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Effects of dehydroepiandrosterone on proliferation of human aortic smooth muscle cells
Life Sciences, Vol. 60, No. 11, pp. 833-838,1997 Coovrieht 0 1597 Elsevier Science Inc. PrinkiJ ii the USA. Ail rights reserved ooz4-3205/w $17.00 + .oo
ELSEVIER
PII SOO24-3205(97)00011-S
EFFECTS OF DEHYDROEPIANDROSTERONE ON PROLIFERATION OF HUMAN AORTIC SMOOTH MUSCLE CELLS Akihiko Yoneyama, Yoshinobu Kamiya, Masanobu Kawaguchi and Takao Fujinami The Third Department of Internal Medicine, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan (Received in final
form December 27,19%)
Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) have been shown to be associated with the progression of coronary atherosclerosis in clinical and in vivo studies. However, the mechanisms responsible for the association have not been determined. In the present study, we found that DHEA influences the in vitro growth of vascular smooth muscle cells obtained from the human aorta (hASMC). The concentrations of DHEA ranging from 10-s M to 10-e M significantly stimulated the mitogenesis of hASMC in serum-free culture. On the other hand, 4 hrs of pretreatment with DHEA attenuated the fetal calf serum induced proliferative effect in a dose-dependent manner. However, the in vitro effects of DHEA on the mitogenesis observed in hASMC were not seen in rat-derived aortic smooth muscle cell lines (A10 cells). With respect to DHEAS, the hormone, at concentrations up to 1O-5 M did not affect the growth of either hASMC or A10 cells in vitro. The growth response of hASMC to DHEA in vitro was markedly affected by the culture conditions. The differential proliferative effects of DHEA on smooth muscle cells between rat and human are of interest. We conclude that the effects of DHEA on mitogenesis of hASMC may, at least in part, explain the association between DHEA and atherosclerosis. Key Word:
dehydroepiandrosterone,
vascular smooth muscle, atherosclerosis,
A10 ~eh.s
Dehydroepiandrosterone sulfate (DHEAS) is an abundant secretory product of the human adrenal gland. However, dehydroepiandrosterone (DHEA) has a more potent biologic activity and higher turnover rate than DHEAS. The conversion of DHEAS to DHEA is mediated by steroid sulfatases that are present in many tissues. To date, the biologic functions of DHEA/DHEAS have not been fully determined. Several investigations, including one prospective study, have demonstrated an inverse correlation between the serum level of DHEAS and the incidence of coronary artery disease in males (1,2,3). It has been hypothesized that the anti-atherogenic effects of DHEA/DHEAS are mediated through improvement in disorders of the lipid or glucose metabolism (4,5). studies, DHEA inhibited In addition, in in vivo atherosclerotic changes in the aorta and coronary arteries of rabbits (6,7). With respect to steroid hormones in general, it has been reported that estrogen or glucocorticoids attenuate the growth of vascular smooth muscle cells (VSMC)in vitro (8,9,10). Thus, it cannot be excluded that DHEA also has direct actions on VSMC. However, to our knowledge, there are no in vitro reports of direct associations between DHEA and the proliferation of human VSMC. We therefore hypothesized that DHEA/DHEAS attenuate the proliferation of vascular intimal cells and prevented the formation of atherosclerotic plaques, resulting in the retardation of atherosclerosis. To test our hypothesis, we examined the effects of DHEA on the growth of human aortic smooth muscle cells (hASMC) in vitro.
834
Effects
of DHEA on Proliferation of VSMC
Vol. 60, No. 11, 1997
Materials DHEA and DHEAS were obtained from Sigma Chem. Co. (St. Louis, MO, USA). hASMC were from Clonetics (San Diego, CA, USA) through Kurabo Co. (Osaka, Japan). The cells were harvested from the aorta of a 21-week-old Caucasian male infant who had no evidence of atherosclerosis. Rat aortic smooth muscle cells (A10 cells) were from the American Type Culture Collection (Rockville, ML, USA). Maintaining medium for hASMC (S-GM) from Kurabo Co. is modified-MCDB131 medium (S-BM) containing 5% fetal calf serum (FCS) and supplemented with human epidermal growth factor (EGF, 10 ng/mL), human basic fibroblast growth factor (bFGF, 2 ng/mL) and dexamethasone (0.39 ug/mL). Cell proliferation assay kit was purchased from Amersham Co. (Tokyo, Japan). Cell culture hASMC were grown in S-GM in a humidified atmosphere of 5% CO* in air at 37°C. Medium was changed every 2 days, and the cells were detached from culture dishes by treating with a solution of 0.025% of trypsin-EDTA. A10 cells, grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FCS (FCWDMEM), were detached from culture dishes by treating with a solution of 0.2% of trypsine-EDTA. The cells were then seeded into 96-well culture dishes (Falcon, NJ, USA) at 2500 cells/cm2 and were grown in S-GM or FCWDMEM. hASMC used for experiments were from passages 4 to 5. hASMC and A10 cells were grown to sub-confluence in each well and then incubated for 24 hrs in S-BM or DMEM containing 0.3% BSA, and these quiescent monolayer cells were used in this study. Mitogenesis in serum-free culture The quiescent hASMC and A10 cells monolayers were then incubated in serum-free medium (hASMC: S-BM, A10 cells: DMEM) containing test materials for 14 and 12 hrs, respectively. The test materials were DHEA or DHEAS ranging from 10-a M to 10-s M. After incubation, cell proliferation was assayed using BrdU incorporation kit. Mitogenesis of DHEA Pretreatment cells The quiescent hASMC and A10 cells monolayers were pretreated with DHEA or DHEAS ranging from 10-s M to lo-5 M for 4 hrs. Then mediums were exchanged to S-GM or FCS/DMEM, and after 14-hr (hASMC) or 12-hr (A10 cells) incubation, cell proliferation was assayed using BrdU incorporation into DNA. These cell monolayers were pretreated with 10-s M of DHEA for various durations ( 2,4, 8 and 24 hrs). After mediums were exchanged for S-GM or FCS/DMEM, cell proliferation was assayed using BrdU incorporation kit. Assay for BrdU incorporation into DNA After 4 hrs of incubation with BrdU, cells were fixed with an acetate-ethanol solution. After the addition of anti-BrdU antibody and horseradish peroxidase-anti IgG antibody to fixed cells, BrdU incorporation into DNA was detected using an ELISA cell proliferation assay kit. BrdU incorporation into DNA is expressed as percent control determined by (sample optical density(OD) - blank OD) /(control OD - blank OD). All values are expressed as mean+SD (n=3) in four separate experiments.
Effect of DHEA on proliferation of MSMC and Al 0 cell in absence of growth stimulants DHEA stimulated the mitogenesis of hASMC in serum-free culture as shown in Figure 1. The significant proliferative effect were observed at DHEA concentrations ranging from 10-s M to 1O-6 M, with the maximal effect seen at about 1O-8 M (138% of control). The stimulatory effects were also seen in hASMC obtained from adult (data not shown). In contrast, DHEA did not affect the mitogenesis of A10 cells. Effect of DHEA pretreatment on proliferation of hASMC and Al 0 cell in presence of growth stimulants The BrdU incorporation for four hrs observed in hASMC in SGM was about lo-fold greater than that in SBM. FCS-induced mitogenesis was also seen in A10 cells. DHEA inhibited
Vol. 60, No. 11, 1997
83.5
Effects of DHEA on Proliferation of VSMC
control
-6
-9
-7
DHEA amcen~ation
Fig. 1 Stimulatory effect of DHEA on mitogenesis incubated in the absence of growth factors.
-6
-5
(Log M)
in smooth
muscle
cells
Monolayers of quiescent hASMC and A10 cells were incubated in medium (hASMC: S-BM; A10 cells: DMEM) with various concentrations of DHEA at 37°C for 14 or 12 hrs, respectively. Thereafter, BrdU incorporation into DNA was assessed. Each data point represents mean?SD for three determinations increase Closed circles: hASMC. Open circles: Al0 cells. over control. *: p < 0.01 compared to control.
control
-10
-9
-6
DHEA concentration
Fig. 2 Inhibitory effect of DHEA on mitogenesis incubated in the absence of growth factors. Monolayers of quiescent hASMC and A10 (hASMC: S-GM, A10 cells: FCSDMEM) with 37°C for 14 or 12 hrs, respectively. Thereafter, Each data point represents mean t SD of control. Closed circles: hASMC. Open circles: *: p < 0.01 compared to control.
-7
-6
(Log M)
in smooth
muscle
cells
cells were incubated in medium various concentrations of DHEA at BrdU incorporation was assessed. three determinations increase over A10 cells.
836
Vol. 60, No. 11, WY7
Effects of DHEA on Proliferation of VSMC
control
2 DHEA
4
a
24
prekeatment time (hours)
Fig. 3 Pretreatment time of DHEA and attenuation of hASMC mitogenesis Monolayers of quiescent hASMC were incubated in S-BM with DHEA of 10-8 M for various durations. Media was exchanged to S-GM and after 14-hr incubation, cell proliferation was assayed Each data point represents mean?SD of three determinations increase over control. *: ~~0.01 compared with control.
S-GM stimulated BrdU incorporation in hASMC. The inhibitory effect was dose-dependent at DHEA concentrations ranging from 10. 10 M to 10-6 M as shown in Figure 2. The growing rate was 52% of control at 10-s M DHEA and 33% at 1O-6 M DHEA. The inhibitory effect was also seen in hASMC obtained from adult (data not shown). In contrast, the inhibitory effects on mitogenesis observed in hASMC were not seen in A10 cells. There was no increase in cell death when hASMC were incubated with DHEA (1O-4 M) for up to 48 hrs, and the growth of hASMC completely recovered after removal of DHEA. Effect of pretreatment with DHEA on proliferation of hASMC An inhibitory effect on the mitogenesis of hASMC was observed at more than 4 hrs DHEA pretreatment time, but not with only 2 hrs of pretreatment time, as shown in Figure 3. Effects of DHEAS on DNA synthesis DHEAS did not stimulate the BrdU incorporation into DNA of hASMC, which was a different finding observed in the addition of DHEA alone. Furthermore, DHEAS at concentrations ranging from 1O-9M up to 10m5M did not attenuate the FCS-induced mitogenesis of hASMC (data not shown).
This is the first report that DHEA influences the growth of hASMC in vitro. The inhibitory action of DHEA on mitotic activity of other cultured cells has been reported in great detail by several authors. This effect has been demonstrated in fibroblast (1 l), T-lymphocytes (12) normal hepatocytes and hepatocarcinoma cells (13). The effects of DHEA on VSMC growth are complicated, based on our finding of distinctive growth responses to DHEA based on the presence or absence of agents that stimulate growth. DHEA alone stimulated the growth of VSMC in serum-free culture. The range of DHEA concentrations stimulating the growth of VSMC in the absence of growth factors is from 10m8M to 10-G M. Several clinical studies have reported that serum DHEAS levels are inversely correlated to mortality from cardiovascular disease in males (1,2,3). Our findings in vitro seem
Vol. 60, No. 11, 1997
Effects of DHEA on Proliferation of VSMC
MI
at odds with these clinical reports. However, there are several reports concerning differences in the growth responses to agonists between in vitro and in vivo studies. We also reported that opposite effects of growth regulatory peptides on the growth of human epidermoid carcinoma cells from comparing in vitro and in vivo studies (14). Therefore, the in vitro findings observed in the present study are not necessarily incompatible with previous clinical findings (1,2,3). On the other hand, in the present study DHEA attenuated the growth of hASMC induced by the addition of FCS which contains various growth mediators, EGF and bFGF. The effects of DHEA were found only at DHEA concentrations greater than 10-9 M. The plasma DHEA concentration of healthy adult male is about 10-s M, so the DHEA levels used in our study are The required pretreatment time for DHEA was similar to human physiologic concentrations. more than 4 hrs, which is similar to a report concerning the effects of glucocorticoids on VSMC (15). It is well established that glucocorticoid and its receptor complex binds to the specific element of DNA. Because of this, we believe that DHEA affects hASMC through the binding to DNA. DHEAS is the major secretory product of the human adrenal gland. The human plasma concentration of DHEAS is in the range of lO-‘j M to 1O-5 M and is about thousand times greater than the concentration of DHEA. In our studies, DHEAS had no effect on hASMC growth regulation in either the presence or absence of growth stimulating agents. In the light of the present findings, hASMC may have no receptor of DHEAS and/or no phenol sulfotransferase to convert DHEAS to DHEA. In contrast to hASMC, we found that DHEA did not affect the growth of rat vasculer smooth muscle cells either in the presence or absence of FCS. The concentrations of DHEA and DHEAS in rat serum are much lower than in humans (16), so the physiologic role of DHEA may be very different in rat compared to human. While it should be determined whether DHEA can affect VSMC obtained from other species, the regulatory effects of DHEA on VSMC growth may be specific to humans. In conclusion, the growth response of human VSMC to DHEA in vitro was modulated by the presence of other growth factors in the culture. Further examination should be performed to determine the mechanisms by which DHEA affects the growth of hASMC in vitro. These findings should help shed light on the role of DHEA in anti-atherogenesis in vivo.
1. E.B. CONNOR, K.T. KHAW, S.S.C. YEN: N. Engl. J. Med. 3l5 1519-1524 (1986) 2. L.E. MITCHELL, D.L. SPRECHER, I.B. BORECKI, T. RICE, P.M. LASKARZEWSKI, D.C. RAO: Circulation 8.989-93 (1994) A.Z.LACROIX, K.Y.DWAYNE, M.REED: Circulation 86 1529-1535 (1992) :: S.M. HAFFNER, R.A. VALDEZ L. MYKKANAN, M.P. STERN, MS. KATZ: Metabolism ti599-603 (1994) 5. A.J. MORALETS, J.J. NOLAN, J.C. NELSON, S.S.C. YEN:J. Clin. Endclinol. Metab. 28 1360-1367 (1994) 6. D.M. EICH, J.E. NESTLER, D.E. JOHNSON, G.H. DWORKIN, D. KO, A.S. WECHSLER, M.L. HESS: Circulation 8;L261-269 (1993) 7. Y. ARAD, J.J. BADIMON, W.C. HEMBREE, H.N. GINSBERG: arteriosclerosis9 159-166 (1989) 8. T. NAKANO,‘E. W. RAINES, J.A. ABRAHAM, F.G. WENZEL IV, S. HIGASHIYAMA, M. KLAGSBRUN, R. ROSS: J. Biol. Chem. 2.6822941-22947 (1993) 9. A. ORIMO, S. INOUE, A. IKEGAMI, T. HOSOI, M. AKISHITA, Y. OUCHI, M. MURAMATSU, H. ORIMO: Biochem. Biophys. Res. Commun. x730-736 (1993) 10. R.H. KARAS, B.L. PATTERSON, M.E. MENDELSOHN: Circulation 89 1943-1950 (1994) 11. P. SAENGER, M. NEW: Experimentia 3966-967 (1976) 12. M.A.W, R.W. DORCHUK, B.A. ARANEO: J. Steroid Biochem. Mol. Biol. 42293-304 (1992)
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Effects of DHEA on Proliferationof VSMC
Vol. 60, No. 11, 1997
13. KALIMI M, J. OPOKU, Q.S. LU: B’IOCh em. Biophys. Res. Commun. X22-29 (1988) 14. Y. KAMIYA, E. OHMUM M. ARAI, T. FUJII, F. HAYAKAWA, J. ITO, M. KAWAGUCHI, T. TSUSHIMA, N. SAKUMA: Biochem. Biophys. Res. Commun. l!X 302-307 (1993) 15. Y. ITO, 0. KOZAWA, H. TOKUDA, A. SUZUKI, Y. WATANABE, J. KOTOYORI, Y. OISO: atherosclerosis 11Q69-76 (1994) 16. W.M. WEERDEN, H.G. BIERINGS, G.J. STEENBRUGGE, F.H. JONG, F.H. SCHRODER: Life Sciences 5Q857-861 (1992)
ELSEVIER
PII SOO24-3205(97)00011-S
EFFECTS OF DEHYDROEPIANDROSTERONE ON PROLIFERATION OF HUMAN AORTIC SMOOTH MUSCLE CELLS Akihiko Yoneyama, Yoshinobu Kamiya, Masanobu Kawaguchi and Takao Fujinami The Third Department of Internal Medicine, Nagoya City University Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan (Received in final
form December 27,19%)
Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEAS) have been shown to be associated with the progression of coronary atherosclerosis in clinical and in vivo studies. However, the mechanisms responsible for the association have not been determined. In the present study, we found that DHEA influences the in vitro growth of vascular smooth muscle cells obtained from the human aorta (hASMC). The concentrations of DHEA ranging from 10-s M to 10-e M significantly stimulated the mitogenesis of hASMC in serum-free culture. On the other hand, 4 hrs of pretreatment with DHEA attenuated the fetal calf serum induced proliferative effect in a dose-dependent manner. However, the in vitro effects of DHEA on the mitogenesis observed in hASMC were not seen in rat-derived aortic smooth muscle cell lines (A10 cells). With respect to DHEAS, the hormone, at concentrations up to 1O-5 M did not affect the growth of either hASMC or A10 cells in vitro. The growth response of hASMC to DHEA in vitro was markedly affected by the culture conditions. The differential proliferative effects of DHEA on smooth muscle cells between rat and human are of interest. We conclude that the effects of DHEA on mitogenesis of hASMC may, at least in part, explain the association between DHEA and atherosclerosis. Key Word:
dehydroepiandrosterone,
vascular smooth muscle, atherosclerosis,
A10 ~eh.s
Dehydroepiandrosterone sulfate (DHEAS) is an abundant secretory product of the human adrenal gland. However, dehydroepiandrosterone (DHEA) has a more potent biologic activity and higher turnover rate than DHEAS. The conversion of DHEAS to DHEA is mediated by steroid sulfatases that are present in many tissues. To date, the biologic functions of DHEA/DHEAS have not been fully determined. Several investigations, including one prospective study, have demonstrated an inverse correlation between the serum level of DHEAS and the incidence of coronary artery disease in males (1,2,3). It has been hypothesized that the anti-atherogenic effects of DHEA/DHEAS are mediated through improvement in disorders of the lipid or glucose metabolism (4,5). studies, DHEA inhibited In addition, in in vivo atherosclerotic changes in the aorta and coronary arteries of rabbits (6,7). With respect to steroid hormones in general, it has been reported that estrogen or glucocorticoids attenuate the growth of vascular smooth muscle cells (VSMC)in vitro (8,9,10). Thus, it cannot be excluded that DHEA also has direct actions on VSMC. However, to our knowledge, there are no in vitro reports of direct associations between DHEA and the proliferation of human VSMC. We therefore hypothesized that DHEA/DHEAS attenuate the proliferation of vascular intimal cells and prevented the formation of atherosclerotic plaques, resulting in the retardation of atherosclerosis. To test our hypothesis, we examined the effects of DHEA on the growth of human aortic smooth muscle cells (hASMC) in vitro.
834
Effects
of DHEA on Proliferation of VSMC
Vol. 60, No. 11, 1997
Materials DHEA and DHEAS were obtained from Sigma Chem. Co. (St. Louis, MO, USA). hASMC were from Clonetics (San Diego, CA, USA) through Kurabo Co. (Osaka, Japan). The cells were harvested from the aorta of a 21-week-old Caucasian male infant who had no evidence of atherosclerosis. Rat aortic smooth muscle cells (A10 cells) were from the American Type Culture Collection (Rockville, ML, USA). Maintaining medium for hASMC (S-GM) from Kurabo Co. is modified-MCDB131 medium (S-BM) containing 5% fetal calf serum (FCS) and supplemented with human epidermal growth factor (EGF, 10 ng/mL), human basic fibroblast growth factor (bFGF, 2 ng/mL) and dexamethasone (0.39 ug/mL). Cell proliferation assay kit was purchased from Amersham Co. (Tokyo, Japan). Cell culture hASMC were grown in S-GM in a humidified atmosphere of 5% CO* in air at 37°C. Medium was changed every 2 days, and the cells were detached from culture dishes by treating with a solution of 0.025% of trypsin-EDTA. A10 cells, grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FCS (FCWDMEM), were detached from culture dishes by treating with a solution of 0.2% of trypsine-EDTA. The cells were then seeded into 96-well culture dishes (Falcon, NJ, USA) at 2500 cells/cm2 and were grown in S-GM or FCWDMEM. hASMC used for experiments were from passages 4 to 5. hASMC and A10 cells were grown to sub-confluence in each well and then incubated for 24 hrs in S-BM or DMEM containing 0.3% BSA, and these quiescent monolayer cells were used in this study. Mitogenesis in serum-free culture The quiescent hASMC and A10 cells monolayers were then incubated in serum-free medium (hASMC: S-BM, A10 cells: DMEM) containing test materials for 14 and 12 hrs, respectively. The test materials were DHEA or DHEAS ranging from 10-a M to 10-s M. After incubation, cell proliferation was assayed using BrdU incorporation kit. Mitogenesis of DHEA Pretreatment cells The quiescent hASMC and A10 cells monolayers were pretreated with DHEA or DHEAS ranging from 10-s M to lo-5 M for 4 hrs. Then mediums were exchanged to S-GM or FCS/DMEM, and after 14-hr (hASMC) or 12-hr (A10 cells) incubation, cell proliferation was assayed using BrdU incorporation into DNA. These cell monolayers were pretreated with 10-s M of DHEA for various durations ( 2,4, 8 and 24 hrs). After mediums were exchanged for S-GM or FCS/DMEM, cell proliferation was assayed using BrdU incorporation kit. Assay for BrdU incorporation into DNA After 4 hrs of incubation with BrdU, cells were fixed with an acetate-ethanol solution. After the addition of anti-BrdU antibody and horseradish peroxidase-anti IgG antibody to fixed cells, BrdU incorporation into DNA was detected using an ELISA cell proliferation assay kit. BrdU incorporation into DNA is expressed as percent control determined by (sample optical density(OD) - blank OD) /(control OD - blank OD). All values are expressed as mean+SD (n=3) in four separate experiments.
Effect of DHEA on proliferation of MSMC and Al 0 cell in absence of growth stimulants DHEA stimulated the mitogenesis of hASMC in serum-free culture as shown in Figure 1. The significant proliferative effect were observed at DHEA concentrations ranging from 10-s M to 1O-6 M, with the maximal effect seen at about 1O-8 M (138% of control). The stimulatory effects were also seen in hASMC obtained from adult (data not shown). In contrast, DHEA did not affect the mitogenesis of A10 cells. Effect of DHEA pretreatment on proliferation of hASMC and Al 0 cell in presence of growth stimulants The BrdU incorporation for four hrs observed in hASMC in SGM was about lo-fold greater than that in SBM. FCS-induced mitogenesis was also seen in A10 cells. DHEA inhibited
Vol. 60, No. 11, 1997
83.5
Effects of DHEA on Proliferation of VSMC
control
-6
-9
-7
DHEA amcen~ation
Fig. 1 Stimulatory effect of DHEA on mitogenesis incubated in the absence of growth factors.
-6
-5
(Log M)
in smooth
muscle
cells
Monolayers of quiescent hASMC and A10 cells were incubated in medium (hASMC: S-BM; A10 cells: DMEM) with various concentrations of DHEA at 37°C for 14 or 12 hrs, respectively. Thereafter, BrdU incorporation into DNA was assessed. Each data point represents mean?SD for three determinations increase Closed circles: hASMC. Open circles: Al0 cells. over control. *: p < 0.01 compared to control.
control
-10
-9
-6
DHEA concentration
Fig. 2 Inhibitory effect of DHEA on mitogenesis incubated in the absence of growth factors. Monolayers of quiescent hASMC and A10 (hASMC: S-GM, A10 cells: FCSDMEM) with 37°C for 14 or 12 hrs, respectively. Thereafter, Each data point represents mean t SD of control. Closed circles: hASMC. Open circles: *: p < 0.01 compared to control.
-7
-6
(Log M)
in smooth
muscle
cells
cells were incubated in medium various concentrations of DHEA at BrdU incorporation was assessed. three determinations increase over A10 cells.
836
Vol. 60, No. 11, WY7
Effects of DHEA on Proliferation of VSMC
control
2 DHEA
4
a
24
prekeatment time (hours)
Fig. 3 Pretreatment time of DHEA and attenuation of hASMC mitogenesis Monolayers of quiescent hASMC were incubated in S-BM with DHEA of 10-8 M for various durations. Media was exchanged to S-GM and after 14-hr incubation, cell proliferation was assayed Each data point represents mean?SD of three determinations increase over control. *: ~~0.01 compared with control.
S-GM stimulated BrdU incorporation in hASMC. The inhibitory effect was dose-dependent at DHEA concentrations ranging from 10. 10 M to 10-6 M as shown in Figure 2. The growing rate was 52% of control at 10-s M DHEA and 33% at 1O-6 M DHEA. The inhibitory effect was also seen in hASMC obtained from adult (data not shown). In contrast, the inhibitory effects on mitogenesis observed in hASMC were not seen in A10 cells. There was no increase in cell death when hASMC were incubated with DHEA (1O-4 M) for up to 48 hrs, and the growth of hASMC completely recovered after removal of DHEA. Effect of pretreatment with DHEA on proliferation of hASMC An inhibitory effect on the mitogenesis of hASMC was observed at more than 4 hrs DHEA pretreatment time, but not with only 2 hrs of pretreatment time, as shown in Figure 3. Effects of DHEAS on DNA synthesis DHEAS did not stimulate the BrdU incorporation into DNA of hASMC, which was a different finding observed in the addition of DHEA alone. Furthermore, DHEAS at concentrations ranging from 1O-9M up to 10m5M did not attenuate the FCS-induced mitogenesis of hASMC (data not shown).
This is the first report that DHEA influences the growth of hASMC in vitro. The inhibitory action of DHEA on mitotic activity of other cultured cells has been reported in great detail by several authors. This effect has been demonstrated in fibroblast (1 l), T-lymphocytes (12) normal hepatocytes and hepatocarcinoma cells (13). The effects of DHEA on VSMC growth are complicated, based on our finding of distinctive growth responses to DHEA based on the presence or absence of agents that stimulate growth. DHEA alone stimulated the growth of VSMC in serum-free culture. The range of DHEA concentrations stimulating the growth of VSMC in the absence of growth factors is from 10m8M to 10-G M. Several clinical studies have reported that serum DHEAS levels are inversely correlated to mortality from cardiovascular disease in males (1,2,3). Our findings in vitro seem
Vol. 60, No. 11, 1997
Effects of DHEA on Proliferation of VSMC
MI
at odds with these clinical reports. However, there are several reports concerning differences in the growth responses to agonists between in vitro and in vivo studies. We also reported that opposite effects of growth regulatory peptides on the growth of human epidermoid carcinoma cells from comparing in vitro and in vivo studies (14). Therefore, the in vitro findings observed in the present study are not necessarily incompatible with previous clinical findings (1,2,3). On the other hand, in the present study DHEA attenuated the growth of hASMC induced by the addition of FCS which contains various growth mediators, EGF and bFGF. The effects of DHEA were found only at DHEA concentrations greater than 10-9 M. The plasma DHEA concentration of healthy adult male is about 10-s M, so the DHEA levels used in our study are The required pretreatment time for DHEA was similar to human physiologic concentrations. more than 4 hrs, which is similar to a report concerning the effects of glucocorticoids on VSMC (15). It is well established that glucocorticoid and its receptor complex binds to the specific element of DNA. Because of this, we believe that DHEA affects hASMC through the binding to DNA. DHEAS is the major secretory product of the human adrenal gland. The human plasma concentration of DHEAS is in the range of lO-‘j M to 1O-5 M and is about thousand times greater than the concentration of DHEA. In our studies, DHEAS had no effect on hASMC growth regulation in either the presence or absence of growth stimulating agents. In the light of the present findings, hASMC may have no receptor of DHEAS and/or no phenol sulfotransferase to convert DHEAS to DHEA. In contrast to hASMC, we found that DHEA did not affect the growth of rat vasculer smooth muscle cells either in the presence or absence of FCS. The concentrations of DHEA and DHEAS in rat serum are much lower than in humans (16), so the physiologic role of DHEA may be very different in rat compared to human. While it should be determined whether DHEA can affect VSMC obtained from other species, the regulatory effects of DHEA on VSMC growth may be specific to humans. In conclusion, the growth response of human VSMC to DHEA in vitro was modulated by the presence of other growth factors in the culture. Further examination should be performed to determine the mechanisms by which DHEA affects the growth of hASMC in vitro. These findings should help shed light on the role of DHEA in anti-atherogenesis in vivo.
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