyl esterase (Carb. Est.), adenylpyrophosphatase (APP), S’mononucleotidase @’-Nu),malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) in the male rat aorta. White columns, normotensive rats; black columns, SHR rats. The results are expressed as percentage differences, taking the mean activity in aortas of normotensive rats to be 100%(mean SD).
11 1iI 1
FIGURE 3. Enzyme activity in the female rat aorta. White columns, normotensive rats; black columns, SHR rats. For abbreviations, see FIGURE 2 legend.
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.-c x .-* .-> c
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sions in the vessel wall. Histochemical follow-up and biochemical tests of the vessel wall enzymes in both S H R and Normal rats revealed a similar increase in the activity of ACP and a decline in the activities of MDH and Carb. Est. in hypertensive ani4a & 4b). As shown by the L D H zymogram, the aortas of S H R rats mals. (FIGURES exhibit an increase in the anaerobic LDH, fraction as compared to normal animals
FIGURE5. Representative picture of the electrophoretic separation of LDH isoenzymes in aortas of normotensive and SHR male rats.
(FIGURE 5). Quantitative data expressed as the percentage of individual fractions show that the anaerobic LDH, fraction reaches 10.4% in aortas of S H R rats in contrast to a level of 7% in N rats. Further study of the vascular system during development of S H R rats revealed that significant changes in activities of MDH, Carb. Est., and 5'-Nu occur near Day 40; it is noteworthy that the activity of M D H is increased, although in adult rats, it is
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FIGURE 6. Time course of enzyme activity in SHR rat aortas (at 20, 40, 60, and 80 days) expressed as percentage differences, taking the mean activity in normotensive rats to be 100%.
FIGURE 7 . Time course of blood pressure (BP) and weight (W) of normotensive and SHR rats.
Mrhovi et al.: Vessel Wall Metabolism
permanently significantly lowered as compared with normal animals; this decrease is observed as early as 60 days of age (FIGURE 6). The activity of Carb. Est. in the vessels of S H R rats diminishes continuously with age and corresponds to the value obtained in the study of various models of vascular lesions, for example, calciferol intoxication, alloxane diabetes, accumulation of mucopolysaccharides, and experimental hypertension. The activities of AP and 5'-Nu increase considerably toward the 40th day of life, then gradually decline during further development, and eventually rise again to exceed in adult S H R rats the level found in normal rats (FIGURE 6). The activity of LDH experiences an elevation at 40 days of age, but in adult animals, it falls below the level observed in controls. The above results imply that, during development, aortas of spontaneously hypertensive rats undergo changes of metabolism that are reflected by, among other things, a lowered activity of the Krebs cycle enzymes. The aorta is known to utilize 75% of the glucose consumed, which serves as the main source of energy, by anaerobic glycolysis; the remainder is oxidized.ln Despite this, the main source of useful energy is the aerobic pathway.18 The decreased part of the Krebs cycle in glycolysis is thus reflected by a considerable reduction in the total amount of utilizable energy produced. Another factor is accumulation of lactate, which promotes acidosis; this condition, in turn, represents one of the pathogenetic factors that facilitate the initiation of atherosclerosis.20.2'This idea is substantiated by results of a tentative analysis of LDH isoenzymes; the increased level of the anaerobic LDH, fraction in the aorta of S H R rats indicates an accumulation of lactate and a shift of the vascular wall metabolism toward the anaerobic pathway. Because the most conspicuous BP change in S H R rats occurs at about 40 days of age (FIGURE7), alterations in the activities of vascular wall enzymes during this period can be considered to be closely connected with this factor. However, studies of other aspects of the metabolism in S H R rats suggest that the interconnection is rather complex; thus, a certain S H R specificity was proved to be present in the isoenzymes of the specific esterase that is unigenically controlled and transmitted to the offspring according to the law of codominancy.22 During the initial developmental stages of S H R rats, the vascular wall apparenlly undergoes certain metabolic alterations associated either with the rise in blood pressure or with a certain genetic mechanism. This alteration, or disorder, is further aggravated with age and obviously becomes one of the factors that lead to the appearance of pathologic changes in the vascular wall of these animals. Analogous changes in the activities of the above enzymes were also observed in rats with experimentally induced hypertension.z However, with the data available, we cannot determine which change in either blood pressure or metabolism is primary and to what extent the individual changes are interdependent. The metabolism of S H R rats is clearly affected by numerous genetic and other factors, and the ultimate answer to t h e problem will thus require further study. Despite the above restrictions, it may be concluded that the enzymatic equipment of the vascular wall of S H R rats seems to be less suitable than that of normal animals, and, in light of earlier findings, it may contribute to the greater susceptibility and predisposition of these aortas to pathologic changes. REFERENCES 1.
T.,Z. LOJDA & 0. M R H O V ~1963. . Atherosclerosis and Its Origin. M. Sandler & 0. H. Bourne, Eds.: 459-507. Academic Press, Inc. New York, N.Y.
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