Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle
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VRATISLAVHADRAVA AND JOHANNE TREMBLAY Centre de Recherche H6tel-Dieu de Montreal, 3850 rue Saint-Urbain, Pavillon Marie-de-la-Ferre, Montrkal (QuPbec), Canada H2 W I T8 RAFICK-PIERRESEKALY Clinical Research Institute of MontrPal, MontrPal, Que., Canada H2 W IR7 AND
PAVEL HAMET' Centre de Recherche H6tel-Dieu de Montrkal, 3850 rue Saint-Urbain, Pavillon Marie-de-la-Ferre, MontrPal (QuPbec), Canada H2 W 1 T8 Received November 7, 1991 HADRAVA, V., TREMBLAY, J., SEKALY,R.-P., and HAMET,P. 1992. Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle. Biochem. Cell Biol. 70: 599-604. The present study was designed to characterize the growth kinetics of the exaggerated proliferative response to mitogens of vascular smooth muscle cells from spontaneously hypertensive rats compared with cells from normotensive WistarKyoto controls. Cellular DNA content, analyzed by flow cytometry, demonstrated a 4-h accelerated entry into the S phase of the cell cycle of vascular smooth muscle cells from spontaneously hypertensive rats; the significant (4.5-fold) increase in the percentage of cells in the S phase occurred between 8 and 12 h after calf serum stimulation. A 3.9-fold increase of cells in the S phase was seen in the normotensive controls only between 12 and 16 h. Transit through the cell cycle was quantitated by flow cytometry using the Hoechst 33 342 - bromodeoxyuridine substitution technique. Vascular smooth muscle cells from spontaneously hypertensive rats went through the cell cycle 4 h ahead of cells from normotensive Wistar-Kyoto rats. This accelerated transit of spontaneously hypertensive rat cells was mostly due to an earlier entry into the S phase. Persistence of this new intermediate phenotype in cell culture suggests its primary pathogenetic role in spontaneous hypertension. Key words: hypertension, proliferation, flow cytometry, bromodeoxyuridine substitution, Go/G, phase. J., SEKALY,R.-P., et H ~ E TP., 1992. Accelerated entry of aortic smooth muscle cells HADRAVA,V., TREMBLAY, from spontaneously hypertensive rats into the S phase of the cell cycle. Biochem. Cell Biol. 70 : 599-604. La prtsente ttude veut caracttriser la cinttique de la croissance de la rtponse proliftrative exagtrte aux mitogenes des cellules des muscles lisses vasculaires chez des rats spontankment hypertensifs comparts aux cellules des contr8les Wistar-Kyoto normotensifs. Analyste par cytomktrie a flot continu, la teneur du DNA cellulaire dtmontre une entrke accClCrke de 4 h dans la phase S du cycle cellulaire des cellules des muscles lisses vasculaires chez les rats spontantment hypertensifs; l'augmentation importante (4,s fois) du pourcentage des cellules en phase S survient entre 8 et 12 h a p r b stimulation avec le strum de veau. Chez les contr8les normotensifs, une augmentation de 3,9 fois des cellules en phase S n'est obsewte qu'entre 12 et 16 h. Nous avons quantifik le transit a travers le cycle ceuulaire par cytomktrie a flot continu utilisant la technique de substitution avec le Hoechst 33 342 - bromodksoxyuridine. Les cellules des muscles lisses vasculaires des rats spontantment hypertensifs passent a travers le cycle cellulaire 4 h avant les cellules des rats Wistar-Kyoto normotensifs. Ce transit acctltrC des cellules des rats spontantment hypertensifs est surtout dfi a une entrte plus hfitive dans la phase S. La persistance de ce nouveau phtnotype intermtdiaire dans la culture des cellules suggbe son r81e pathogCnCtique premier dans l'hypertension spontante. Mots clPs : hypertension, proliftration, cytomktrie a flot continu, substitution de la bromodtsoxyuridine, phase Go/G,. [Traduit par la rtdaction]
Introduction Narrowing of the vessel lumen due to arterial wall thickening is a common feature in hypertension. VSMC hyperplasia in the SHR model of essential hypertension has been demonstrated in resistance vessels by histological studies (Mulvany et al. 1978, 1985). Aortic and mesenteric smooth muscle cells from SHR proliferate more rapidly than cells derived from normotensive WKY controls (Yamori et al. ABBREVIATIONS: VSMC, vascular smooth muscle cell(s); SHR, spontaneously hypertensive rat(s); WKY, Wistar-Kyoto rat(s); EGF, epidermal growth factor; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle medium; PBS, phosphatebuffered saline; PBS', Dulbecco's PBS containing 320 pM c a 2 + and 60 pM M~''; BrdU, bromodeoxyuridine; NP-40, Nonidet P-40. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprim4 au Canada
1981; Hamet et al. 1988; Blennerhassett et al. 1989; Hadrava et al. 1989; Scott-Burden et al. 1989; Paquet et al. 1989; Bukoski 1990; Hamada et al. 1990a, 1990b) in vitro, supporting the possibility of an abnormal intermediate phenotype that is not a consequence of elevated blood pressure. Even though experimentally induced hypertension may lead to the enhanced migration of cells from aortic explants, the shorter interdivision time afterwards does not persist over six subcultures (Mey et al. 1980; Haudenschild et al. 1985; Hamada et al. 1990b). We and others have previously shown that the greater proliferation of SHR VSMC, persisting under culture conditions until the 19th passage studied, is not due to varying cell survival or attachment ability after passage, and that SHR cells manifest an exaggerated maximal response to calf serum, EGF, and P D G F (Hadrava et al. 1989; Scott-Burden et al.
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0
g
G 2 * M = 16
G2*M = 12
(I
rm
zm
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im
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Propidium iodide fluorescence intensity 1. Cell cycle analysis of aortic smooth muscle cells from WKY and SHR. Cells were made quiescent by serum starvation and then stimulated with 10% calf serum. They were harvested at various times (between 0 and 24 h), treated with RNAse, stained with propidium iodide, and analyzed on a FACScan flow cytometer. A total of 10 x 10' cells were accumulated for each DNA histogram. The proportion of cells in each phase of the cell cycle was determined using the Baisch mathematical model (Baisch et al. 1975). FIG.
1989). Characterization of VSMC growth kinetics is therefore essential for an understanding of these abnormalities, as well as for the ulterior orientation of therapeutic strategies in hypertension. The present study evaluated by flow cytometry the cell cycle transition kinetics of cultured VSMC from SHR and their normotensive WKY controls. Methods Cell culture Cultured VSMC were obtained by an explant method (Ross 1971) from WKY and SHR aortae as described previously (Franks et a/. 1984; Hadrava et al. 1989; Hamet et al. 1989). In preparation for the experiments, the sparsely seeded, nonconfluent cells were made quiescent by serum deprivation; after 16 to 20 h of attachment, the 10% calf serum-supplemented culture medium was replaced by defined serum-free medium (DMEM containing 2.5 pg insulin/mL, 2.5 pg transferrin/mL, and 2.5 ng selenium/mL or 0.2% calf serum in DMEM for 72 h.
Flow cytometric analysis of BrdU substitution Cells were inoculated in 80-cmZ flasks at a concentration of 7.5 x lo5 celldflask and made quiescent as above. Cell suspension samples were prepared and processed for cell cycle analysis as described (Sekaly et al. 1983). Briefly, the cells were stimulated by DMEM containing 10% calf serum to which BrdU (Boehringer Mannheim Corp., Dorval, Que.) was added at a concentration of 10 pg/mL. To overcome BrdU's cytotoxic effect due to inhibition of ribonucleotide reductase (Meuth and Green 1974), deoxycytidine (Boehringer Mannheim Corp.) was added in equimolar concentrations (8 pg/mL) to BrdU. Cell suspensions were obtained by trypsinization at various time periods after calf serum stimulation and the addition of BrdU and deoxycytidine. The cells were then resuspended in PBS', fixed in 50% ethanol, and kept at 4OC. After washing in PBS', they were stained with 1 pg/mL of the adenine- and thymidine-specific binding dye, Hoechst 33 342 (Molecular Probes, Inc., Eugene, Oreg.), in the presence of 0.05% NP-40.Fluorescence analysis was performed on a FACStar (Becton Dickinson ImrnunocytometrySystems) with an argon laser adjusted to emit 50 mW at 365-nm wavelengths. For each Hoechst fluorescence histogram, 5 x 10' cells were accumulated. In two control experiments, half of each sample was stained by propidium iodide and analyzed by FACScan as described above.
Cellular DNA content analysis by flow cytometry Cells were inoculated in 80-cm2 flasks at a concentration of 5 x 10' cells/flask and made quiescent, as described above. They were stimulated with 10% calf serum and arrested by trypsinizaStatistical analysis tion at various times from 0 to 24 h. Cells resuspended in Values are given as mean SEM. The level of significance of Dulbecco's PBS containig 320 pM c a 2 + and 60 pM M ~ ~ differences + between the means was evaluated by Student's t-test (PBS+) were fixed in 50% ethanol and kept at 4°C. Immediately for unpaired data and by two-way and multivariate ANOVA before cell cycle analysis, they were washed in PBS' and (Morrison 1976). resuspended in 2 mL PBS'. DNA was stained with propidium iodide (20 pg/mL), a specific dye for double-stranded nucleic acid Results (Sigma, St. Louis, Mo.). To prevent RNA staining by propidium iodide, 10 pg/mL of ribonuclease A (Sigma) was added to each Analysis of cellular DNA content sample. After 30 min of incubation at 37"C, the cells were analyzed Quiescent cells were stimulated at time 0 with 10% calf in a FACScan flow cytometer (Becton Dickinson Immunocytometry serum. After different periods, they were trypsinized, fixed, Systems, Mountain View, Calif.) with an argon laser adjusted to and stained with the DNA-binding dye propidium iodide, emit 15 mW at 488-nm wavelengths. From each cell sample, and processed by FACScan for cellular DNA analysis. The 10 x 10' events were accumulated for each histogram. The prointensity of fluorescence emitted was directly proportional portion of cells in cell cycle phases was determined from each to DNA content. The time course of analysis is presented histogram, using the model described by Baisch et al. (1975). in Fig. 1 (typical experiment) and Table 1 (data from five VSMC size was estimated simultaneously from the same cell independent experiments). Quiescent cells were mostly seen samples by analysis of forward light scatter.
*
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Fig. 3. Since each cell in Go/GI gives rise to two daughter cells after mitotic division, the numbers of G I ' cells were divided by two to obtain the real transition kinetics (Bohmer 1979). The distribution of Go/Gl minus G11/2was plotted against time after stimulation and BrdU addition. A linear (SHR, r = 0.992; WKY, r = 0.994) relationship appeared between 12 and 28 h. At earlier times (between 0 and 8 h), the apparent increase in the proportion of Go/Gl cells corresponded to entry into this peak of original G2 + M cells that had undergone mitosis without BrdU incorporation. The intercepts of the curves with line 0 corresponded to the time when half of the cells that were initially in the Go/Gl phase had progressed through the cell cycle and had divided. The curves were parallel, showing no measurable difference in transition kinetics through the S phase. The shift of the SHR VSMC curve to the left is explained by 4-h-accelerated Go/Gl-S phase transition.
0
4
8
12
16
20
24
28
32
36
(Hours) FIG. 3. Determination of the transition kinetics of WKY and SHR aortic smooth muscle cells. The proportions of cells in G, and GI' compartments were obtained from histograms similar to those shown in Fig. 2. The proportions of cells in the GIf peak were divided by a factor of two, since each cell gives rise to two daughter cells after mitotic division. Differences of G, minus GI' /2 were plotted against time after calf serum stimulation and BrdU addition. Means + SEM are from three independent experiments. Initial values are normalized to 100. Line 0 represents equal proportions of GI and GI' / 2 cells. The intercepts of the curves with line 0 correspond to the time when half of the cells have progressed through the cell cycle and have divided. 0,WKY (r = 0.994, slope = 0.105); a , SHR (r = 0.992, slope = 0.098). TIME
therefore appear in a new peak of quenched fluorescence. This permitted us to follow the decrease of Go/Gl cells and progression through the cell cycle, as recorded by the appearance and increase of daughter cells in the GI ' peak that have passed through the S and G2 + M phases. After stimulation with 10% calf serum and the addition of BrdU, the S compartment, to the right of the major Go/GI peak, remained empty (Fig. 2). From 8 h on, we observed the quenched fluorescence of GI ' cells to the left of the Go/GI peak with higher proportions in the case of SHR cells; WKY cells followed the SHR pattern with approximately a 4-h lag period (Figs. 2 and 3). The proportion of Go/GI cells continued to decrease until 32 h, demonstrating that with time, almost all cells entered the cell cycle for both WKY and SHR. It clearly demonstrates that the increased proliferation of cultured VSMC from SHR is not due to a greater proportion of cells entering the cell cycle. The transition kinetics of VSMC from WKY and SHR in three independent experiments are summarized in
Discussion The early rise in blood pressure in genetic models of hypertension (Gray 1984) makes it difficult to distinguish between primary and secondary events occurring in adulthood in these models. In previous studies, we demonstrated increased proliferation in heart, kidney, and aorta already in SHR neonates in vivo (Walter and Hamet 1986). These results are sustained by greater numbers of laminae in SHR aortae, even in the fetal stage (Eccleston-Joyner and Gray 1988). The notion that cardiovascular hyperplasia may be causally related to spontaneous hypertension is supported by studies demonstrating that cardiac and renal hyperplasia associated with genetic hypertension is present in newborns of four different spontaneously hypertensive models, but absent in offspring of parents with renal and experimental hypertension (Pang et al. 1986). The increased proliferation of VSMC represents one of the anomalies potentially pathogenetically linked with hypertension (Yamori et al. 1981; Hamet et al. 1988; Blennerhassett et al. 1989; Hadrava et al. 1989; ScottBurden et al. 1989; Paquet et al. 1989; Bukoski 1990; Hamada et al. 1990b). The careful description of cell cycle parameters in SHR and WKY with an accurate, rapid and reliable technique should allow the evaluation of that linkage using F2, backcrosses, and (or) recombinant inbred progenies of genetically hypertensive strains and their normotensive controls. It has been shown that SHR VSMC during the logarithmic phase of growth present a shorter doubling time, as well as time to complete one cell cycle (Harnada et al. 1990a). It was suggested that cells from SHR either spend less time traversing the Gl phase of the cell cycle or that a lesser proportion of cells enter Go compared with WKY. In the present study, by two different and complementary flow cytometry methods and using synchronized cultured cells, we showed that SHR VSMC present a 4-h accelerated entry into the S phase of the cell cycle with a minor additional defect in the faster G2-M phase transition. There was no difference between WKY and SHR in proportions of cells entering the cell cycle and no difference in S phase transition kinetics. The 4-h accelerated GI-S phase transition may represent a new intermediate phenotype in genetic hypertension, which may now be tested in genetic linkage studies. At a density comparable to that used in the present studies, quiescent VSMC from WKY and SHR present the same
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NOTES
very low mRNA levels of c-fos and c-myc protooncogenes typical of noncompetent cells (Hamet et al. 1991; Tremblay et al. 1992), and calf serum stimulation induces the mRNA expression of these two protooncogenes at the same levels and kinetics. The response to low physiologic concentrations of PDGF, the competence-inducing factor, has been reported t o be the same in VSMC from WKY (Scott-Burden et al. 1988; Hadrava et al. 1989). Both WKY and SHR VSMC seem, therefore, to be arrested at the same point of the Go/GI subphase. The fact that VSMC from SHR present a greater response t o EGF (the most important progression factor acting downstream of competence) than WKY cells (Hadrava et al. 1989; Scott-Burden et al. 1989) suggests that the major defect of SHR VSMC resulting in accelerated entry into the S phase is situated within the progression subphase. This hypothesis is strengthened by the finding of a calmodulin activator in SHR VSMC (Huang et al. 1988), conferring greater activity t o t h e ca2+-calmodulin system. Indeed, calmodulin has to be elevated during the progression subphase (Chafouleas et al. 1982, 1984). The possibility that the explant method selected subpopulations of cells that are more adapted t o in vitro conditions cannot be ruled out. Nevertheless, the experimental conditions were identical for explants derived from both WKY and SHR aortae and their VSMC growth in subcultures. The pathophysiological role of VSMC proliferation in vivo in genetic hypertension is not fully understood. In both genetic and secondary hypertension, structural changes in the vessel wall are predominant pathologic features together with a higher contractile response of the vascular mass. SHR appear t o have both hyperplastic and hypertrophic components contributing t o aortic structural changes in the established phase of the disease (Bucher et al. 1984). Owens and Schwartz (1982) have shown that SHR aortae contained more polyploid cells. Hypertrophy accompanied by polyploidy is observed in experimentally induced hypertension and seems t o be dominant in the adult aorta while hyperplastic changes are more evident in the resistant vasculature (Owens and Schwartz 1983; Owens and Reidy 1985). In long-term cultures, however, it is hyperproliferation that is the predominant phenotype in both aortic (Hadrava et al. 1989) and mesenteric (Bukoski 1990) VSMC from SHR. Moreover, long-term treatment of essentially hypertensive patients as well as SHR with antihypertensive drugs does not eliminate all the structural changes, suggesting further that high blood pressure is not the sole deterrnining factor (Jespersen et al. 1985; Owens 1987; Aalkjaer et al. 1989; Christensen et al. 1989). Thus, a tendency towards hyperplasia is present in both aortic and resistance vessels of SHR. In conclusion, the hyperproliferation of cultured VSMC from SHR may be explained by a 4-h accelerated entry into the S phase of the cell cycle. This well-defined abnormality can be evaluated for its linkage with hypertension in future genetic studies.
Acknowledgements These studies were supported by grants from the Medical Research Council of Canada (MA-10803) and the Heart and Stroke Foundation of Canada. V.H. is a fellow from The Diabetic Children's Foundation, and J.T. is a scholar of Fonds de la Recherche en SantC du Quebec. The authors
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acknowledge the technical assistance of Carole Long and Claude Cantin, the editorial work of Ovid Da Silva, and the secretarial help of Louise Chevrefils. Aalkjaer, C., Eiskjaer, H., Mulvany, M.J., et al. 1989. Abnormal structure and function of isolated subcutaneous resistance vessels from essential hypertensive patients despite antihypertensive treatment. J. Hypertens. 7: 305-3 10. Baisch, H., Gohde, W., and Linden, W.A. 1975. Analysis of PCPdata to determine the fraction of cells in the various phases of cell cycle. Radiat. Environ. Biophys. 12: 31-39. Blennerhassett, M.G., Kannan, M.S., and Garfield, R.E. 1989. Density-dependent hyperpolarization in cultured aortic smooth muscle cells. Am. J. Physiol. 256: C644-C65 1. Bohmer, R.M. 1979. Flow cytometric cell cycle analysis using the quenching of 33258 Hoechst fluorescence by bromodeoxyuridine incorporation. Cell Tissue Kinet. 12: 101-1 10. Bolzon, B.J., and Cheung, D.W. 1989. Isolation and characterization of single vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension (Dallas), 14: 137-144. Bucher, B., Travo, P., and Stoclet, J.C. 1984. Smooth muscle cell hypertrophy and hyperplasia in the thoracic aorta of spontaneously hypertensive rats. Cell Biol. Int. Rep. 8: 567-577. Bukoski, R.D. 1990. Intracellular c a 2 + metabolism of isolated resistance arteries and cultured vascular myocytes of spontaneously hypertensive and Wistar-Kyoto normotensive rats. J. Hypertens. 8: 37-43. Chafouleas, J.G., Bolton, W.E., Hidaka, H., et al. 1982. Calmodulin and the cell cycle: involvement in regulation of cellcycle progression. Cell, 28: 41-50. Chafouleas, J.G., Lagace, L., Bolton, W.E., et al. 1984. Changes in calmodulin and its mRNA accompany reentry of quiescent (Go) cells into the cell cycle. Cell, 36: 73-81. Christensen, K.L., Jespersen, L.T., and Mulvany, M.J. 1989. Development of blood pressure in spontaneously hypertensive rats after withdrawal of long-term treatment related to vascular structure. J. Hypertens. 7: 83-90. Eccleston-Joyner, C.A., and Gray, S.D. 1988. Arterial hypertrophy in the fetal and neonatal spontaneously hypertensive rat. Hypertension (Dallas), 12: 513-518. Franks, D.J., Plamondon, J., and Hamet, P. 1984. An increase in adenylate cyclase activity precedes DNA synthesis in cultured vascular smooth muscle cells. J. Cell. Physiol. 119: 41 -45. Gray, S.D. 1984. Pressure profiles in neonatal spontaneously hypertensive rats. Biol. Neonate 45: 25-32. Hadrava, V., Tremblay, J., and Hamet, P. 1989. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension (Dallas), 13: 589-597. Hadrava, V., Tremblay, J., and Hamet, P. 1991. Intrinsic factors involved in vascular smooth muscle cell proliferation in hypertension. Clin. Invest. Med. 14: 535-544. Hamada, M., Harris. E.L., Millar, J.A., and Simpson, F.O. 1990~. Temporal differences in the cell cycles of cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. J. Vasc. Med. Biol. 2: 136-141. Hamada. M., Nishio. I., Baba, A., et al. 1990b. Enhanced DNA synthesis of cultured vascular smooth muscle cells from spontaneously hypertensive rats-difference of response to growth factor, intracellular free calcium concentration and DNA synthesizing cell cycle. Atherosclerosis (Shannon, Irel.), 81: 191-198. Hamet, P., Hadrava, V., Kruppa, U., and Tremblay, J. 1988. Vascular smooth muscle cell hyperresponsivenessto growth factors in hypertension. J. Hypertens. 6(Suppl. 4): S36-S39. Hamet, P., Pang, S.C., and Tremblay, J. 1989. Atrial natriuretic factor-induced egression of cyclic guanosine 3 ' : 5 ' monophosphate in cultured vascular smooth muscle and endothelial cells. J. Biol. Chem. 264: 12 364 - 12 369. Hamet, P., Hadrava, V., Kruppa, U., and Tremblay, J. 1991.
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Transforming growth factor Dl expression and effect in aortic smooth muscle cells from spontaneously hypertensive rats. Hypertension (Dallas), 17: 896-901. Haudenschild, C.C., Grunwald, J., and Chobanian, A.V. 1985. Effects of hypertension on migration and proliferation of smooth muscle in culture. Hypertension (Dallas), 7 (Suppl. I): 1-101-1-104. Huang, S.L., Wen, Y.I., Kupranycz, D.B., et al. 1988. Abnormality of calmodulin in hypertension: evidence of the presence of an activator. J. Clin. Invest. 82: 276-281. Jespersen, L.T., Nyborg, N.C.B., Pedersen, O.L., el al. 1985. Cardiac mass and peripheral vascular structure in hydralazine-treated spontaneously hypertensive rats. Hypertension (Dallas), 7: 734-741. Meuth, M., and Green, H. 1974. Induction of a deoxycytidineless state in cultured mammalian cells by bromodeoxyuridine. Cell, 2: 109-112. Mey, J., Grunwald, J., and Hauss, W.H. 1980. Growth rate differences between arterial smooth muscle cells cultivated from rat impaired by short- or long-term hypertension respectively. Artery (Fulton, Mich.), 8: 348-354. Morrison, D.F. 1976. Multivariate statistical methods. 2nd ed. McGraw-Hill, New York. Mulvany, M.J., Hansen, P.K., and Aalkjaer, C. 1978. Direct evidence that the greater contractility of resistance vessels in spontaneously hypertensive rats is associated with narrowed lumen, thickened media and an increased number of smooth muscle cell layers. Circ. Res. 43: 854-864. Mulvany, M.J., Baandrup, U., and Gundersen, H.J.G. 1985. Evidence for hyperplasia in mesenteric resistance vessels of spontaneously hypertensive rats using a three-dimensional disector. Circ. Res. 57: 794-800. Owens, G.K. 1987. Influence of blood pressure on development of aortic medial smooth muscle hypertrophy in spontaneously hypertensive rats. Hypertension (Dallas), 9: 178-187. Owens, G.K., and Reidy, M.A. 1985. Hyperplastic growth response of vascular smooth muscle cells following induction of acute hypertension in rats by aortic coarctation. Circ. Res. 57: 695-705.
Owens, G.K., and Schwartz, S.M. 1982. Alterations in vascular smooth muscle mass in the spontaneously hypertensive rat-role of cellular hypertrophy, hyperploidy, and hyperplasia. Circ. Res. 51: 280-289. Owens, G.K., and Schwartz, S.M. 1983. Vascular smooth muscle hypertrophy and hyperploidy in the Goldblatt hypertensive rat. Circ. Res. 53: 491-501. Pang, S.C., Long, C., Poirier, M., et al. 1986. Cardiac and renal hyperplasia in newborn genetically hypertensive rats. J. Hypertens. 4 (Suppl. 3): S119-S122. Paquet, J.L., Baudouin-Legros, M., Marche, P., and Meyer, P. 1989. Enhanced proliferating activity of cultured smooth muscle cells from SHR. Am. J. Hypertens. 2: 108-1 10. Ross, R. 1971. The smooth muscle cell. 11. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell Biol. 50: 172-186. Scott-Burden, T., Resink, T.J., and Buhler, F.R. 1988. Growth regulation in smooth muscle cells from normal and hypertensive rats. J. Cardiovasc. Pharmacol. 12 (Suppl. 5): S124-S127. Scott-Burden, T., Resink, T.J., Baur, U., et al. 1989. Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats. Hypertension (Dallas), 13: 295-304. Sekaly, R.P., Ceredig, R., and MacDonald, H.R. 1983. Generation of thyrnocyte subpopulations in organ culture: correlated analysis of LYT-2 phenotype and cell cycle status by flow microfluorometry. J. Immunol. 131: 1085-1089. Tremblay, J., Hadrava, V., Kruppa, U., et al. 1992. Enhanced growth-dependent expression of TGFD, and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats. Can. J. Physiol. Pharmacol. 70: 565-572. Walter, S.V., and Hamet, P. 1986. Enhanced DNA synthesis in heart and kidney of newborn spontaneously hypertensive rats. Hypertension (Dallas), 8: 520-525. Yamori, Y., Igawa, T., Kanbe, T., et al. 1981. Mechanisms of structural vascular changes in genetic hypertension: analyses of cultured vascular smooth muscle cells from spontaneously hypertensive rats. Clin. Sci. 61: 121s-123s.
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VRATISLAVHADRAVA AND JOHANNE TREMBLAY Centre de Recherche H6tel-Dieu de Montreal, 3850 rue Saint-Urbain, Pavillon Marie-de-la-Ferre, Montrkal (QuPbec), Canada H2 W I T8 RAFICK-PIERRESEKALY Clinical Research Institute of MontrPal, MontrPal, Que., Canada H2 W IR7 AND
PAVEL HAMET' Centre de Recherche H6tel-Dieu de Montrkal, 3850 rue Saint-Urbain, Pavillon Marie-de-la-Ferre, MontrPal (QuPbec), Canada H2 W 1 T8 Received November 7, 1991 HADRAVA, V., TREMBLAY, J., SEKALY,R.-P., and HAMET,P. 1992. Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle. Biochem. Cell Biol. 70: 599-604. The present study was designed to characterize the growth kinetics of the exaggerated proliferative response to mitogens of vascular smooth muscle cells from spontaneously hypertensive rats compared with cells from normotensive WistarKyoto controls. Cellular DNA content, analyzed by flow cytometry, demonstrated a 4-h accelerated entry into the S phase of the cell cycle of vascular smooth muscle cells from spontaneously hypertensive rats; the significant (4.5-fold) increase in the percentage of cells in the S phase occurred between 8 and 12 h after calf serum stimulation. A 3.9-fold increase of cells in the S phase was seen in the normotensive controls only between 12 and 16 h. Transit through the cell cycle was quantitated by flow cytometry using the Hoechst 33 342 - bromodeoxyuridine substitution technique. Vascular smooth muscle cells from spontaneously hypertensive rats went through the cell cycle 4 h ahead of cells from normotensive Wistar-Kyoto rats. This accelerated transit of spontaneously hypertensive rat cells was mostly due to an earlier entry into the S phase. Persistence of this new intermediate phenotype in cell culture suggests its primary pathogenetic role in spontaneous hypertension. Key words: hypertension, proliferation, flow cytometry, bromodeoxyuridine substitution, Go/G, phase. J., SEKALY,R.-P., et H ~ E TP., 1992. Accelerated entry of aortic smooth muscle cells HADRAVA,V., TREMBLAY, from spontaneously hypertensive rats into the S phase of the cell cycle. Biochem. Cell Biol. 70 : 599-604. La prtsente ttude veut caracttriser la cinttique de la croissance de la rtponse proliftrative exagtrte aux mitogenes des cellules des muscles lisses vasculaires chez des rats spontankment hypertensifs comparts aux cellules des contr8les Wistar-Kyoto normotensifs. Analyste par cytomktrie a flot continu, la teneur du DNA cellulaire dtmontre une entrke accClCrke de 4 h dans la phase S du cycle cellulaire des cellules des muscles lisses vasculaires chez les rats spontantment hypertensifs; l'augmentation importante (4,s fois) du pourcentage des cellules en phase S survient entre 8 et 12 h a p r b stimulation avec le strum de veau. Chez les contr8les normotensifs, une augmentation de 3,9 fois des cellules en phase S n'est obsewte qu'entre 12 et 16 h. Nous avons quantifik le transit a travers le cycle ceuulaire par cytomktrie a flot continu utilisant la technique de substitution avec le Hoechst 33 342 - bromodksoxyuridine. Les cellules des muscles lisses vasculaires des rats spontantment hypertensifs passent a travers le cycle cellulaire 4 h avant les cellules des rats Wistar-Kyoto normotensifs. Ce transit acctltrC des cellules des rats spontantment hypertensifs est surtout dfi a une entrte plus hfitive dans la phase S. La persistance de ce nouveau phtnotype intermtdiaire dans la culture des cellules suggbe son r81e pathogCnCtique premier dans l'hypertension spontante. Mots clPs : hypertension, proliftration, cytomktrie a flot continu, substitution de la bromodtsoxyuridine, phase Go/G,. [Traduit par la rtdaction]
Introduction Narrowing of the vessel lumen due to arterial wall thickening is a common feature in hypertension. VSMC hyperplasia in the SHR model of essential hypertension has been demonstrated in resistance vessels by histological studies (Mulvany et al. 1978, 1985). Aortic and mesenteric smooth muscle cells from SHR proliferate more rapidly than cells derived from normotensive WKY controls (Yamori et al. ABBREVIATIONS: VSMC, vascular smooth muscle cell(s); SHR, spontaneously hypertensive rat(s); WKY, Wistar-Kyoto rat(s); EGF, epidermal growth factor; PDGF, platelet-derived growth factor; DMEM, Dulbecco's modified Eagle medium; PBS, phosphatebuffered saline; PBS', Dulbecco's PBS containing 320 pM c a 2 + and 60 pM M~''; BrdU, bromodeoxyuridine; NP-40, Nonidet P-40. ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprim4 au Canada
1981; Hamet et al. 1988; Blennerhassett et al. 1989; Hadrava et al. 1989; Scott-Burden et al. 1989; Paquet et al. 1989; Bukoski 1990; Hamada et al. 1990a, 1990b) in vitro, supporting the possibility of an abnormal intermediate phenotype that is not a consequence of elevated blood pressure. Even though experimentally induced hypertension may lead to the enhanced migration of cells from aortic explants, the shorter interdivision time afterwards does not persist over six subcultures (Mey et al. 1980; Haudenschild et al. 1985; Hamada et al. 1990b). We and others have previously shown that the greater proliferation of SHR VSMC, persisting under culture conditions until the 19th passage studied, is not due to varying cell survival or attachment ability after passage, and that SHR cells manifest an exaggerated maximal response to calf serum, EGF, and P D G F (Hadrava et al. 1989; Scott-Burden et al.
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0
g
G 2 * M = 16
G2*M = 12
(I
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Propidium iodide fluorescence intensity 1. Cell cycle analysis of aortic smooth muscle cells from WKY and SHR. Cells were made quiescent by serum starvation and then stimulated with 10% calf serum. They were harvested at various times (between 0 and 24 h), treated with RNAse, stained with propidium iodide, and analyzed on a FACScan flow cytometer. A total of 10 x 10' cells were accumulated for each DNA histogram. The proportion of cells in each phase of the cell cycle was determined using the Baisch mathematical model (Baisch et al. 1975). FIG.
1989). Characterization of VSMC growth kinetics is therefore essential for an understanding of these abnormalities, as well as for the ulterior orientation of therapeutic strategies in hypertension. The present study evaluated by flow cytometry the cell cycle transition kinetics of cultured VSMC from SHR and their normotensive WKY controls. Methods Cell culture Cultured VSMC were obtained by an explant method (Ross 1971) from WKY and SHR aortae as described previously (Franks et a/. 1984; Hadrava et al. 1989; Hamet et al. 1989). In preparation for the experiments, the sparsely seeded, nonconfluent cells were made quiescent by serum deprivation; after 16 to 20 h of attachment, the 10% calf serum-supplemented culture medium was replaced by defined serum-free medium (DMEM containing 2.5 pg insulin/mL, 2.5 pg transferrin/mL, and 2.5 ng selenium/mL or 0.2% calf serum in DMEM for 72 h.
Flow cytometric analysis of BrdU substitution Cells were inoculated in 80-cmZ flasks at a concentration of 7.5 x lo5 celldflask and made quiescent as above. Cell suspension samples were prepared and processed for cell cycle analysis as described (Sekaly et al. 1983). Briefly, the cells were stimulated by DMEM containing 10% calf serum to which BrdU (Boehringer Mannheim Corp., Dorval, Que.) was added at a concentration of 10 pg/mL. To overcome BrdU's cytotoxic effect due to inhibition of ribonucleotide reductase (Meuth and Green 1974), deoxycytidine (Boehringer Mannheim Corp.) was added in equimolar concentrations (8 pg/mL) to BrdU. Cell suspensions were obtained by trypsinization at various time periods after calf serum stimulation and the addition of BrdU and deoxycytidine. The cells were then resuspended in PBS', fixed in 50% ethanol, and kept at 4OC. After washing in PBS', they were stained with 1 pg/mL of the adenine- and thymidine-specific binding dye, Hoechst 33 342 (Molecular Probes, Inc., Eugene, Oreg.), in the presence of 0.05% NP-40.Fluorescence analysis was performed on a FACStar (Becton Dickinson ImrnunocytometrySystems) with an argon laser adjusted to emit 50 mW at 365-nm wavelengths. For each Hoechst fluorescence histogram, 5 x 10' cells were accumulated. In two control experiments, half of each sample was stained by propidium iodide and analyzed by FACScan as described above.
Cellular DNA content analysis by flow cytometry Cells were inoculated in 80-cm2 flasks at a concentration of 5 x 10' cells/flask and made quiescent, as described above. They were stimulated with 10% calf serum and arrested by trypsinizaStatistical analysis tion at various times from 0 to 24 h. Cells resuspended in Values are given as mean SEM. The level of significance of Dulbecco's PBS containig 320 pM c a 2 + and 60 pM M ~ ~ differences + between the means was evaluated by Student's t-test (PBS+) were fixed in 50% ethanol and kept at 4°C. Immediately for unpaired data and by two-way and multivariate ANOVA before cell cycle analysis, they were washed in PBS' and (Morrison 1976). resuspended in 2 mL PBS'. DNA was stained with propidium iodide (20 pg/mL), a specific dye for double-stranded nucleic acid Results (Sigma, St. Louis, Mo.). To prevent RNA staining by propidium iodide, 10 pg/mL of ribonuclease A (Sigma) was added to each Analysis of cellular DNA content sample. After 30 min of incubation at 37"C, the cells were analyzed Quiescent cells were stimulated at time 0 with 10% calf in a FACScan flow cytometer (Becton Dickinson Immunocytometry serum. After different periods, they were trypsinized, fixed, Systems, Mountain View, Calif.) with an argon laser adjusted to and stained with the DNA-binding dye propidium iodide, emit 15 mW at 488-nm wavelengths. From each cell sample, and processed by FACScan for cellular DNA analysis. The 10 x 10' events were accumulated for each histogram. The prointensity of fluorescence emitted was directly proportional portion of cells in cell cycle phases was determined from each to DNA content. The time course of analysis is presented histogram, using the model described by Baisch et al. (1975). in Fig. 1 (typical experiment) and Table 1 (data from five VSMC size was estimated simultaneously from the same cell independent experiments). Quiescent cells were mostly seen samples by analysis of forward light scatter.
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Fig. 3. Since each cell in Go/GI gives rise to two daughter cells after mitotic division, the numbers of G I ' cells were divided by two to obtain the real transition kinetics (Bohmer 1979). The distribution of Go/Gl minus G11/2was plotted against time after stimulation and BrdU addition. A linear (SHR, r = 0.992; WKY, r = 0.994) relationship appeared between 12 and 28 h. At earlier times (between 0 and 8 h), the apparent increase in the proportion of Go/Gl cells corresponded to entry into this peak of original G2 + M cells that had undergone mitosis without BrdU incorporation. The intercepts of the curves with line 0 corresponded to the time when half of the cells that were initially in the Go/Gl phase had progressed through the cell cycle and had divided. The curves were parallel, showing no measurable difference in transition kinetics through the S phase. The shift of the SHR VSMC curve to the left is explained by 4-h-accelerated Go/Gl-S phase transition.
0
4
8
12
16
20
24
28
32
36
(Hours) FIG. 3. Determination of the transition kinetics of WKY and SHR aortic smooth muscle cells. The proportions of cells in G, and GI' compartments were obtained from histograms similar to those shown in Fig. 2. The proportions of cells in the GIf peak were divided by a factor of two, since each cell gives rise to two daughter cells after mitotic division. Differences of G, minus GI' /2 were plotted against time after calf serum stimulation and BrdU addition. Means + SEM are from three independent experiments. Initial values are normalized to 100. Line 0 represents equal proportions of GI and GI' / 2 cells. The intercepts of the curves with line 0 correspond to the time when half of the cells have progressed through the cell cycle and have divided. 0,WKY (r = 0.994, slope = 0.105); a , SHR (r = 0.992, slope = 0.098). TIME
therefore appear in a new peak of quenched fluorescence. This permitted us to follow the decrease of Go/Gl cells and progression through the cell cycle, as recorded by the appearance and increase of daughter cells in the GI ' peak that have passed through the S and G2 + M phases. After stimulation with 10% calf serum and the addition of BrdU, the S compartment, to the right of the major Go/GI peak, remained empty (Fig. 2). From 8 h on, we observed the quenched fluorescence of GI ' cells to the left of the Go/GI peak with higher proportions in the case of SHR cells; WKY cells followed the SHR pattern with approximately a 4-h lag period (Figs. 2 and 3). The proportion of Go/GI cells continued to decrease until 32 h, demonstrating that with time, almost all cells entered the cell cycle for both WKY and SHR. It clearly demonstrates that the increased proliferation of cultured VSMC from SHR is not due to a greater proportion of cells entering the cell cycle. The transition kinetics of VSMC from WKY and SHR in three independent experiments are summarized in
Discussion The early rise in blood pressure in genetic models of hypertension (Gray 1984) makes it difficult to distinguish between primary and secondary events occurring in adulthood in these models. In previous studies, we demonstrated increased proliferation in heart, kidney, and aorta already in SHR neonates in vivo (Walter and Hamet 1986). These results are sustained by greater numbers of laminae in SHR aortae, even in the fetal stage (Eccleston-Joyner and Gray 1988). The notion that cardiovascular hyperplasia may be causally related to spontaneous hypertension is supported by studies demonstrating that cardiac and renal hyperplasia associated with genetic hypertension is present in newborns of four different spontaneously hypertensive models, but absent in offspring of parents with renal and experimental hypertension (Pang et al. 1986). The increased proliferation of VSMC represents one of the anomalies potentially pathogenetically linked with hypertension (Yamori et al. 1981; Hamet et al. 1988; Blennerhassett et al. 1989; Hadrava et al. 1989; ScottBurden et al. 1989; Paquet et al. 1989; Bukoski 1990; Hamada et al. 1990b). The careful description of cell cycle parameters in SHR and WKY with an accurate, rapid and reliable technique should allow the evaluation of that linkage using F2, backcrosses, and (or) recombinant inbred progenies of genetically hypertensive strains and their normotensive controls. It has been shown that SHR VSMC during the logarithmic phase of growth present a shorter doubling time, as well as time to complete one cell cycle (Harnada et al. 1990a). It was suggested that cells from SHR either spend less time traversing the Gl phase of the cell cycle or that a lesser proportion of cells enter Go compared with WKY. In the present study, by two different and complementary flow cytometry methods and using synchronized cultured cells, we showed that SHR VSMC present a 4-h accelerated entry into the S phase of the cell cycle with a minor additional defect in the faster G2-M phase transition. There was no difference between WKY and SHR in proportions of cells entering the cell cycle and no difference in S phase transition kinetics. The 4-h accelerated GI-S phase transition may represent a new intermediate phenotype in genetic hypertension, which may now be tested in genetic linkage studies. At a density comparable to that used in the present studies, quiescent VSMC from WKY and SHR present the same
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NOTES
very low mRNA levels of c-fos and c-myc protooncogenes typical of noncompetent cells (Hamet et al. 1991; Tremblay et al. 1992), and calf serum stimulation induces the mRNA expression of these two protooncogenes at the same levels and kinetics. The response to low physiologic concentrations of PDGF, the competence-inducing factor, has been reported t o be the same in VSMC from WKY (Scott-Burden et al. 1988; Hadrava et al. 1989). Both WKY and SHR VSMC seem, therefore, to be arrested at the same point of the Go/GI subphase. The fact that VSMC from SHR present a greater response t o EGF (the most important progression factor acting downstream of competence) than WKY cells (Hadrava et al. 1989; Scott-Burden et al. 1989) suggests that the major defect of SHR VSMC resulting in accelerated entry into the S phase is situated within the progression subphase. This hypothesis is strengthened by the finding of a calmodulin activator in SHR VSMC (Huang et al. 1988), conferring greater activity t o t h e ca2+-calmodulin system. Indeed, calmodulin has to be elevated during the progression subphase (Chafouleas et al. 1982, 1984). The possibility that the explant method selected subpopulations of cells that are more adapted t o in vitro conditions cannot be ruled out. Nevertheless, the experimental conditions were identical for explants derived from both WKY and SHR aortae and their VSMC growth in subcultures. The pathophysiological role of VSMC proliferation in vivo in genetic hypertension is not fully understood. In both genetic and secondary hypertension, structural changes in the vessel wall are predominant pathologic features together with a higher contractile response of the vascular mass. SHR appear t o have both hyperplastic and hypertrophic components contributing t o aortic structural changes in the established phase of the disease (Bucher et al. 1984). Owens and Schwartz (1982) have shown that SHR aortae contained more polyploid cells. Hypertrophy accompanied by polyploidy is observed in experimentally induced hypertension and seems t o be dominant in the adult aorta while hyperplastic changes are more evident in the resistant vasculature (Owens and Schwartz 1983; Owens and Reidy 1985). In long-term cultures, however, it is hyperproliferation that is the predominant phenotype in both aortic (Hadrava et al. 1989) and mesenteric (Bukoski 1990) VSMC from SHR. Moreover, long-term treatment of essentially hypertensive patients as well as SHR with antihypertensive drugs does not eliminate all the structural changes, suggesting further that high blood pressure is not the sole deterrnining factor (Jespersen et al. 1985; Owens 1987; Aalkjaer et al. 1989; Christensen et al. 1989). Thus, a tendency towards hyperplasia is present in both aortic and resistance vessels of SHR. In conclusion, the hyperproliferation of cultured VSMC from SHR may be explained by a 4-h accelerated entry into the S phase of the cell cycle. This well-defined abnormality can be evaluated for its linkage with hypertension in future genetic studies.
Acknowledgements These studies were supported by grants from the Medical Research Council of Canada (MA-10803) and the Heart and Stroke Foundation of Canada. V.H. is a fellow from The Diabetic Children's Foundation, and J.T. is a scholar of Fonds de la Recherche en SantC du Quebec. The authors
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acknowledge the technical assistance of Carole Long and Claude Cantin, the editorial work of Ovid Da Silva, and the secretarial help of Louise Chevrefils. Aalkjaer, C., Eiskjaer, H., Mulvany, M.J., et al. 1989. Abnormal structure and function of isolated subcutaneous resistance vessels from essential hypertensive patients despite antihypertensive treatment. J. Hypertens. 7: 305-3 10. Baisch, H., Gohde, W., and Linden, W.A. 1975. Analysis of PCPdata to determine the fraction of cells in the various phases of cell cycle. Radiat. Environ. Biophys. 12: 31-39. Blennerhassett, M.G., Kannan, M.S., and Garfield, R.E. 1989. Density-dependent hyperpolarization in cultured aortic smooth muscle cells. Am. J. Physiol. 256: C644-C65 1. Bohmer, R.M. 1979. Flow cytometric cell cycle analysis using the quenching of 33258 Hoechst fluorescence by bromodeoxyuridine incorporation. Cell Tissue Kinet. 12: 101-1 10. Bolzon, B.J., and Cheung, D.W. 1989. Isolation and characterization of single vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension (Dallas), 14: 137-144. Bucher, B., Travo, P., and Stoclet, J.C. 1984. Smooth muscle cell hypertrophy and hyperplasia in the thoracic aorta of spontaneously hypertensive rats. Cell Biol. Int. Rep. 8: 567-577. Bukoski, R.D. 1990. Intracellular c a 2 + metabolism of isolated resistance arteries and cultured vascular myocytes of spontaneously hypertensive and Wistar-Kyoto normotensive rats. J. Hypertens. 8: 37-43. Chafouleas, J.G., Bolton, W.E., Hidaka, H., et al. 1982. Calmodulin and the cell cycle: involvement in regulation of cellcycle progression. Cell, 28: 41-50. Chafouleas, J.G., Lagace, L., Bolton, W.E., et al. 1984. Changes in calmodulin and its mRNA accompany reentry of quiescent (Go) cells into the cell cycle. Cell, 36: 73-81. Christensen, K.L., Jespersen, L.T., and Mulvany, M.J. 1989. Development of blood pressure in spontaneously hypertensive rats after withdrawal of long-term treatment related to vascular structure. J. Hypertens. 7: 83-90. Eccleston-Joyner, C.A., and Gray, S.D. 1988. Arterial hypertrophy in the fetal and neonatal spontaneously hypertensive rat. Hypertension (Dallas), 12: 513-518. Franks, D.J., Plamondon, J., and Hamet, P. 1984. An increase in adenylate cyclase activity precedes DNA synthesis in cultured vascular smooth muscle cells. J. Cell. Physiol. 119: 41 -45. Gray, S.D. 1984. Pressure profiles in neonatal spontaneously hypertensive rats. Biol. Neonate 45: 25-32. Hadrava, V., Tremblay, J., and Hamet, P. 1989. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension (Dallas), 13: 589-597. Hadrava, V., Tremblay, J., and Hamet, P. 1991. Intrinsic factors involved in vascular smooth muscle cell proliferation in hypertension. Clin. Invest. Med. 14: 535-544. Hamada, M., Harris. E.L., Millar, J.A., and Simpson, F.O. 1990~. Temporal differences in the cell cycles of cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. J. Vasc. Med. Biol. 2: 136-141. Hamada. M., Nishio. I., Baba, A., et al. 1990b. Enhanced DNA synthesis of cultured vascular smooth muscle cells from spontaneously hypertensive rats-difference of response to growth factor, intracellular free calcium concentration and DNA synthesizing cell cycle. Atherosclerosis (Shannon, Irel.), 81: 191-198. Hamet, P., Hadrava, V., Kruppa, U., and Tremblay, J. 1988. Vascular smooth muscle cell hyperresponsivenessto growth factors in hypertension. J. Hypertens. 6(Suppl. 4): S36-S39. Hamet, P., Pang, S.C., and Tremblay, J. 1989. Atrial natriuretic factor-induced egression of cyclic guanosine 3 ' : 5 ' monophosphate in cultured vascular smooth muscle and endothelial cells. J. Biol. Chem. 264: 12 364 - 12 369. Hamet, P., Hadrava, V., Kruppa, U., and Tremblay, J. 1991.
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