Atherosclerosis, 81 (1990) 191-198 Elsevier Scientific Publishers Ireland,
ATHERO
191 Ltd.
04442
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
Masanori Hamada *, Ichiro Nishio, Akira Baba, Kazuya Fukuda, Jirou Takeda, Masako Ura, Takuzo Hano, Masato Ku&ii and Yoshiaki Masuyama Division of Cardiology, Department of Medicine, Wakayama Medical College, Wakayama (Japan) (Received 17 April, 1989) (Revised, received 27 November, 1989) (Accepted 18 December, 1989)
Summary It is widely reported that cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) show enhanced proliferation compared with cells from Wistar-Kyoto rats (WKY). The present studies were designed to find out whether this exaggerated proliferation in SHR is determined genetically and, if so, to evaluate the mechanism on the cell cycle. (1) Incorporation of [3H]thymidine into DNA was enhanced in CVSMCs from 3- and 12-week-old SHR compared with WKY but not in CVSMCs from DOCA-salt hypertensive rats compared with the cells from sham-operated rats. (2) DNA synthesis in SHR cells was enhanced further by addition of insulin (which is considered to be a progression factor) but not by arginine-vasopressin (AVP; considered to be a competence factor) or by angiotensin II (AII). On the other hand, insulin, AVP and AI1 significantly augmented DNA synthesis in WKY cells. (3) Intracellular free calcium concentration was slightly, but significantly, higher in SHR cells. (4) An increase in the population of DNA-synthesizing S-phase cells and decrease in (G2 + M)-phase cells in SHR were observed by flowcytometry. These data suggest (1) that enhanced DNA synthesis in CVSMCs from SHR is determined genetically, (2) that enhanced DNA synthesis in CVSMCs from SHR is largely dependent on an increased proportion of S-phase cells and (3) that this increase in S-phase cells in CVSMCs from SHR could be due to enhanced competence gene expression in SHR cells. (4) The increased intracellular free calcium concentration is compatible with an activation of the inositoltrisphosphate pathway.
Correspondence to: Dr. Masanori Hamada, Division of Cardiology, Dept. of Internal Medicine, Wakayama Medical College, Wakayama, Japan.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
* Present address: Wellcome Medical Research Otago Medical School, University of Otago, P.O. Dunedin, New Zealand.
Ltd.
Institute, Box 913,
192 Key words:
Muscle smooth vascular; Cell proliferation; Growth factors; Hypertension
Introduction Hypertrophy, hyperploidy and hyperplasia of vascular smooth muscle cells (VSMCs) have been reported in rats with experimental and genetic hypertension [l-3]. These findings have been attributed to the effect of high blood pressure or to enhanced sympathetic nervous activity or other factors [4-61. On the other hand, some experiments, using cell culture systems, suggest a genetic abnormality in cell proliferation of cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) [7,8]. Recent advances in the concept of growth factors indicate that both ‘competence factors’ and ‘progression factors’ are needed for the CVSMCs to complete the cell cycle [9]. In short, competence factors push GO-phase cells into Gl-phase while progression factors enable the cells in Gl-phase to move into S-phase and synthesize DNA. In the context of VSMCs, vasopressin is considered to be purely a competence factor [lo], and insulin to be purely a progression factor [9]. The aim of this study was (1) to find out whether the exaggerated proliferation of CVSMCs from SHR is determined genetically, and (2), if so, to determine the mechanism in the cell cycle by using specific growth factors and flowcytometry. Materials and methods Stock culture Vascular smooth muscle cells were prepared by the explant method [ll]. In brief, the thoracic aorta was excised under sterile conditions from WKY and SHR (Wakayama Medical College experimental animal colony) at age 3 or 12 weeks. After careful removal of adventitia and intima, the aortic media was cut into pieces and incubated in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo) supplemented with 10% fetal calf serum (FCS, Gibco, New York) at 37” C under
Spontaneously
hypertensive
rats; Flowcytometry;
conditions of 95% air and 5% CO, for 4 weeks. As a form of experimental hypertension, lo-week-old DOCA-salt hypertensive Wistar rats and shamimplanted controls were used [12]; CVSMCs were prepared as described above 2 weeks after DOCA implants. Cells in primary culture were separated with 0.25% trypsin (2.0 ml) and dispersed into 6-cm flasks (Terumo, Tokyo) at an approximate cm2 for subculture. density of 4.0 X lo5 cells/27 At the 4th passage of subculture, the nature of the cells was identified by monoclonal antibody which recognizes VSMCs specifically. The CVSMCs were used at the pre-confluent stage of the fifth passage. Measurement of doubling time CVSMCs were seeded at an approximate density of 4.0 x lo5 cells/27 cm2 in DMEM supplemented with 10% FCS and incubated at 37 o C and 95% sir/5% CO, for 72 h. CVSMCs from 2 or 3 flasks were separated with 0.25% trypsin (2.0 ml) and the cell number of 1 flask was counted by hemocytometer. The medium of the other flask was replaced by fresh 10% FCS( +) DMEM, and the cell number was counted every 24 h for 5 days. Doubling time was determined in the exponential growth phase by the following equation. DT = (t - t,,)log 2/(log
iV - log N,)
where t, is the time at first counting, t is the time at second counting, N is cell number at time t, and N,, is cell number at time t,. Measurement of basal levels of DNA synthesis DNA synthesis was evaluated by measurement of incorporation of [3H]thymidine into DNA. 2.0 PCi [3H]thymidine (NEN, specific activity 20 Ci/mmol) was added to 4 ml medium, and incubated at 37’C, 95% sir/5% CO, for a maximum of 6 h. After incubation with radioactive tracer, CVSMCs were rinsed twice with ice-cold phosphate-buffered saline (PBS), and sonicated
193 with 5 ml of PBS. 2 ml of suspension was used to measure labeled DNA by the method of Berns [13]. To compare control levels of DNA synthesis of the 2 strains of rats, [3H]thymidine incorporation was expressed in terms of DNA content, which was determined by a fluorescent technique using NUCRESAN”’ (Funakoshi, Tokyo) [14]. DNA-synthesizing cell cycle analysis by flowcytometry In this experiment, 8 different cell lines of each strain of rats were examined. CVSMCs were used in exponential growth phase. The medium was replaced by fresh 10% FCS( +) DMEM, and the CVSMCs were preincubated for 12 h at 37 o C in 95% sir/5% CO,. 5 pg/ml of bromodeoxyuridine (BrdU) was added to each flask and the flasks were incubated for 30 min. Incorporation of BrdU into the CVSMCs was then stopped by washing twice with ice-cold PBS. CVSMCs were separated by the addition of 0.25% trypsin in 0.05% EGTA, fixed with 70% ethanol and stored at 4°C. After DNA denaturation with 4.0 N HCl, samples were mixed with fluorescein isothiocyanate (FITC)labeled anti-BrdU antibody (Becton Dickinson, CA, U.S.A.) and incubated at 25” C for 20 min. Propidium iodide (PI) was added to the sample to stain double-stranded DNA. Thereafter with excitation at 488 nm, double-stranded DNA was measured by emission at 610 nm and single-stranded DNA was determined by emission at 525 nm by flow cytometry (FACStarTM). For each cell strain, lo4 CVSMCs were measured [15]. Effects of insulin, vasopressin and angiotensin II on DNA synthesis CVSMCs were seeded at an approximate density of 4.0 x lo5 cells/27 cm2 in DMEM supplemented with 10% FCS and incubated at 37” C, 95% sir/5% CO, for 72 h. Then, to avoid the effect of other growth factors, CVSMCs were subcultured without FCS for 24 h before preincubation with these chemicals. In FCS( - ) DMEM, there was almost complete inhibition of cell division for at least 48 h. Insulin (26.5 IU/mg, Sigma) was added to the medium at concentrations of 6.25, 12.5, 25, 50 and 100 mu/ml. Argininevasopressin (AVP) and angiotensin II (AH) (Sigma) were added at concentrations of 10p9, lo-*, lo-‘, 10e6 and lop5 M.
After 12 h preincubation insulin, AVP or AH, 2.0 added to 4 ml medium further incubated for 6 h. ration into DNA was then as described above.
of the CVSMCs with PCi [3H]thymidine was and the CVSMCs were [3H]Thymidine incorpomeasured by the method
Basal level of cytosolic free calcium concentration CVSMCs were dispersed by 1.0% collagenase (Sigma) for 5 min and suspended in FCS( -) DMEM. They were washed with medium A (145 mM NaCl, 5 mM KCl, 1 mM NaHPO,, 1 mM CaCl,, 0.5 mM MgSO,, 5 mM glucose, 10 mM Hepes, pH 7.4) and resuspended in medium A at an approximate concentration of lo6 cells/ml. Then 15 PM of Quin 2/AM (Sigma) was loaded for 50 min at 37 o C to complete hydrolysis of the ester. After centrifuging at 800 rpm for 5 min, CVSMCs were resuspended in medium A at 30 o C. Fluorescence (F) was recorded at 340 nm excitation and 490 nm emission wavelength. Thereafter, F max was determined by adding 20 PM digitonin. Fmin was measured by adding Tris-based 3 mM EGTA. Intracellular free calcium concentration ([Ca2+]i nM) was calculated by the following equation [ 161:
Effect of SHR-conditioned medium on [-‘H]thymidine incorporation into DNA SHR-conditioned medium was prepared as follows. CVSMCs from SHR in the exponential growth phase were put into new FCS ( -) DMEM, and cultured for 24 h. Then the medium was collected and passed through 0.22~pm pore filter (Advantec, Tokyo). WKY-conditioned medium was made by the same method. CVSMCs were preincubated in FCS (-) DMEM supplemented with 2.5-30% of the conditioned medium for 12 h. After preincubation, [ 3H]-thymidine (2.0 PCi) was added to each flask, the flasks were incubated for 6 h and [3H]thymidine incorporation into DNA was measured as described before. Statistical evaluation The data were analyzed tailed Student’s t-test.
by ANOVA
and two-
194 Results
TABLE 1
Rates of outgrowth from explants
BASAL LEVEL OF [3H]THYMIDINE INCORPORATION INTO DNA OF CVSMCs FROM SHR AND WKY OF DIFFERENT AGES
The outgrowth of VSMCs from aortic explants was observed by light microscopy from the 5th to the 14th day of primary culture. Explants from SHR showed somewhat earlier cell appearance but the difference was not significant. At 4 weeks after preparation, the percentage of the explants from which VSMCs migrated was significantly greater in SHR cultures (SHR: 34.4%, 4 rats, n = 64 dishes, WKY: 13.2%, 4 rats, n = 76 dishes, x2 = 8.9, P < 0.01) and in cultures from DOCA-treated Wistar rats (DOCA-salt: 23.9%, 5 rats, n = 67 dishes, sham-implanted Wistar: 6.5%, 5 rats, n = 77 dishes, x2 = 8.7, P < 0.01). Doubling time
The cell growth curve for 5th passage CVSMCs was almost exponential from the third to the sixth day, after which growth slowed as confluence was reached (Fig. 1). Doubling time was calculated from the cell numbers on the 4th and the 6th days. Doubling time for 12-week-old SHR cells was 33.8 k 1.3 h and for 12-week-old WKY 60.9 + 2.8 h (mean 1_ SEM, n = 13 rats, P < O.Ol), showing accelerated cell division by SHR CVSMCs. Doubling time for the cells from DOCA-salt rats was 39.2 f 2.9 h and for cells from sham-operated rats 45.8 * 3.0 h (mean + SEM, n = 8 cell lines), showing no significant difference between DOCA-salt rats and sham-operated rats.
I
I
I
It
5day2
I
cpm/%
DNA
5 146 k 1411* 7385+1621746+ 98~~ 3432+ 30-
WKY (12 weeks) SHR (12 weeks) WKY (3 weeks) SHR (3 weeks)
n 8 8 5 5
* P
ATHERO
191 Ltd.
04442
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
Masanori Hamada *, Ichiro Nishio, Akira Baba, Kazuya Fukuda, Jirou Takeda, Masako Ura, Takuzo Hano, Masato Ku&ii and Yoshiaki Masuyama Division of Cardiology, Department of Medicine, Wakayama Medical College, Wakayama (Japan) (Received 17 April, 1989) (Revised, received 27 November, 1989) (Accepted 18 December, 1989)
Summary It is widely reported that cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) show enhanced proliferation compared with cells from Wistar-Kyoto rats (WKY). The present studies were designed to find out whether this exaggerated proliferation in SHR is determined genetically and, if so, to evaluate the mechanism on the cell cycle. (1) Incorporation of [3H]thymidine into DNA was enhanced in CVSMCs from 3- and 12-week-old SHR compared with WKY but not in CVSMCs from DOCA-salt hypertensive rats compared with the cells from sham-operated rats. (2) DNA synthesis in SHR cells was enhanced further by addition of insulin (which is considered to be a progression factor) but not by arginine-vasopressin (AVP; considered to be a competence factor) or by angiotensin II (AII). On the other hand, insulin, AVP and AI1 significantly augmented DNA synthesis in WKY cells. (3) Intracellular free calcium concentration was slightly, but significantly, higher in SHR cells. (4) An increase in the population of DNA-synthesizing S-phase cells and decrease in (G2 + M)-phase cells in SHR were observed by flowcytometry. These data suggest (1) that enhanced DNA synthesis in CVSMCs from SHR is determined genetically, (2) that enhanced DNA synthesis in CVSMCs from SHR is largely dependent on an increased proportion of S-phase cells and (3) that this increase in S-phase cells in CVSMCs from SHR could be due to enhanced competence gene expression in SHR cells. (4) The increased intracellular free calcium concentration is compatible with an activation of the inositoltrisphosphate pathway.
Correspondence to: Dr. Masanori Hamada, Division of Cardiology, Dept. of Internal Medicine, Wakayama Medical College, Wakayama, Japan.
0021-9150/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
* Present address: Wellcome Medical Research Otago Medical School, University of Otago, P.O. Dunedin, New Zealand.
Ltd.
Institute, Box 913,
192 Key words:
Muscle smooth vascular; Cell proliferation; Growth factors; Hypertension
Introduction Hypertrophy, hyperploidy and hyperplasia of vascular smooth muscle cells (VSMCs) have been reported in rats with experimental and genetic hypertension [l-3]. These findings have been attributed to the effect of high blood pressure or to enhanced sympathetic nervous activity or other factors [4-61. On the other hand, some experiments, using cell culture systems, suggest a genetic abnormality in cell proliferation of cultured vascular smooth muscle cells (CVSMCs) from spontaneously hypertensive rats (SHR) [7,8]. Recent advances in the concept of growth factors indicate that both ‘competence factors’ and ‘progression factors’ are needed for the CVSMCs to complete the cell cycle [9]. In short, competence factors push GO-phase cells into Gl-phase while progression factors enable the cells in Gl-phase to move into S-phase and synthesize DNA. In the context of VSMCs, vasopressin is considered to be purely a competence factor [lo], and insulin to be purely a progression factor [9]. The aim of this study was (1) to find out whether the exaggerated proliferation of CVSMCs from SHR is determined genetically, and (2), if so, to determine the mechanism in the cell cycle by using specific growth factors and flowcytometry. Materials and methods Stock culture Vascular smooth muscle cells were prepared by the explant method [ll]. In brief, the thoracic aorta was excised under sterile conditions from WKY and SHR (Wakayama Medical College experimental animal colony) at age 3 or 12 weeks. After careful removal of adventitia and intima, the aortic media was cut into pieces and incubated in Dulbecco’s modified Eagle’s medium (DMEM, Nissui, Tokyo) supplemented with 10% fetal calf serum (FCS, Gibco, New York) at 37” C under
Spontaneously
hypertensive
rats; Flowcytometry;
conditions of 95% air and 5% CO, for 4 weeks. As a form of experimental hypertension, lo-week-old DOCA-salt hypertensive Wistar rats and shamimplanted controls were used [12]; CVSMCs were prepared as described above 2 weeks after DOCA implants. Cells in primary culture were separated with 0.25% trypsin (2.0 ml) and dispersed into 6-cm flasks (Terumo, Tokyo) at an approximate cm2 for subculture. density of 4.0 X lo5 cells/27 At the 4th passage of subculture, the nature of the cells was identified by monoclonal antibody which recognizes VSMCs specifically. The CVSMCs were used at the pre-confluent stage of the fifth passage. Measurement of doubling time CVSMCs were seeded at an approximate density of 4.0 x lo5 cells/27 cm2 in DMEM supplemented with 10% FCS and incubated at 37 o C and 95% sir/5% CO, for 72 h. CVSMCs from 2 or 3 flasks were separated with 0.25% trypsin (2.0 ml) and the cell number of 1 flask was counted by hemocytometer. The medium of the other flask was replaced by fresh 10% FCS( +) DMEM, and the cell number was counted every 24 h for 5 days. Doubling time was determined in the exponential growth phase by the following equation. DT = (t - t,,)log 2/(log
iV - log N,)
where t, is the time at first counting, t is the time at second counting, N is cell number at time t, and N,, is cell number at time t,. Measurement of basal levels of DNA synthesis DNA synthesis was evaluated by measurement of incorporation of [3H]thymidine into DNA. 2.0 PCi [3H]thymidine (NEN, specific activity 20 Ci/mmol) was added to 4 ml medium, and incubated at 37’C, 95% sir/5% CO, for a maximum of 6 h. After incubation with radioactive tracer, CVSMCs were rinsed twice with ice-cold phosphate-buffered saline (PBS), and sonicated
193 with 5 ml of PBS. 2 ml of suspension was used to measure labeled DNA by the method of Berns [13]. To compare control levels of DNA synthesis of the 2 strains of rats, [3H]thymidine incorporation was expressed in terms of DNA content, which was determined by a fluorescent technique using NUCRESAN”’ (Funakoshi, Tokyo) [14]. DNA-synthesizing cell cycle analysis by flowcytometry In this experiment, 8 different cell lines of each strain of rats were examined. CVSMCs were used in exponential growth phase. The medium was replaced by fresh 10% FCS( +) DMEM, and the CVSMCs were preincubated for 12 h at 37 o C in 95% sir/5% CO,. 5 pg/ml of bromodeoxyuridine (BrdU) was added to each flask and the flasks were incubated for 30 min. Incorporation of BrdU into the CVSMCs was then stopped by washing twice with ice-cold PBS. CVSMCs were separated by the addition of 0.25% trypsin in 0.05% EGTA, fixed with 70% ethanol and stored at 4°C. After DNA denaturation with 4.0 N HCl, samples were mixed with fluorescein isothiocyanate (FITC)labeled anti-BrdU antibody (Becton Dickinson, CA, U.S.A.) and incubated at 25” C for 20 min. Propidium iodide (PI) was added to the sample to stain double-stranded DNA. Thereafter with excitation at 488 nm, double-stranded DNA was measured by emission at 610 nm and single-stranded DNA was determined by emission at 525 nm by flow cytometry (FACStarTM). For each cell strain, lo4 CVSMCs were measured [15]. Effects of insulin, vasopressin and angiotensin II on DNA synthesis CVSMCs were seeded at an approximate density of 4.0 x lo5 cells/27 cm2 in DMEM supplemented with 10% FCS and incubated at 37” C, 95% sir/5% CO, for 72 h. Then, to avoid the effect of other growth factors, CVSMCs were subcultured without FCS for 24 h before preincubation with these chemicals. In FCS( - ) DMEM, there was almost complete inhibition of cell division for at least 48 h. Insulin (26.5 IU/mg, Sigma) was added to the medium at concentrations of 6.25, 12.5, 25, 50 and 100 mu/ml. Argininevasopressin (AVP) and angiotensin II (AH) (Sigma) were added at concentrations of 10p9, lo-*, lo-‘, 10e6 and lop5 M.
After 12 h preincubation insulin, AVP or AH, 2.0 added to 4 ml medium further incubated for 6 h. ration into DNA was then as described above.
of the CVSMCs with PCi [3H]thymidine was and the CVSMCs were [3H]Thymidine incorpomeasured by the method
Basal level of cytosolic free calcium concentration CVSMCs were dispersed by 1.0% collagenase (Sigma) for 5 min and suspended in FCS( -) DMEM. They were washed with medium A (145 mM NaCl, 5 mM KCl, 1 mM NaHPO,, 1 mM CaCl,, 0.5 mM MgSO,, 5 mM glucose, 10 mM Hepes, pH 7.4) and resuspended in medium A at an approximate concentration of lo6 cells/ml. Then 15 PM of Quin 2/AM (Sigma) was loaded for 50 min at 37 o C to complete hydrolysis of the ester. After centrifuging at 800 rpm for 5 min, CVSMCs were resuspended in medium A at 30 o C. Fluorescence (F) was recorded at 340 nm excitation and 490 nm emission wavelength. Thereafter, F max was determined by adding 20 PM digitonin. Fmin was measured by adding Tris-based 3 mM EGTA. Intracellular free calcium concentration ([Ca2+]i nM) was calculated by the following equation [ 161:
Effect of SHR-conditioned medium on [-‘H]thymidine incorporation into DNA SHR-conditioned medium was prepared as follows. CVSMCs from SHR in the exponential growth phase were put into new FCS ( -) DMEM, and cultured for 24 h. Then the medium was collected and passed through 0.22~pm pore filter (Advantec, Tokyo). WKY-conditioned medium was made by the same method. CVSMCs were preincubated in FCS (-) DMEM supplemented with 2.5-30% of the conditioned medium for 12 h. After preincubation, [ 3H]-thymidine (2.0 PCi) was added to each flask, the flasks were incubated for 6 h and [3H]thymidine incorporation into DNA was measured as described before. Statistical evaluation The data were analyzed tailed Student’s t-test.
by ANOVA
and two-
194 Results
TABLE 1
Rates of outgrowth from explants
BASAL LEVEL OF [3H]THYMIDINE INCORPORATION INTO DNA OF CVSMCs FROM SHR AND WKY OF DIFFERENT AGES
The outgrowth of VSMCs from aortic explants was observed by light microscopy from the 5th to the 14th day of primary culture. Explants from SHR showed somewhat earlier cell appearance but the difference was not significant. At 4 weeks after preparation, the percentage of the explants from which VSMCs migrated was significantly greater in SHR cultures (SHR: 34.4%, 4 rats, n = 64 dishes, WKY: 13.2%, 4 rats, n = 76 dishes, x2 = 8.9, P < 0.01) and in cultures from DOCA-treated Wistar rats (DOCA-salt: 23.9%, 5 rats, n = 67 dishes, sham-implanted Wistar: 6.5%, 5 rats, n = 77 dishes, x2 = 8.7, P < 0.01). Doubling time
The cell growth curve for 5th passage CVSMCs was almost exponential from the third to the sixth day, after which growth slowed as confluence was reached (Fig. 1). Doubling time was calculated from the cell numbers on the 4th and the 6th days. Doubling time for 12-week-old SHR cells was 33.8 k 1.3 h and for 12-week-old WKY 60.9 + 2.8 h (mean 1_ SEM, n = 13 rats, P < O.Ol), showing accelerated cell division by SHR CVSMCs. Doubling time for the cells from DOCA-salt rats was 39.2 f 2.9 h and for cells from sham-operated rats 45.8 * 3.0 h (mean + SEM, n = 8 cell lines), showing no significant difference between DOCA-salt rats and sham-operated rats.
I
I
I
It
5day2
I
cpm/%
DNA
5 146 k 1411* 7385+1621746+ 98~~ 3432+ 30-
WKY (12 weeks) SHR (12 weeks) WKY (3 weeks) SHR (3 weeks)
n 8 8 5 5
* P
