Tobacco Documents Online

Authors: Yenning Cui', William H Gutstein', Sana Jabr', Tze-chen HsiehZ and Joseph M. Wu2°* Title: Control of human vascular smooth muscle cell proliferation by sera derived from "experimentally stressed" individuals Affiliation: Departments of Pathology' and Biochemistry and Molecular BiologyZ, New York Medical College, Valhalla, New York 10595 Running Title: c-myc and smooth muscle cell proliferation Address of Correspondence: Dr. Joseph M. Wu Department of Biochemistry and Molecular Biology New York Medical College Basic Sciences Building, Valhalla, NY 10595 Telephone: 914-594-4062 Fax: - 914-594-4058 1

Abstract Recently we reported that individuals with high scores in standardized hostility evaluation tests, when placed in a "relaxed" environment, may have an association with increases in a blood- borne mitogenic substance(s) for arterial smooth muscle cells (1). To further investigate the molecular basis for such an association, PDS (plasma derived serum with platelet derived growth factor (PDGF) removed) from individuals showing the greatest differential pre/post-stress mitogenic activity, were tested for ability to modulate changes in the steady state of the c-myc mRNA in cultured VSMC (vascular smooth muscle cells) by Northern blot analysis. Post-stress PDS resulted in a significant increase in c-myc mRNA, when compared with pre-stress PDS of the same individual. These results give further experimental support for the notion that stress (even in the form of transient, episodic psychological challenges) may affect the cardiovascular system 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 (CHD), commonly recognized as a complication of a more fundamental vascular pathology, i.e., atherosclerosis (AS). 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 (2-5). A key feature in the pathogenesis of AS is the proliferation of vascular smooth muscle cells (VSMC) in the arterial wall into the intima. Although this proliferation is known to be influenced in vitro and in vivo by a number of blood-borne substances, designated as either "mitogens" or "growth factors" (6), in humans the stimulus for this phenomenon is unknown. We had previously reported that when young (3 months) rats on normal diets were exposed to brief episodes of hypothalamic stimulation (HS), after removing platelets and preparing platelet derived serum (PDS), the latter was found to contain a heat-stable, non- dialyzable substance(s) capable of promoting growth of cultured VSMC. This mitogenic activity appeared to be unrelated to PDGF (1,7,18). These findings 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. Support that a similar relationship might exist in human populations came from our recent study, in which we reported that individuals with high scores in standardized hostility evaluation tests, when placed in a "relaxed" environment, may have an associated increase in a blood-borne mitogenic substance(s) for arterial smooth muscle cells (1). These results were interpreted in the 3

context of endogenous stresses purportedly associated with "high hostility" scoring individuals that elicits a blood-borne substance(s), entirely distinct from PDGF, which increases the proliferation of VSMC (1). In this report, we show, based on analysis using PDS prepared from six subjects with the greatest differential pre/post-stress proliferative response, that the PDGF- independent mitogen resulted in a corresponding increase in the steady state concentration of mRNA for the c-myc protooncogene. 4

Materials and Methods These were identical to a previous report (1,8), except as noted below. PDS from six subjects, whose sera gave the most pronounced differential pre/post stress increase in cell growth and ['H] thymidine incorporation, were used in Northern blot analysis. One PDS carne from the "Frustrated" group, defined as individuals challenged with unreasonably high score requirements in playing an electronic game. The remaining five PDS were derived from the "Harassed" Group, who had to perform a task identical to those in the "Harassed" group, except that the electronic game challenge was superimposed with disparaging and critical verbal remarks. RNA Isolation and Northern Blot Analysis Normal VSMC (human) were seeded in 75 cm2 flasks containing 20% FBS until 80%- 85% confluence was reached. They were switched to a serum free media for 2 days, after which they were cultured in 5% FBS or 5% PDS for 15 min, 30 min, 1, 2, and 24h, respectively, and harvested. Total RNA was prepared from VSMC using commercial reagents (TRSOLVTm, BIOTECS LABORATORIES, INC). Fifteen micrograms of RNA were denatured with glyoxaVdimethyl sulfoxide, electrophoresed in 1% agarose, and transferred to nitrocellulose membrane (OptiBind, 0.45 µm, Schleicher and Schuell) using a LKB Vacugene system, and baked for 2 h at 80°C. Prehybridization was performed overnight at 42°C in 50% formamide, 5X SSC, 5X Denhardt's solution, 50 mM phosphate buffer, pH 6.5 and 250 µg/mI denatured salmon sperm DNA. Following prehybridization, hybridizations were carried out at 42°C for 16-20 h in 50% formamide, 5X SSC, 1X Denhardt's solution, 20 mM phosphate buffer, pH 6.5, 10% dextran sulfate and either the 32P-labeled c-myc or 18S rRNA probes, prepared by using the 5

random primer labeling method. The membranes were washed twice with 2X SSC, 0.1"/° SDS at room temperature for 10 min, followed by a 45 min wash at 55°C in the above solution, and another wash for 45 min at 55°C in 0.1 x SSC/0.1% SDS. The filters were dried and exposed to Kodak XAR-5 film sandwiched between two intensifying screens. Scanning was performed on the autoradiogram using a Hoefer Scientific GS-300 scanning densitometer (San Francisco, CA). Statistics Statistical calculations were done using the Student's t-test to analyze differences in the measured variable. Among groups, comparisons were made by contrasting paired means of the variable in question, between before and after the test session.

Results and Discussion Growth of VSMC in Human PDS Confluent primary VSMC showed characteristic multi-layered, spindle-shaped "hills and valleys" pattern and were confirmed to be smooth muscle cells by immunofluorescent staining using anti-a-smooth muscle actin. When cultured in media supplemented with heat-inactivated FBS, VSMC grew exponentially from the third to the ninth day, with an average doubling time of 33.1 t2.3 h(n=3). The effects of pre-stress PDS on proliferation of VSMC are shown in Figure 1. Compared to FBS, PDS was much less capable of supporting the growth of VSMC. However, compared to cultures maintained in media supplemented with 0.5% BSA only, growth of the PDS-fortified cultures was substantially stimulated, reaching peak of growth after 25-30 h. Effect of Sera of "Psychologically-Stressed" Subiects on Growth, DNA Synthesis, and c- Mvc Protooncogene Expression in.Cultured VSMC To determine whether PDS prepared from pre- and post-stress subjects had differential effects on cultured VSMC, cells were incubated with various concentrations of pre- and post- stress PDS prepared from the same individual, for up to 5 days. Results of cultures maintained with 5% PDS are presented in Figure 2A. Clearly the post-stress PDS was more effective in inducing VSMC proliferation, as measured by increases in cell count. The difference was statistically significant for the day 5 cultures, P<0.05 (Figure 2A, n = 6). Similar results were obtained when proliferation was measured by incorporation of radioactive thymidine into DNA, P<0.05 (Figure 2B, n = 6). The cell count increase correlated well with changes in thymidine uptake (correlation coefficient, r= 0.745, P< 0.0005). 7

Since previous studies have shown a correlation between cellular proliferation, DNA synthesis, and the expression of c-myc in a number of mammalian cells (9-12), we determined c- myc expression in control and treated-cells using Northern blot analysis. Total RNA was isolated from VSMC and probed with a labeled c-myc exon-IIL The 18S rRNA was used as an internal control to verify the precision of RNA loading. VSMC cultured in serum free medium and switched to 5% FBS had peak c-myc expression at 0.5 h (Figure 3). As expected, no corresponding change in the expression of the 18S rRNA was observed. When c-myc changes in cells cultured with pre- and post-stress PDS were compared, a significant difference, as evidenced by the greater post-PDS-elicited c-myc mRNA increase, was observed (Figure 4,5). In the majority of the cases tested (5/6), the c-myc mRNA expression was consistently less when cells were maintained in the pre-stress sera (Figure 4, n = 5, P<0.05). Such a result coincided with the observed cellular proliferation and radioactive thymidine incorporation data. In one individual whose sera elicited a large differential thymidine incorporation response in VSMC, no corresponding increase in c-myc was observed. In fact, the pre-stress PDS for this individual gave a higher c-myc mRNA value, compared with the post-stress PDS. The data show that, among those individuals with altered emotional states (as exemplified by frustrated feelings that may include anger, aggressive inclinations and rage), there emerged a relationship between a subpopulation in this group and increases in the serum concentration of a non-PDGF mitogen for cultured human VSMC in their blood samples, whose ability to induced mitogenesis is presumably related to the up-regulation of the proto-oncogene, c-myc. The data presented here further reinforce the idea that one way in which these psychological factors may act is by elevating substances in the blood which promote proliferation of vascular smooth muscle 8

cells, a key component of atherogenesis. It is of interest to note that in the same study there was little change in the traditional risk factors, such as serum cholesterol concentrations or blood pressure. Results of the present studies, although preliminary, tend to support findings from epidemiologic studies, indicating that "stressed" individuals are prone to the development of coronary heart disease (1,14,15). The nature of the substance or substances in the serum responsible for the increase of c- myc activity of cultured VSMC cannot be determined from the data of this study. Serum contains a number of growth factors for VSMC, of which PDGF is the most potent (6, 16). In this investigation, PDGF could be ruled out. Substances other than growth factors in the serum also possess mitogenic properties for these cells (14, 17-21). The exact molecular identity of this substance awaits further work. The precise mechanism by which these altered emotional states could result in an increase in serum mitogenic activity for cells which may participate in the atherogenic process remains to be determined. Work in experimental animals (rats and the nonhuman primate, S. sciureus) suggests that the diencephalon may be involved. In these experiments, electrical stimulation of the hypothalamus gives rise to a series of complex steps which culminate in the proliferation of VSMC, both in vitro and in vivo and in arterial lesions similar to those of atherosclerosis, as well as in agitated behavior when the animals are not under anesthesia. These results have been reviewed in earlier publications (14, 17-20). Assuming a similarity in the human situation, the present investigation focuses on the increase of serum mitogenic activity alone - although the means by which this response is achieved is currently unknown. 9

REFERENCES 1. Gutstein W.H., Teresi, LA., Wu J.M., Salimian, F., Cui Y., Paul, I., and Jaba, S. (1998) Increased serum mitogenic activity for arterial smooth muscle cells associated with relaxation and low educational lever in human subjects with high but not low hostility traits: implications for atherogenesis. Journal of Psychosomatic research. In press. 2. Eliot R.S. (1987) Coronary artery heart disease: Biobehavioral factors, overview. In: Circulation Vol. 76 Suppl. (Part 2) J.T. Shepherd and S.M. Weiss, eds. 3. Strong J.P., Solberg L.A., Restrapo C. (1968) Atherosclerosis in persons with coronary heart disease. Lab. Invest. 18, 527. 4. Ross R., Glomset J.A. (1976) The pathogenesis of atherosclerosis. N. Engl. J. Med. 295, 369. 5. Benfante R.J., Reed D.M., MacLean C.J., et al. (1989) Risk factors in middle age that predict early and late onset of coronary heart disease. J. Clin. Epidemiol. 42, 95. 6. Antoniades H.N., Pantazis, P., Owen, A.J. (1987) Human platelet-derived growth factor and the sis/PDGF-2 gene. In: Guroff, G. Ed. Onco eg nes, genes and growth factors. P.1-40, New York: John Wiley and Sons. 7. Gutstein W.H. (1988) The central nervous system and atherogenesis: Endothelial injury. Atherosclerosis 70, 145. 8. Cui, Y., An, S., Jabr, S., Maturana, J., Wu, J.M., and Gutstein, W.H. The anti-proliferative effects of nicotinamide and 3-aminobenzamide on human smooth muscle cells in vitro. Biochem. & Mol. Biol. International. 31, 935. 9. Wu C.C., Chen S.J., Yen M.H. (1992) Effects of noise on blood pressure and vascular reactivities. Clin. Exptl. Pharmacol. Physiol. 19, 833. 10