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Growth of human aortic smooth muscle cells cultured with human serum is retarded when serum lipids are lowered by medroxyprogesterone
Atherosclerosis, 61 (1987) 223-228 Elsevier Scientific Publishers Ireland,
223 Ltd.
ATH 04003
Growth of human aortic smooth muscle cells cultured with human serum is retarded when serum lipids are lowered by medroxyprogesterone T. Rijnnemaa, H. J;irvel&nen, A. Lehtonen, M. Grijnroos, J. Mamiemi and A. Rautio Rehabilitation Research Centre of the Social Insurance Institution, 20720 Turku 72, Departments of Medical Chemistry, Medicine, and Obstetrics and Gynecology, University of Turku, 20520 Turku 52, Department of Pharmacology, University of Oulu, 90220 Oulu 22 (Finland) (Received 2 December, 1986) (Revised, received 1 April 1987) (Accepted 8 April, 1987)
Human aortic smooth muscle cells were cultured in the presence of sera from 7 normolipidemic women before and after treatment with high-dose medroxyprogesterone acetate, which caused 16% and 25% decreases in serum cholesterol and HDL-cholesterol concentrations, respectively. As assessed by cell counting and by DNA determination the growth of the cells was retarded significantly in the presence of sera taken after the treatment. At the same time, there were no marked changes in the incorporation rate of [ 3H]proline into collagen or [ 3H]glucosamine into hyaluronic acid by the cells. The results indicate that: (1) the mitogenicity of human serum can be altered by drug treatment of serum donors, (2) simultaneously with a lowering of serum lipids in man in vivo, a decreased mitogenicity of sera occurs in vitro.
Key words: Cell proliferation; muscle cells
Collagen; Hyaluronic
Introduction
High concentration of serum cholesterol and low concentration of high density lipoprotein
Tbis study was supported by a research grant from the Medical Council of the Academy of Finland. Correspondence to: T. Romremaa, M.D., Rehabilitation Research Centre of the Social Insurance Institution 20720 Turku 72, Finland. 0021-9150/87/$03.50
0 1987 Elsevier
Scientific
Publishers
Ireland,
acid; Medroxyprogesterone;
Serum lipids; Smooth
(HDL) cholesterol are well-known risk factors for coronary heart disease [l]. Much research has been focused on the factors that affect the concentration of these lipids. However, it is poorly known what happens in arterial cells of an individual when the concentration of his serum lipoproteins changes due to various manipulations. One approach to the problem is via arterial cell cultures. The situation in vivo can be mimicked by adding to the cultures isolated lipoprotein fracLtd.
224
tions at various concentrations. Another and perhaps a more physiological way is to culture the cells with sera taken from individuals before and after manipulations affecting their serum lipids. Medroxyprogesterone acetate (MPA) is a widely used progestin in gynecology. At high doses it causes a drastic decrease in serum cholesterol and especially in HDL-cholesterol [2]. In this study we wanted to determine whether the change in serum lipids caused by MPA is associated with changes in the effects of the sera on some functions of cultured human aortic smooth muscle cells (HSMCs) that have been considered to be important for atherogenesis. The functions studied included proliferation, and synthesis of two extracellular matrix components, collagen and hyaluronic acid. Materials and methods Patients
Seven postmenopausal women (mean age 66 _+ 10 years) with endometrial cancer received medroxyprogesterone acetate (Lutopolar”, Medipolar, Finland) perorally 100 mg daily. Fasting blood samples were drawn before and after 2 and 14 days of treatment. Sera for cell cultures were prepared conventionally, sterilized, heat-inactivated at 56 o C for 30 min, and stored frozen at - 75 o C until use. During progestin treatment the patients received no other therapy for endometrial cancer. Cells
Smooth muscle cells (SMCs) were isolated from the intima-media of the aorta of a 20-week-old human fetus using trypsin-collagenase digestion [3]. The cells were cultured at 37 o C in 95% sir/5% CO, in Dulbecco’s MEM (Flow Laboratories, Irvine, U.K.), pH 7.3, containing 23 mmol/l sodium bicarbonate, 20 mmol/l Hepes, 50 pg/ml streptomycin sulphate, 100 units/ml penicillin and 10% fetal calf serum (FCS, Flow). Cells from the 7th to the 8th passage were used in the experiments. Cell proliferation
HSMCs were trypsinized and pipetted into plastic flasks with a 25-cm2 growth area (Nunc,
Roskilde, Denmark) at a density of about 6000 cells/cm’. After 24 h the old medium was sucked off and the cells were washed once with serum-free medium. Four ml of Dulbecco’s MEM without FCS but containing 15% test serum was added into the flasks. The medium was changed after 3 and 6 days. After 7 days the HSMCs were detached with 0.25% trypsin and aliquots of the cell suspension were taken for cell counting with a hemocytometer and for the determination of DNA 141. Incorporation of [3H]glucosamine
HSMCs were grown in cell culture tubes with a 5.5~cm2 growth area (Nunc) until confluent. The old medium was replaced by 2 ml of Dulbecco’s MEM without FCS but containing 15% test serum and 3 pCi of [ 3H]glucosamine (TRK 375, spec. act. 3.2 Ci/mmol, The Radiochemical Centre, Amersham, U.K.). After incubation for 24 h the 3H radioactivity incorporated into hyaluronic acid in the medium was measured after papain digestion [5]. Because serum lipoproteins might interfere with this analytical procedure [6], aliquots of the samples were also analyzed after delipidation [6]. Incorporation of [3H]proline
For the study of incorporation rate of [3H]proline into collagen the same HSMC cultures as for the proliferation measurements were used. At day 6, 10 PCi of L-[G-3H]proline (TRA 82, The Radiochemical Centre) plus 50 pg/ml ascorbate and 20 pg/ml proline were added to the cultures. After 24 h the medium was poured off, the cells were washed twice with saline containing 0.2% proline and the washings added to the medium. The samples were dialyzed against cold running tap water for 3 days, hydrolyzed, and analyzed for radioactive hydroxyproline [7], which was taken as the measure of [ 3H]proline incorporation rate into collagen. Serum lipids and plasma hormones
Serum cholesterol was determined enzymatitally (Nycotesta, Nyegaard & Co. A/S, Oslo, Norway). For the determination of HDLcholesterol and triglycerides plasma lipids were extracted with chloroform/methanol (1 : 1, v/v)
225 TABLE
[8]. The extracts were washed [9] and triglycerides and HDL-cholesterol were determined as described earlier [lO,ll]. Concentrations of plasma insulin (Novo Industri antiserum), growth hormone (Sorin Biomedica antiserum) and somatomedin-C (Nichols Institute Diagnostics, Los Angeles, U.S.A.) were estimated by radioimmunoassays.
2
EFFECT OF MEDROXYPRGGESTERONE ACETATE ON FASTING PLASMA INSULIN, GROWTH HORMONE AND SOMATOMEDIN-C CONCENTRATIONS Mean + SD, n = 7. Initial
acetate
(/%/l)
6.1
12.3+
5.3
12.7k
5.7
2.1 f
1.7
1.5+
0.5
0.9*
0.1
Somatomedin-C 590
(U/l)
f230
490
All differences between ment are non-significant
Results
Mean plasma MPA was 2.1 pg/l after 2 days’ and 3.5 pg/l after 14 days of treatment (Table 1). At day 2 serum total cholesterol and HDLcholesterol concentrations were both decreased by 7% and at day 14 by 16% and 25%, respectively. The ratio of HDL-cholesterol to serum cholesterol was slightly but significantly lower at day 14 than initially. The concentration of triglycerides was not affected by MPA treatment, and neither were mean fasting plasma insulin and somatomedin-C concentrations by MPA treatment (Table 2). Mean fasting plasma growth hormone concentration was decreased during MPA treatment but the decrease was not statistically significant (paired t-test). As compared to sera taken before MPA treat-
f270
520
initial values and values (paired r-test).
+250 after
treat-
ment sera taken after 14 days’ treatment retarded the growth of HSMCs significantly, by 20% (Fig. 1). This was shown both by cell number calculations and by DNA determinations. The retardation in HSMC growth was seen both in relatively rapidly growing cultures (Fig. 1, Exp. 2) and in more slowly growing cultures (Fig. 1, Exp. 1). The various sera had no toxic effect on HSMCs as judged by phase-contrast microscopy. MPA itself had no effect on the proliferation of HSMCs when added directly into cell culture flasks (Fig. 2). Sera taken at the various points in time of MPA treatment did not differ in their effects on the incorporation rate of [3H]glucosamine into hyaluronic acid by HSMCs (Fig. 3). The incorporation rate of [ 3Hlproline into collagen (calculated per DNA) by HSMCs was sig-
1
CONCENTRATION Means
12.5*
Growth hormone
Plasma MPA was measured by radioimmunoassay (12). The direct effect of MPA on cell proliferation was studied by adding MPA (DepoProvera, Upjohn Company, Michigan, U.S.A.) at various concentrations to the culture medium containing 15% pooled human female serum. The experimental conditions were the same as described in the section ‘cell proliferation’.
TABLE
After 14 days
Insulin (mu/l)
Medroxyprogesterone
After two days
*SD,
OF SERUM
LIPIDS
AND
MEDROXYPROGESTERONE
(MPA)
IN WOMEN
TREATED
WITH
n = 7.
FS-cholesterol (mmol/l) FS-HDL-cholesterol (mmol/l) FS-HDL-cholesterol/F%cholesterol FS-triglycerides (mmol/l) FS-MPA @g/l) * P c 0.05, * * * P < 0.001 as compared
Initial
After two days
After 14 days
5.72 f 1.15 1.02 kO.27 0.18&-0.03 2.21+ 0.87 0
5.32 f 0.91 * 0.95 f 0.24 * 0.18 f 0.03 1.96kO.76 2.11 f 1.38
4.78 kO.76 * * * 0.77 f 0.24 * * * 0.16 +0.03 * 2.04kO.89 3.54+ 2.41
to the values before
treatment
(paired
t-test)
MPA
226 CELL
NUMBER EXP n=7
x lo-‘/FLASK
1
EXP n=6
DNA
pg/FLASK
2
1L
NO
EXP2 n=5
DELIPIDATION
DPM x ldL
DELIPIDATION
/TUBE
rlL
F 12
DPM x 1O-L /TUBE
10
l_-stL * +
a
*
6
L
2
0 21.4
TIME
AFTER
0 2 1L
0 2 1c
STARTING
TREATMENT
0 2 1L
(DAYS)
Fig. 1. Proliferation of human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means+ SD. Expts. 1 and 2 were performed similarly but the cells in the 2 experiments were derived from different batches containing cells with a relatively slow (Expt. 1) or rapid (Expt. 2) growth rate. * P < 0.05, * * *** P -c0.001 as compared to values before treatP < 0.01, ment (paired r-test).
TIME
AFTER
TREATMENT
DPMxlO-‘/ FLASK (n=6)
OPMxlO-‘1 vg DNA (n-5) *
r6
I 5 L 3
1 DNA pg /FLASK
:
0 2 IL TIME
0 2 1L AFTER
+ I iI Y
COLLAGEN
PROTEINS
2 CELL NUMBER x lO-5/FLASK
(DAYS)
Fig. 3. Incorporation rate of [ 3H]glucosamine into hyaluronic acid by human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means+SD of values of 7 women. See Material and Methods for the reason for delipidation.
ALL
nificantly higher in the presence of sera taken after 2 weeks treatment than in the presence of sera taken initially (Fig. 4). There was, however, no difference when the incorporation rate was calculated per culture flask. Because almost all radioactivity in the proteins secreted into the medium was found in collagen, the incorporation
0 2 1L
STARTING
STARTING
r6
DPMxlO-; FLASK (n =6)
0 21.4
TREATMENT
DPMxlO-l/ i.q DNA (n = 5) +
0 21L
(DAYS)
Fig. 4. Incorporation rate of [3H]proline into collagen and other proteins by human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means + SD. * P < 0.05as compared to values before treatment (paired f-test).
00.51 CONCENTRATION ()lg/Ll
510
0 05
1
5 10
OF MEDROXYPROGESTERONE
Fig. 2. The direct effect of medroxyprogesterone on the pro liferation of human aortic smooth muscle cells in culture. Bars represent means + SD of 3 replicates. For details of experimental procedure, see Material and Methods.
rate of [ 3H]proline into non-collagenous could not be determined reliably.
proteins
Discussion
of
Increased proliferation of arterial SMCs is one the key events in the pathogenesis of
227 atherosclerosis [13]. Several factors affect the proliferation of these cells in culture [14,15], including hyperlipidemic serum, various lipoprotein fractions, diabetic serum, insulin, growth hormone, somatomedins, platelet-derived growth factor (PDGF), endothelial cell-derived growth factor, macrophage-derived growth factor and epidermal growth factor (EGF). Most of the growth-promoting activity of normal serum is known to reside in the non-lipoprotein fraction of serum, PDGF being the most potent of the various growth factors [16]. Other serum mitogens appear to act in a coordinated fashion with PDGF [14,17]. Low density lipoproteins (LDL) have been shown to act as mitogens for aortic SMCs from monkeys [18]. Both LDL and HDL are mitogenic for cultured swine [19] and bovine aortic SMCs [20]. There are no studies concerning the effect of human lipoproteins on the proliferation of human vascular cells in culture and the results of studies on the effect of human hypercholesterolemic serum on the growth of HSMCs are conflicting [21-231. The apparent weakness in these studies has been their cross-sectional nature as they have not examined the mitogenicity of sera from the same individuals at various time points and various cholesterol concentrations. Recently, we have shown that the growth-promoting effect of human serum on HSMCs is a constant donor-dependent property since mitogenicity was similar in sera taken at 3-week intervals from the same subjects [24]. The main result of the present study was the finding that the mitogenicity of human sera can be altered by manipulation of the serum donors in vivo, in this case by treatment with MPA. However, it remains to be established whether a reduced mitogenicity of serum in vitro is associated with a slower rate of atherogenesis in vivo. The reduction in cell growth during MPA treatment in our study could not be imputed to MPA itself since MPA added directly to the culture medium had no effect on cell proliferation. The effect of MPA, therefore, must be mediated by its effect on the composition of human serum. MPA caused decreases in serum LDL- and HDLcholesterol concentrations, as has been shown earlier by Grijnroos and Lehtonen [2], and it also caused a decrease in plasma growth hormone level;
however, it had no effect on plasma insulin and somatomedin-C levels. In our recent study we have shown that growth hormone at concentrations of l-10 pg/l does not affect the proliferation of HSMCs in culture [25]. Therefore, the serum cholesterol-lowering effect of MPA treatment is a possible mechanism by which MPA treatment reduces the mitogenicity of human serum. This suggestion is supported by the fact that cholesterol, as a major component of cell membranes, is known to play an important role in cell proliferation [26]. Of course, it cannot be ruled out that the reduced mitogenicity of serum after MPA treatment is caused by the effect of MPA on the growth factors not determined in this study, e.g., growth factors of platelet origin, transforming growth factor-beta and EGF. Unfortunately, no assays for these growth factors were available at the time of this study. The synthesis of collagen is increased in the early stage of atherosclerosis [27]. Hyperlipidemic serum does not affect the synthesis of collagen by rat [3], rabbit [28] or human [22] aortic SMCs in culture. In the present study there was no difference in the incorporation rate of [3H]proline into collagen per culture in the presence of the various sera strengthening the idea that the synthesis of collagen by cultured vascular cells is not dependent on the concentration of serum lipoproteins in the culture medium. Atherosclerosis is associated with an increase in the synthesis of sulphated glycosaminoglycans and a decrease in the relative rate of hyaluronate synthesis [29]. Human hypercholesterolemic serum decreases the synthesis of hyaluronate by HSMCs in culture [21]. In similar cell culture conditions in the present study no variation could be observed in the incorporation rate of [3H]glucosamine into hyaluronic acid in the presence of sera before and after MPA treatment. This suggests that moderate reduction of serum lipids in normolipidemic subjects has no harmful effect on the synthesis of glycosaminoglycans in respect to atherosclerosis. Our results show that changes in serum lipids are associated with alterations in the effects of the sera on the proliferation of cultured arterial SMCs. As the changes in lipids were produced by MPA, further studies in man using other means of manipulating serum lipids (e.g., changing the com-
228 position of diet), as has already been done in monkeys [30], are needed to evaluate the more general relevance of the present results. 18
References Gordon, T., Kannel, W.B., Castelli, W.P. and Dawber, T.R., Lipoproteins, cardiovascular disease and death. The Framingham Study, Arch. Intern. Med., 141 (1981) 1128. Grtinroos, M. and Lehtonen, A., Effect of high dose progestin on serum lipids, Atherosclerosis, 47 (1983) 101. Riinnemaa, T. and Doherty, N.S., Effect of serum and liver extracts from hypercholesterolemic rats on the synthesis of collagen by isolated aortas and cultured aortic smooth muscle cells, Atherosclerosis, 26 (1977) 261. Jalkanen, M., Larjava, H., Turakainen, H. and Tammi, M., Improved application of the diphenylamine reaction for the determination of DNA, J. B&hem. Biophys. Methods, 3 (1980) 195. Saarni, H. and Tammi, M., A rapid method for separation and assay of radiolabelled mucopolysaccharides from cell culture medium, Anal. Biochem., 81 (1977) 40. Pietill, K. and Nikkari, T., Hyperlipidemic rabbit serum reduces recovery of acidic glycosaminoglycans from tissue culture medium, Anal. B&hem., 116 (1981) 317. Juva, K. and Prockop, D.J., Modified procedure for the assay of 3H- or r4C-labeled hydroxyproline, Anal. Biothem., 15 (1966) 77. 8 Folch, J., Lees, M. and Sloane Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 9 Svennerholm, L., Distribution and fatty acid composition of phosphoglycerides in normal human brain, J. Lipid Res., 9 (1968) 570. 10 Carlson, L.A., Determination of serum triglycerides, J. Atheroscler. Res., 3 (1963) 334. 11 Viikari, J., Precipitation of plasma lipoproteins by PEG6000 and its evaluation with electrophoresis and ultracentrifugation, Stand. J. Clin. Lab. Invest., 36 (1976) 265. 12 Rautio, A., Liver function and medroxyprogesterone acetate elimination in man, Biomed. Pharmacother., 38 (1984) 199. 13 Ross, R. and Glomset, J.A., Atherosclerosis and the arterial smooth muscle cell, Science, 180 (1973) 1332. 14 Charnley-Campbell, J., Campbell, G.R. and Ross, R., The smooth muscle cell in culture, Physiol. Rev. 59 (1979) 1. 15 Ross, R., Atherosclerosis: a problem of the biology of arterial wall cells and their interactions with blood components, Arteriosclerosis, 1 (1981) 293. 16 Ross, R., Glomset, J., Kariya, B. and Harker, L., A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro, Proc. Natl. Acad. Sci. USA, 71 (1974) 1207. 17 Assoian, R.K., Grotendorst, G.R., Miller, D.M. and Spom,
19
20
21
22
23
24
25
26 27 28
29
30
M.B., Cellular transformation by coordinated action of three peptide growth factors from human platelets, Nature, 309 (1984) 804. Fischer-Dzoga, K. and Wissler, R.W., Stimulation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2 (Effects of varying concentrations of hyperlipemic serum and low density lipoproteins of varying fat origins), Atherosclerosis, 24 (1976) 515. Brown, B.C., Mahley, R. and Assmann, G., Swine aortic smooth muscle in tissue culture. Some effects of purified swine lipoproteins on cell growth and morphology, Circ. Res., 39 (1976) 415. Gospodarowicz, D., Hirabayashi, K., Gig&e, L. and Tauber, J.-P., Factors controlling the proliferative rate, final cell density, and life span of bovine vascular smooth muscle cells in culture, J. Cell. Biol. 89 (1981) 568. Jibvel&inen, H., Rannemaa, T., Tammi, M., Vihersaari, T., Lehtonen, A. and Viikari, J., Type IIA hyperlipoproteinemic sera decrease the synthesis of hyaluronic acid by cultured human aortic smooth muscle cells, Atherosclerosis, 39 (1981) 61. JZrveltien, H., Halme, T., Lehtonen, A. and Ronnemaa, T., Serum from type IIA hyperlipoproteinemic patients does not stimulate proliferation of and collagen synthesis in human fetal aortic smooth muscle cells in culture, Atherosclerosis, 56 (1985) 199. Koschinsky, T., Btinting, C.E., RiJtter, R. and Gries, F.A., Increased growth stimulation of human vascular cells by serum from patients with primary hyper-LDL-cholesterolemia, Atherosclerosis, 63 (1987) 7. Jarvel~en, H., Peltonen, J. and Riinnemaa, T., The growth-promoting effect of human whole blood serum on cultured human aortic smooth muscle cells is a constant serum donor-dependent property, In Vitro, 22 (1986) 515. Jlrveliiinen, H., Rtinnemaa, T. and Lehtonen, A., Effect of sera from male type 1 (insulin-dependent) diabetics on human aortic smooth muscle cells in culture, Acta Endocrinol., 114 (1987) 362. Chen, H.W., Role of cholesterol metabolism in cell growth, Fed. Proc., 43 (1984) 126. Lindner, J., Injury and repair of arterial tissue. Histochemical alterations, Angiology, 25 (1974) 628, 672. Pietill, K. and Nikkari, T., Effect of phase of growth and hyperlipidemic serum on the synthesis of collagen in rabbit aortic smooth muscle cells in culture, Med. Biol., 56 (1978) 11. Tammi, M., SeppBlB, P.O., Lehtonen, A. and Mottiinen, M., Connective tissue components in normal and atherosclerotic human coronary arteries, Atherosclerosis, 29 (1978) 191. Fless, G.M., Fischer-Dzoga, K., J&n, D.J., Bates, S. and Scanu, A.M., Structural and functional changes of Rhesus serum low density lipoproteins during cycles of diet-induced hypercholesterolemia, Arteriosclerosis, 2 (1982) 475.
223 Ltd.
ATH 04003
Growth of human aortic smooth muscle cells cultured with human serum is retarded when serum lipids are lowered by medroxyprogesterone T. Rijnnemaa, H. J;irvel&nen, A. Lehtonen, M. Grijnroos, J. Mamiemi and A. Rautio Rehabilitation Research Centre of the Social Insurance Institution, 20720 Turku 72, Departments of Medical Chemistry, Medicine, and Obstetrics and Gynecology, University of Turku, 20520 Turku 52, Department of Pharmacology, University of Oulu, 90220 Oulu 22 (Finland) (Received 2 December, 1986) (Revised, received 1 April 1987) (Accepted 8 April, 1987)
Human aortic smooth muscle cells were cultured in the presence of sera from 7 normolipidemic women before and after treatment with high-dose medroxyprogesterone acetate, which caused 16% and 25% decreases in serum cholesterol and HDL-cholesterol concentrations, respectively. As assessed by cell counting and by DNA determination the growth of the cells was retarded significantly in the presence of sera taken after the treatment. At the same time, there were no marked changes in the incorporation rate of [ 3H]proline into collagen or [ 3H]glucosamine into hyaluronic acid by the cells. The results indicate that: (1) the mitogenicity of human serum can be altered by drug treatment of serum donors, (2) simultaneously with a lowering of serum lipids in man in vivo, a decreased mitogenicity of sera occurs in vitro.
Key words: Cell proliferation; muscle cells
Collagen; Hyaluronic
Introduction
High concentration of serum cholesterol and low concentration of high density lipoprotein
Tbis study was supported by a research grant from the Medical Council of the Academy of Finland. Correspondence to: T. Romremaa, M.D., Rehabilitation Research Centre of the Social Insurance Institution 20720 Turku 72, Finland. 0021-9150/87/$03.50
0 1987 Elsevier
Scientific
Publishers
Ireland,
acid; Medroxyprogesterone;
Serum lipids; Smooth
(HDL) cholesterol are well-known risk factors for coronary heart disease [l]. Much research has been focused on the factors that affect the concentration of these lipids. However, it is poorly known what happens in arterial cells of an individual when the concentration of his serum lipoproteins changes due to various manipulations. One approach to the problem is via arterial cell cultures. The situation in vivo can be mimicked by adding to the cultures isolated lipoprotein fracLtd.
224
tions at various concentrations. Another and perhaps a more physiological way is to culture the cells with sera taken from individuals before and after manipulations affecting their serum lipids. Medroxyprogesterone acetate (MPA) is a widely used progestin in gynecology. At high doses it causes a drastic decrease in serum cholesterol and especially in HDL-cholesterol [2]. In this study we wanted to determine whether the change in serum lipids caused by MPA is associated with changes in the effects of the sera on some functions of cultured human aortic smooth muscle cells (HSMCs) that have been considered to be important for atherogenesis. The functions studied included proliferation, and synthesis of two extracellular matrix components, collagen and hyaluronic acid. Materials and methods Patients
Seven postmenopausal women (mean age 66 _+ 10 years) with endometrial cancer received medroxyprogesterone acetate (Lutopolar”, Medipolar, Finland) perorally 100 mg daily. Fasting blood samples were drawn before and after 2 and 14 days of treatment. Sera for cell cultures were prepared conventionally, sterilized, heat-inactivated at 56 o C for 30 min, and stored frozen at - 75 o C until use. During progestin treatment the patients received no other therapy for endometrial cancer. Cells
Smooth muscle cells (SMCs) were isolated from the intima-media of the aorta of a 20-week-old human fetus using trypsin-collagenase digestion [3]. The cells were cultured at 37 o C in 95% sir/5% CO, in Dulbecco’s MEM (Flow Laboratories, Irvine, U.K.), pH 7.3, containing 23 mmol/l sodium bicarbonate, 20 mmol/l Hepes, 50 pg/ml streptomycin sulphate, 100 units/ml penicillin and 10% fetal calf serum (FCS, Flow). Cells from the 7th to the 8th passage were used in the experiments. Cell proliferation
HSMCs were trypsinized and pipetted into plastic flasks with a 25-cm2 growth area (Nunc,
Roskilde, Denmark) at a density of about 6000 cells/cm’. After 24 h the old medium was sucked off and the cells were washed once with serum-free medium. Four ml of Dulbecco’s MEM without FCS but containing 15% test serum was added into the flasks. The medium was changed after 3 and 6 days. After 7 days the HSMCs were detached with 0.25% trypsin and aliquots of the cell suspension were taken for cell counting with a hemocytometer and for the determination of DNA 141. Incorporation of [3H]glucosamine
HSMCs were grown in cell culture tubes with a 5.5~cm2 growth area (Nunc) until confluent. The old medium was replaced by 2 ml of Dulbecco’s MEM without FCS but containing 15% test serum and 3 pCi of [ 3H]glucosamine (TRK 375, spec. act. 3.2 Ci/mmol, The Radiochemical Centre, Amersham, U.K.). After incubation for 24 h the 3H radioactivity incorporated into hyaluronic acid in the medium was measured after papain digestion [5]. Because serum lipoproteins might interfere with this analytical procedure [6], aliquots of the samples were also analyzed after delipidation [6]. Incorporation of [3H]proline
For the study of incorporation rate of [3H]proline into collagen the same HSMC cultures as for the proliferation measurements were used. At day 6, 10 PCi of L-[G-3H]proline (TRA 82, The Radiochemical Centre) plus 50 pg/ml ascorbate and 20 pg/ml proline were added to the cultures. After 24 h the medium was poured off, the cells were washed twice with saline containing 0.2% proline and the washings added to the medium. The samples were dialyzed against cold running tap water for 3 days, hydrolyzed, and analyzed for radioactive hydroxyproline [7], which was taken as the measure of [ 3H]proline incorporation rate into collagen. Serum lipids and plasma hormones
Serum cholesterol was determined enzymatitally (Nycotesta, Nyegaard & Co. A/S, Oslo, Norway). For the determination of HDLcholesterol and triglycerides plasma lipids were extracted with chloroform/methanol (1 : 1, v/v)
225 TABLE
[8]. The extracts were washed [9] and triglycerides and HDL-cholesterol were determined as described earlier [lO,ll]. Concentrations of plasma insulin (Novo Industri antiserum), growth hormone (Sorin Biomedica antiserum) and somatomedin-C (Nichols Institute Diagnostics, Los Angeles, U.S.A.) were estimated by radioimmunoassays.
2
EFFECT OF MEDROXYPRGGESTERONE ACETATE ON FASTING PLASMA INSULIN, GROWTH HORMONE AND SOMATOMEDIN-C CONCENTRATIONS Mean + SD, n = 7. Initial
acetate
(/%/l)
6.1
12.3+
5.3
12.7k
5.7
2.1 f
1.7
1.5+
0.5
0.9*
0.1
Somatomedin-C 590
(U/l)
f230
490
All differences between ment are non-significant
Results
Mean plasma MPA was 2.1 pg/l after 2 days’ and 3.5 pg/l after 14 days of treatment (Table 1). At day 2 serum total cholesterol and HDLcholesterol concentrations were both decreased by 7% and at day 14 by 16% and 25%, respectively. The ratio of HDL-cholesterol to serum cholesterol was slightly but significantly lower at day 14 than initially. The concentration of triglycerides was not affected by MPA treatment, and neither were mean fasting plasma insulin and somatomedin-C concentrations by MPA treatment (Table 2). Mean fasting plasma growth hormone concentration was decreased during MPA treatment but the decrease was not statistically significant (paired t-test). As compared to sera taken before MPA treat-
f270
520
initial values and values (paired r-test).
+250 after
treat-
ment sera taken after 14 days’ treatment retarded the growth of HSMCs significantly, by 20% (Fig. 1). This was shown both by cell number calculations and by DNA determinations. The retardation in HSMC growth was seen both in relatively rapidly growing cultures (Fig. 1, Exp. 2) and in more slowly growing cultures (Fig. 1, Exp. 1). The various sera had no toxic effect on HSMCs as judged by phase-contrast microscopy. MPA itself had no effect on the proliferation of HSMCs when added directly into cell culture flasks (Fig. 2). Sera taken at the various points in time of MPA treatment did not differ in their effects on the incorporation rate of [3H]glucosamine into hyaluronic acid by HSMCs (Fig. 3). The incorporation rate of [ 3Hlproline into collagen (calculated per DNA) by HSMCs was sig-
1
CONCENTRATION Means
12.5*
Growth hormone
Plasma MPA was measured by radioimmunoassay (12). The direct effect of MPA on cell proliferation was studied by adding MPA (DepoProvera, Upjohn Company, Michigan, U.S.A.) at various concentrations to the culture medium containing 15% pooled human female serum. The experimental conditions were the same as described in the section ‘cell proliferation’.
TABLE
After 14 days
Insulin (mu/l)
Medroxyprogesterone
After two days
*SD,
OF SERUM
LIPIDS
AND
MEDROXYPROGESTERONE
(MPA)
IN WOMEN
TREATED
WITH
n = 7.
FS-cholesterol (mmol/l) FS-HDL-cholesterol (mmol/l) FS-HDL-cholesterol/F%cholesterol FS-triglycerides (mmol/l) FS-MPA @g/l) * P c 0.05, * * * P < 0.001 as compared
Initial
After two days
After 14 days
5.72 f 1.15 1.02 kO.27 0.18&-0.03 2.21+ 0.87 0
5.32 f 0.91 * 0.95 f 0.24 * 0.18 f 0.03 1.96kO.76 2.11 f 1.38
4.78 kO.76 * * * 0.77 f 0.24 * * * 0.16 +0.03 * 2.04kO.89 3.54+ 2.41
to the values before
treatment
(paired
t-test)
MPA
226 CELL
NUMBER EXP n=7
x lo-‘/FLASK
1
EXP n=6
DNA
pg/FLASK
2
1L
NO
EXP2 n=5
DELIPIDATION
DPM x ldL
DELIPIDATION
/TUBE
rlL
F 12
DPM x 1O-L /TUBE
10
l_-stL * +
a
*
6
L
2
0 21.4
TIME
AFTER
0 2 1L
0 2 1c
STARTING
TREATMENT
0 2 1L
(DAYS)
Fig. 1. Proliferation of human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means+ SD. Expts. 1 and 2 were performed similarly but the cells in the 2 experiments were derived from different batches containing cells with a relatively slow (Expt. 1) or rapid (Expt. 2) growth rate. * P < 0.05, * * *** P -c0.001 as compared to values before treatP < 0.01, ment (paired r-test).
TIME
AFTER
TREATMENT
DPMxlO-‘/ FLASK (n=6)
OPMxlO-‘1 vg DNA (n-5) *
r6
I 5 L 3
1 DNA pg /FLASK
:
0 2 IL TIME
0 2 1L AFTER
+ I iI Y
COLLAGEN
PROTEINS
2 CELL NUMBER x lO-5/FLASK
(DAYS)
Fig. 3. Incorporation rate of [ 3H]glucosamine into hyaluronic acid by human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means+SD of values of 7 women. See Material and Methods for the reason for delipidation.
ALL
nificantly higher in the presence of sera taken after 2 weeks treatment than in the presence of sera taken initially (Fig. 4). There was, however, no difference when the incorporation rate was calculated per culture flask. Because almost all radioactivity in the proteins secreted into the medium was found in collagen, the incorporation
0 2 1L
STARTING
STARTING
r6
DPMxlO-; FLASK (n =6)
0 21.4
TREATMENT
DPMxlO-l/ i.q DNA (n = 5) +
0 21L
(DAYS)
Fig. 4. Incorporation rate of [3H]proline into collagen and other proteins by human aortic smooth muscle cells cultured with sera from women treated with medroxyprogesterone. Bars represent means + SD. * P < 0.05as compared to values before treatment (paired f-test).
00.51 CONCENTRATION ()lg/Ll
510
0 05
1
5 10
OF MEDROXYPROGESTERONE
Fig. 2. The direct effect of medroxyprogesterone on the pro liferation of human aortic smooth muscle cells in culture. Bars represent means + SD of 3 replicates. For details of experimental procedure, see Material and Methods.
rate of [ 3H]proline into non-collagenous could not be determined reliably.
proteins
Discussion
of
Increased proliferation of arterial SMCs is one the key events in the pathogenesis of
227 atherosclerosis [13]. Several factors affect the proliferation of these cells in culture [14,15], including hyperlipidemic serum, various lipoprotein fractions, diabetic serum, insulin, growth hormone, somatomedins, platelet-derived growth factor (PDGF), endothelial cell-derived growth factor, macrophage-derived growth factor and epidermal growth factor (EGF). Most of the growth-promoting activity of normal serum is known to reside in the non-lipoprotein fraction of serum, PDGF being the most potent of the various growth factors [16]. Other serum mitogens appear to act in a coordinated fashion with PDGF [14,17]. Low density lipoproteins (LDL) have been shown to act as mitogens for aortic SMCs from monkeys [18]. Both LDL and HDL are mitogenic for cultured swine [19] and bovine aortic SMCs [20]. There are no studies concerning the effect of human lipoproteins on the proliferation of human vascular cells in culture and the results of studies on the effect of human hypercholesterolemic serum on the growth of HSMCs are conflicting [21-231. The apparent weakness in these studies has been their cross-sectional nature as they have not examined the mitogenicity of sera from the same individuals at various time points and various cholesterol concentrations. Recently, we have shown that the growth-promoting effect of human serum on HSMCs is a constant donor-dependent property since mitogenicity was similar in sera taken at 3-week intervals from the same subjects [24]. The main result of the present study was the finding that the mitogenicity of human sera can be altered by manipulation of the serum donors in vivo, in this case by treatment with MPA. However, it remains to be established whether a reduced mitogenicity of serum in vitro is associated with a slower rate of atherogenesis in vivo. The reduction in cell growth during MPA treatment in our study could not be imputed to MPA itself since MPA added directly to the culture medium had no effect on cell proliferation. The effect of MPA, therefore, must be mediated by its effect on the composition of human serum. MPA caused decreases in serum LDL- and HDLcholesterol concentrations, as has been shown earlier by Grijnroos and Lehtonen [2], and it also caused a decrease in plasma growth hormone level;
however, it had no effect on plasma insulin and somatomedin-C levels. In our recent study we have shown that growth hormone at concentrations of l-10 pg/l does not affect the proliferation of HSMCs in culture [25]. Therefore, the serum cholesterol-lowering effect of MPA treatment is a possible mechanism by which MPA treatment reduces the mitogenicity of human serum. This suggestion is supported by the fact that cholesterol, as a major component of cell membranes, is known to play an important role in cell proliferation [26]. Of course, it cannot be ruled out that the reduced mitogenicity of serum after MPA treatment is caused by the effect of MPA on the growth factors not determined in this study, e.g., growth factors of platelet origin, transforming growth factor-beta and EGF. Unfortunately, no assays for these growth factors were available at the time of this study. The synthesis of collagen is increased in the early stage of atherosclerosis [27]. Hyperlipidemic serum does not affect the synthesis of collagen by rat [3], rabbit [28] or human [22] aortic SMCs in culture. In the present study there was no difference in the incorporation rate of [3H]proline into collagen per culture in the presence of the various sera strengthening the idea that the synthesis of collagen by cultured vascular cells is not dependent on the concentration of serum lipoproteins in the culture medium. Atherosclerosis is associated with an increase in the synthesis of sulphated glycosaminoglycans and a decrease in the relative rate of hyaluronate synthesis [29]. Human hypercholesterolemic serum decreases the synthesis of hyaluronate by HSMCs in culture [21]. In similar cell culture conditions in the present study no variation could be observed in the incorporation rate of [3H]glucosamine into hyaluronic acid in the presence of sera before and after MPA treatment. This suggests that moderate reduction of serum lipids in normolipidemic subjects has no harmful effect on the synthesis of glycosaminoglycans in respect to atherosclerosis. Our results show that changes in serum lipids are associated with alterations in the effects of the sera on the proliferation of cultured arterial SMCs. As the changes in lipids were produced by MPA, further studies in man using other means of manipulating serum lipids (e.g., changing the com-
228 position of diet), as has already been done in monkeys [30], are needed to evaluate the more general relevance of the present results. 18
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