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Stress and Atherogenesis. Smooth Muscle Cell Mitogenic Activity and other Biochemical Changes Associated with Sera of "Stressed" Subjects Joseph M. Wu' amd William H. Gutstein2 Departments of Biochemistry and Molecular Biology' and Pathology2, New York Medical College, Valhalla, New York 10595, U.S.A. 1

Abstract The proliferation of smooth muscle cells in the arterial wall (VSMC) is considered to play a key role in the development of atherosclerosis. To investigate the possible contribution of "stress" (experimentally-induced) to this process, blood from healthy volunteers, ages 21 to 65, screened to exclude major risk factors for coronary heart disease, was assayed for mitogenic activity after the subjects were exposed to one of 2 "stress" conditions. These consisted of a cognitive task with superimposed verbal harassment (group 1), and the cognitive task without harassment (group 2). Mitogenic activity was determined by studying the growth stimulatory effects of PDGF-depleted plasma derived serum (PDS) from "stressed" subjects added to cultured VSMC, as measured by incorporation of radioactive thymidine into DNA or increase in cell number. In addition, changes in the steady state of the mRNA for the c-myc protooncogene were also assayed in VSMC by Northern blot analysis, using sera showing the greatest differential "pre/post stress" mitogenic activity. Blood pressure (BP), heart rate (HR), cortisol, and serum total and HDL cholesterol were also evaluated. All measurements were made immediately before (baseline) and after a 30 min interval. Analysis of the data revealed that there were 33 % of subjects in group 1 with an increase of thymidine incorporation 15 % or greater than baseline, versus 21 % in group 2. The average increases were 45% and 30%. A higher percentage (35-42%) of subjects in group 1 responded with increases in systolic and diastolic blood pressure, compared to subjects in group 2 (15-20%); the average in blood pressure was 10-15%. Similarly, more subjects (52%) in group 1 had an elevated (average 10-15%) serum cortisol, compared to the 42% in group 2 subjects. HR, total HDL cholesterol showed slight changes only. These results suggest that psychoactive factors may affect cardiovascular systems via rapid elicited rises in serum mitogenic activity for VSMC. 2

Introduction Over the last several decades, significant progress have been made in identifying risk factors for coronary heart disease (CHI)), still considered the leading cause of morbidity and mortality in many countries. Some 200 coronary risk factors (CRF), ranging from modifiable lifestyle characteristics such as diet and weight, as well as non-modifiable personal characteristics such as age, sex and family history have been identified, based on the presence of statistically significant numerical association. Traditionally risk factors, including the CRFs, are identified by means of observational, epidemiological studies. Because such studies lack the rigorous scientific controls characterizing experiments performed in the laboratory, often they cannot definitively distinguish between the impact of a particular risk factor and the confounding effects of other risk factors. Such considerations are especially noteworthy in epidemiological studies focusing on CHD because of the large number of suspected risk factors for this disease. In this context, it is worth noting that even the three most important risk factors for CHD -(i) cigarette smoking, (ii) hypercholesterolemia and (iii) hypertension - that have been traditionally cited for their predisposing roles leading to CI-ID, are unable to act as predictors of most new cases (1). Accordingly, results of epidemiological studies identifying CHD risk factors should only be seriously considered when they are supported by biologically plausible mechanisms that adequately explain the relationship between the purported risk factor and the onset of CHD. Indeed, a more practical way to consider CRF is that they merely provide a conceptual framework for arriving at a coherent, practical and balanced approach for the prevention and management of CHD. CHD is a complication of a more fundamental vascular pathology, i.e., atherosclerosis 3

(AS), especially as it affects the coronary circulation. AS is responsible for more morbidity and mortality than any other single degenerative disease, including cancer. Its clinical expression in the form of myocardial infarction, stroke and peripheral vascular disease accounts for about 50% of all deaths in developed countries (2-4). A key feature in the pathogenesis of this fundamental disease is the proliferation of vascular smooth muscle cells (VSMC) in the arterial intima. The end-stage atherosclerotic lesion is characterized by an abnormal focal proliferation in VSMC, which have migrated into the intima and there undergone active replication. Although this replication - or proliferation - is known to be influenced in vitro and in vivo by a number of blood-borne factors, designated as either "mitogens" or "growth factors" (5), in humans the stimulus for this phenomenon is unknown. In recent years, studies have shown that various psychoactive factors may play an important, independent, role in the development of this pathology (6-9). More recent evidence suggests that anger and "hostility" (HO) are some of the main factors, not only in coronary circulation, but also in connection with cerebrovascular disease and peripheral arterial disease, as well (10-15). Interactions between the nervous and cardiovascular systems have been extensively studied in animal experiments as well as in human physiological laboratories. The possible role of mental arousal in provoking changes in the fundamental properties of the cardiovascular system and possibly leading to pathological events is detailed recently (16). However, the means by which these characteristics translate into pathology at the level of the arterial wall are not understood. In both animals and humans, electrical stimulation of the diencephalon (hypothalamus) and structures closely connected with it, such as the limbic system and tegmentum of the midbrain, may elicit "aggression" and aggressive feelings - forms of behavior which are closely linked to the 4

underlying trait of hostility in humans (17-21). Electrical stimulation of the diencephalon also produces lesions in arteries (aorta) of rodents and non-human primates which bear the essential characteristics of AS plaques in humans, i.e., proliferation of VSMC, collagen and elastin synthesis and, in squirrel monkeys, lipid deposition even when diets are not high in fat content (22-24). When young (3 months) rats on normal diets experiencing brief episodes of HS were investigated for a substance in the serum with growth-promoting properties for cells of the arterial wall which participate in the formation of the AS plaque, i.e., VSMC, such a substance(s) was often present. This mitogenic activity appeared to be unrelated to PDGF. More recent work has shown that when the serum of HS animals is fractionated by different concentrations of ammonium sulfate, followed by binding of the fractions to heparin-agarose columns. SDS-PAGE and silver staining of the heparin-agarose bound proteins revealed that in the 30-60% amonium sulfate fraction, there were several distinct protein bands, which were absent in the non-stimulated controls treated in the same way (25). This finding suggested a possible link between aggressive behavior - emanating from the subcortical regions of the brain - and a cellular response in arteries contributing to atherogenesis via a serum factor which was mitogenic for these cells. Reasoning that a similar relationship might exist in human populations, the present study was undertaken among normal subjects. Our results show that upon application of a defined psychological stress, there was a significant increase of two substances in the circulating blood, which are widely believed to be atherogenic, since they induce proliferation of VSMC. These substances are: PDGF, known to be a potent growth factor for cells of mesenchymal origin, and a mitogen, entirely distinct from PDGF. The PDGF-independent mitogen was shown to induce an increase in the steady state concentration of the c-myc mRNA in the "experimentally-stressed" sera-treated VSMC. 5

Materials and Methods Materials [Methyl-'H]thymidine (67-77 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). The 1.8Kb Cla I/Eco RI human c-myc exon III probe was purchased from Oncor, and the rat 18S rRNA probe was provided by Dr. Ira G. Wool of the University of Chicago. Methods Characteristics of the subjects. The subjects consisted of 175 healthy volunteers (56% males and 44% females), aged 21-65 (mean = 41.8±10.6 SD), recruited from the general public. Most (96.5%) were White and 2.7% were Black. All individuals were telephone-screened for established risk factors for atherosclerosis, current use of any medication, including aspirin, occurrence of a major life event within the past 6 months, and past history of chronic and/or debilitating disease. Ten percent of the individuals initially recruited had to be dropped for various reasons, such as not meeting the above criteria, inability to keep necessary appointments, etc. Marital status was reported as 28.3 % single, 58.4 % married and 13.8% divorced, separated or widowed. Exercise was reported by 50.7% as being performed more than 3 times/week and by 49.3% as less than 3 times/week. A history of smoking at least 20 cigarettes/day for 5 years or more was present in 22 % of the total. Psvchological instxuments. Hostility was assessed using the Cooke-Medley subscale of the MMPI (26), which is composed of fifty dichotomous (yes/no) items such as: "I have often had to take orders from someone who did not know as much as I did." The scale alpha was 0.82. Procedures. Prior to acceptance into the study, informed written consent to participate in each 6

phase was obtained from all subjects. The Institutional Review Board of New York Medical College approved the study design. All participants were required to pass a complete physical examination, routine clinical laboratory procedures and an exercise stress test for cardiovascular performance. Individuals whose basal systolic blood pressure was greater than 140 mm Hg or whose diastolic blood pressure exceeded 90 mm Hg and those whose fasting serum cholesterol concentration was greater than 220 mg/dl were excluded. Upon arrival, subjects completed the MMPI questionnaire, and were allowed to rest for a period of 15 minutes. An IV catheter was then placed in the antecubital vein of the left forearm. A BP cuff was placed around the right arm. Catheter and cuff were arranged so as not to interfere with hand movements during the test session. Following another 15-minute period of adjustment, BP was recorded 3x from an automatic monitor at 2-minute intervals and the results (systolic/diastolic) averaged. Pulse rate was simultaneously measured. Blood samples were obtained from the antecubital vein by the same individual using indwelling catheter. Slight pressure was used for venous occlusion, with the subject seated comfortably in a relaxed position for 30 min prior to venipuncture, which was performed with a 21/gauge needle. The first few ml were discarded and the remainder set aside to clot for 30 min at room temperature (2(°C). The test session described was then administered for 30 minutes. During this period all subjects were videofilmed. At the end of the test session BP and PR were recorded as before and a second blood sample obtained. Participants then completed a self-rated questionnaire dealing with the degree of frustration and anger experienced during the session. All subjects were then debriefed and notified of the "purpose" of the study. All samples were obtained at least 3 hours after a light brealdast consisting of 1 slice of dry toast and one unsweetened beverage of tea or black coffee. 7

Test 3ession. Subjects were assigned to one of two groups. Group 1: Harassed (n=120). Individuals were asked to play a handhold electronic game (Mario Brothers) in the presence of one of the members of the research team within the time period of the session and to achieve a score, known by the team to be extremely difficult or impossible to obtain. None of the participants were familiar with the game, so did not question this objective. During the session, the interviewer periodically uttered "subtly" disparaging and critical remarks concerning the accomplishments of the participants during the session. Comments such as "I expected you to do better than that," or "you're not even performing at the level of a ten year old child," were said in an "exasperating" manner. Most of the subjects later reported some degree of "irritation" or "anger" at these remarks. Group 2: Frustrated' (n=53). The same strategy as used for group 1 was employed except that the interviewer (the same individual as in group 1) encouraged the subject to improve his/her score, and scrupulously avoided criticism of any kind. All subjects later reported some degree of "frustration" with the task. Prenaration of plasrna derived serum (PDS). All blood samples were drawn from a vein in the right cubital fossa, before and after administration of psychological test. Each 10-m1 of blood was immediately placed into a chilled 50 ml Falcon centrifuge tube containing 1 ml mixture of sodium citrate, EDTA, theophylline and PGE, (antiplatelet antiaggregating agents). Plasma was first separated by centrifugation at 3500 rpm 4°C for 15 min. and spun another 30 min at 4°C 16,000 rpm in rotor SS34, RC Sorvall centrifuge in order to remove platelets. Supernatant was transferred "'Frustrated/Harassed" are descriptive terms used to characterize individuals playing the game alone (group 2) or those who in addition to playing the game, were also exposed to verbal harassment (group 1). 8

to 50 ml Kimax glass tubes and 4% of 1 M CaC12 was added with incubation at 37°C for 2 hours in a water bath. After clot developed, the solution was spun for 30 min at 4°C/16000 rpm. The supernatant was transferred to prepared dialysis tubing and dialyzed in the cold room at 4°C against freshly prepared Dulbecco's PBS containing CaC12, with one buffer change after 2 hours. After dialysis, the supernatant was heat-inactivated at 560C for 30 min. The supernatant was centrifuged and filtered through sterile 0.45 µm Millex-HA units, aliquoted, and stored at -70°C. PDS samples prepared in this way have been shown to be free of PDGF by radioimmunoassay. Determination of serum PDGF. Serum concentrations of PDGF were measured with a sensitive radioimmunoassay procedure (Amersham International Pic, Amersham, UK). The assay was based on competitive binding between serum PDGF and radioactively labeled recombinant PDGF, for a PDGF-specific antibody. As reported by the manufacturer, cross-reactivity with other growth factors (EGF, TGF-O, bFGF) was less than 0.1 %. Reproducibility was given as coefficient of variation = 6.5% for intra-assay and 10.3-23.5% for inter-assay determination, depending on the concentration assayed. Consistency in accuracy of PDGF determination with different lots of the commercial kit used was evaluated on three separate occasions by measuring samples of known PDGF concentration and agreed with each other within a range of 7-10%. Stability of serum PDGF was ascertained on two occasions by adding a pre-determined amount of PDGF to sera, freezing and storing the "supplemented" sera at -85°C, followed by thawing the stored samples at monthly intervals (up to 4 months), assaying PDGF by RIA and showing that PDGF recovery was within the error limits established for the assay. Using this procedure, a total of 6 samples were tested at random. The presence of inhibitors of PDGF or 9

substances that interfere with the PDGF assay was essentially ruled out. Senim Cortisol Concentration. Cortisol was assayed by a solid-phase radioimmunoassay, based on competition of cortisol in subject serum with 'uI-labeled cortisol for antibody sites over a fixed time (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA 90045). This assay was used as a stress marker. Arterial Blood Pressure (BP) and Pulse Rate (PR). The measurements were taken in the right arm with an Automated Blood Pressure and Pulse Rate SD-700A Series Monitor System (Industrial and Biomedical Sensor Corp, Waltham, MA) based on the low frequency vibration detection principle for BP, and conversion to oscillometric pressure surges to average values, for PR. Three separate measurements for systolic and diastolic BP and PR were obtained at 2 min intervals with the subjects in a relaxed sitting position and averages computed for the data. Total cholesterol and HDL-cholesterol assays. Values were obtained using the Abbot Vision System (Abbot Laboratories, Abbot Park, IL 60064) with total cholesterol assayed by the coupled cholesterol oxidase-peroxidase method (27), and HDL cholesterol, via initial precipitation of LDL and VLDL by magnesium and dextran sulfate, based on the method of Artiss and Zak (28). Cell culture. Pritnary cultures of VSMC were obtained by outgrowth of explants from human thoracic aorta immediately obtained from young accident victims (20-30 years) within 6 hours of death, in cooperation with the organ donor service of this institution, as follows: adventitial layers were first removed with sharp forceps, and the exposed endothelium was scraped using a blade. The strips of the middle third of the tunica media was dissected and incubated for 35 min in Minimal Essential Medium supplemented with 0.2 mM Ca, plus elastase (type III, 15 units/ml), collagenase (type I, 200 units/ml), soy bean trypsin inhibitor (0.4 mg/ml), bovine serum albumin (2 10