Increased serum mitogenic activity for arterial smooth muscle cells associated with relaxation and low educational level in human subjects with high but not low hostility traits: implications for atherogenesis.

Proliferation of arterial smooth muscle cells (aSMC) is a key component of atherogenesis. A sample of 225 volunteers, aged 21-65 years, was exposed to "frustration," "harassment," or "relaxation," after completing the 50-item Hostility subscale of the Minnesota Multiphasic Personality Inventory (MMPI). Whole blood was measured before and after exposure for platelet-derived growth factor (PDGF), and sera were evaluated for total and HDL cholesterol concentrations and PDGF-independent mitogenic activity (SMA). Blood pressure and pulse rate were also evaluated. Analyses of SMA (i.e., serum independent of PDGF) revealed an increase in mitogenic effect for cultured human aSMC when hostility was treated as a dichotomous modifier. Among high-hostility subjects, surprisingly, those in the relaxed group and those with a lower educational level were found to have a significant mitogen response; no significant effects were observed for the low-hostility groups. The data suggest that endogenous stresses may occur in high-hostility individuals when "relaxed," to influence proliferation of arterial smooth muscle cells, as a contribution to atherogenesis. In individuals with lower educational levels and higher hostility scores, lifestyle changes may play a role.

Growth retardation in senescent arterial smooth muscle cells and its reversal following brain stimulation: implications for atherogenesis.

Advancing age and psychosocial stress are each associated with a rising incidence of atherosclerosis. In this investigation we attempted to answer the question of whether they are independent of each other or not. Since a key feature of atherosclerosis is the proliferation of arterial smooth muscle cells (ASMC), we transplanted aortic tissue from aged rats, half of which had received hypothalamic stimulation, as a model for stress, to growth supporting medium, immediately after stimulation and observed their growth behavior for a period of 4 months. Similar observations were carried out on young animals for comparison. Although there was little difference in outgrowth frequency of explants from young animals between stimulated and non-stimulated subjects, in the case of the older rats, explants from animals which were not stimulated were considerably retarded in their growth, whereas those from subjects which had received HS, grew as well as those of the younger ones. These results show that HS can reverse the growth decline in aortic tissues explanted from senescent rats. They also suggest that age per se is not atherogenic in terms of proliferative behavior of ASMC, whereas when interacted with a stressful condition, this may be the case. Since in the elderly there is a decreased tolerance to stress, the 'atherogenic' effects of age in these individuals may be mediated through the stress response.

Age differences in the proliferative response of cultured arterial smooth muscle cells induced by sera of hypothalamically-stimulated rats.

In an earlier report, it was shown that arterial smooth muscle cells (ASMC) cultured from normal rat aorta, proliferated in response to homologous sera from young rats which had received hypothalamic stimulation (HS), in contrast to the effect of sera from non-stimulated age-matched controls (sham-operated). In the present study, this proliferative response was compared in young and old rats. Isolated target cells were subcultured from primary explants of aortic tissue obtained from young, male, Fischer 344 rats, which were electrode-implanted in the hypothalamus but not stimulated. After an initial quiescence period (growth arrest), target cells were exposed to plasma derived serum (PDS) from 4 experimental groups: young stimulated rats; young sham-operated controls; aged stimulated rats and aged sham-operated controls, at PDS concentrations of 2.5% and 5.0% and counted at days 2 and 5. Proliferative responses of ASMC were found to be influenced by concentration of the PDS, age of the donor animal contributing the serum and the presence of HS. The greatest responses were observed in relation to sera derived from aged stimulated rats, especially at the higher concentration, suggesting an interaction of HS with advanced age. These results are discussed in reference to their possible bearing on the pathogenesis of atherosclerosis.

The central nervous system and atherogenesis: role of the arterial smooth muscle cell.

Within the last 2-3 decades neuropsychological factors have assumed a role of importance in atherogenesis implicating a contribution from the CNS to the development of arterial lesions. Despite extensive documentation in clinical studies, little work has been performed with experimental animals which could elucidate underlying mechanisms. Employing hypothalamic stimulation (HS) to model the role of the CNS, experiments in the author's laboratory have shown that atherosclerotic lesions develop in time. Evidence is presented that the underlying mechanism is due to vasospasm associated with proliferation of intimal arterial smooth muscle cells (ASMC). Both, the vasospastic and proliferative responses may originate with the same hypothalamic signal. The vasospastic response compromises perfusion, but by injuring the vessel produces pathology which increases the resistance of the artery to further spasm. The proliferation of ASMC which provides the elements for this 'stiffening' process also helps restore the vascular tone necessary to maintain the important function of perfusion. Atherosclerosis, therefore, may represent an adaptive response to excessive spasm which in many instances has advanced too far.

Spasm of small coronary arteries and ischemic myocardial injury induced by hypothalamic stimulation in the rat.

Electrical stimulation of the lateral hypothalamus resulted in electrocardiographic evidence of acute myocardial ischemia in 35% of normal adult rats under anesthesia. Mean arterial blood pressure was also elevated. Study of vascular corrosion casts disclosed that spasm of smaller branches of the coronary circulation, rather than the major epicardial arteries, was the main cause of the ischemic response. The histologic changes of the same experimental treatment in a separate group of animals revealed multiple focal areas of tissue damage throughout the myocardium, which were quantitatively assessed. The results may be relevant for the clinical problem of various forms of ischemic heart disease in which little evidence is found for organic (atherosclerosis) or dynamic (spasm) stenosis involving the major coronary arteries.

Proliferation of arterial smooth muscle cells incubated in serum from brain-stimulated rats.

Platelet deficient serum prepared from rats subjected to acute electrical stimulation of the lateral hypothalamus demonstrated mitogenic activity when added to incubating media supporting growth of homologous arterial smooth muscle cells in vitro. This activity did not appear to be related to the presence of platelet-derived growth factor, hyperlipidemic lipoproteins or increased amounts of insulin. Plasma arginine vasopressin concentration was elevated in these animals, but further investigation is required to determine if this elevation is causally related. Since hypothalamic stimulation is also associated with severe endothelial injury in vivo, the mitogenic activity of the blood of such animals could induce proliferation of SMC which have migrated into the arterial intima. Such features have been observed in chronically stimulated animals and may be of relevance for the role of neural factors in atherogenesis.

Coronary artery spasm in the rat induced by hypothalamic stimulation.

Anesthetized rats were sterotaxically implanted with electrodes and electrically stimulated in the lateral hypothalamus. During elevation of the S-T segment on simultaneous precordial electrocardiograms, the heart was perfused with glutaraldehyde-paraformaldehyde fixative and the major coronary arteries prepared for morphometry of luminal dimensions. A similar procedure was performed in a second group receiving intravenous arginine vasopressin (AVP) in place of hypothalamic stimulation. Elevation of the S-T segment was present in these animals as well. Control animals were implanted, not stimulated and otherwise treated in the same way. Morphometry showed that reductions of mean luminal diameter and cross-sectional area of statistical significance occurred in the two experimental groups compared to controls, suggesting that coronary spasm was the cause of the elevated S-T segments. Pooled plasma from separate groups of implanted control and hypothalamically-stimulated animals revealed substantial elevation of AVP levels in the latter raising the possibility that the neuroendocrine was involved in eliciting coronary artery spasm.

Endothelial morphology and plasma total and high density lipoprotein cholesterol changes in hypothalamically stimulated squirrel monkeys fed a modified atherogenic diet.

Experimental animals fed atherogenic diets show endothelial damage, impairment of endothelial regeneration and plasma lipid changes characterized by elevation of LDL and decrease of HDL cholesterol concentrations. Previous studies in this laboratory disclosed that chronic electrical stimulation of the lateral hypothalamus was associated with electron-microscopic evidence of endothelial injury in rats and squirrel monkeys maintained on basal (low fat/cholesterol-free) diets. In the present investigation squirrel monkeys fed similar diets supplemented with "modest" amounts of caloric fat and cholesterol were subjected to chronic lateral hypothalamic stimulation for periods as long as 20 months with the expectation that endothelial injury would be greater than in the absence of the supplements. The expectations were not substantiated. Endothelium was found to be surprisingly intact by electron microscopy and similar to that of implanted nonstimulated controls. A further observation of interest was the cholesterolemic response, notably in the HDL fraction, observed in both groups, but more striking in experimental animals. The data suggest that an interaction between a modified lipid/cholesterol diet and hypothalamic stimulation may lead to elevation of plasma HDL cholesterol concentration and preservation of endothelial integrity. Further investigation is required to determine whether these two events are causally related.

Effect of hypothalamic stimulation on the endothelial morphology of the aorta in the conscious squirrel monkey.

The role of neurogenic factors in the development of atherosclerosis has not previously been studied in detail. In recent years evidence has accumulated to implicate endothelial injury as a primary stimulus for the proliferation of myo-intimal cells resulting in the formation of the early morphologic lesion. In the present investigation, the effect on aortic endothelial morphology of repetitive electrical stimulation of the lateral hypothalamus in the conscious, unrestrained squirrel monkey, maintained on a cholesterol-free low-fat diet, has been studied. Stimulation was performed with a self-powered, miniaturized electronic stimulator connected to indwelling electrodes. Implanted nonstimulated animals served as controls. Endothelial injury in the form of cell degeneration, denudation, with plasma insudation and partial junctional separation were observed electron-microscopically in stimulated animals compared with controls. These alterations were found to be independent of hypercholesterolemia and/or hypertension. Possible pathways for the induction of injury in this neurogenic model are: (1) direct, through neural circuits from the brain to the vessel wall, and (2) indirect, by elaboration of angiopathic substances inside or outside of the CNS, released into the circulation and transported to the vessel wall where they exert their effects. Reversibility of the endothelial injury progression to established lesions and mechanisms involved remain to be determined in further investigations.

Neural factors in experimental degenerative arteriopathy.

Intermittent electrical stimulation of the lateral hypothalamus of rats performed for 30 min to 6 hr, results in hyperlipidemia and endothelial cell damage of the aorta and coronary arteries. Hyperlipidemia is related to transient biliary obstruction elicited by hypothalamic stimulation and is characterized by elevation of the cholesterol, phospholipid, and triglyceride fractions. Endothelial cell damage is observed ultrastructurally as plasma membrane degeneration with detachment and the formation of large spaces ("vacuoles"). Thus, neural factors may be implicated in inducing conditions associated with early atherogenesis. Stimulation carried out for longer time intervals would be expected to produce more advanced lesions. However, the role of neural transmission per se (i.e., without hyperlipidemia) in producing arteriopathy is not clearly defined from these experiments. In rats, the lesser splanchnic nerve forms the major innervation of the abdominal aorta. In animals fed normal diets, chronic intermittent stimulation of this nerve (up to 3 weeks) resulted in advanced arteriosclerotic changes with intimal fibrosis and calcification. On histologic examination, lipid deposits appeared to be absent from these lesions. Animals stimulated for shorter periods of time exhibited earlier changes associated with atherogenesis, such as endothelial damage, elastic reduplication, and adherent microthrombi. Thus, direct neural transmission, especially if excessive, plays a role in producing arteriopathy. Hyperlipidemia, if persistent, could modify these lesions so that they would accumulate plasma lipids. Experiments to test this hypothesis are currently in progress.

Neural factors contribute to atherogenesis.

Electron microscopic evidence of early atherogenic changes in the aorta and coronary arteries was obtained in normal fed, conscious, unrestrained rats receiving electrical stimulation in the lateral hypothalamus for periods of up to 62 days. Hypertension and hypercholesterolemia were not etiologic factors. In view of recent observations concerning neuropsychological mechanisms in human ischemic heart disease, the findings raise the possibility that the human central nervous system has a role in the development of atherosclerotic lesions.