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Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells
Atherosclerosis 130 (1997) 1 – 10
Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells Masahiro Akishita, Yasuyoshi Ouchi *, Hideyuki Miyoshi, Koichi Kozaki, Satoshi Inoue, Michiro Ishikawa, Masato Eto, Kenji Toba, Hajime Orimo Department of Geriatrics, Faculty of Medicine, Uni6ersity of Tokyo, 7 -3 -1, Hongo, Bunkyo-ku, Tokyo 113, Japan Received 16 April 1996; received in revised form 9 September 1996; accepted 11 November 1996
Abstract The present study was performed to elucidate the mechanism underlying the anti-atherogenic action of estrogen. We investigated the effect of estrogen on intimal thickening of the rat femoral artery induced by cuff placement and further examined the effect of estrogen on migration and proliferation of vascular smooth muscle cells (VSMCs) in culture. Intimal thickening was significantly greater in males than in control females. Intimal thickening in females was increased to the level in males by ovariectomy. Estrogen replacement to ovariectomized rats reversed this effect. Proliferating cell nuclear antigen immunohistochemistry showed that in vivo proliferation of VSMCs contributed to the difference in intimal thickening. There was no difference in blood pressure and serum lipids, suggesting that estrogen directly acted on artery and inhibited intimal thickening. 17b-Estradiol (E2, 1–100 nmol/l) inhibited migration of cultured rat VSMCs, assayed using a microchemotaxis chamber, in a concentration-dependent manner. E2 (0.01–100 nmol/l), but not progesterone or testosterone, also inhibited [3H]thymidine incorporation in rat VSMCs in a concentration-dependent manner. Indomethacin, N G-monomethyl-L-arginine and methylene blue did not influence the inhibitory action of E2 on [3H]thymidine incorporation, suggesting that prostanoids and nitric oxide are not involved in the action of E2. E2 did not provoke VSMC injury, as measured by the release of incorporated [3H]2-deoxy-D-glucose. These results suggest that the inhibition of migration and proliferation of VSMCs contributes to the inhibitory effect of estrogen on intimal thickening. © 1997 Elsevier Science Ireland Ltd. Keywords: Estrogen; Smooth muscle cell; Migration; Proliferation; Atherosclerosis
1. Introduction It is an established epidemiological observation that the incidence of atherosclerosis is lower in premenopausal women than in men and increases after menopause [1,2]. Estrogen replacement therapy is effective to protect women from coronary heart disease [2 – 4]. Estrogen has also been reported to inhibit the development of atherosclerosis in animal experiments [5 – 7]. These epidemiological and experimental findings suggest that estrogen has an anti-atherogenic action. The mechanism, however, is not fully understood. * Corresponding author. Tel.: +81 3 38155411 ext. 3223; fax: +81 3 38122097.
A number of investigators have reported that lipid metabolism is improved by estrogen administration [5,6,8–10]. Estrogen increases serum high-density lipoprotein cholesterol level and decreases serum lowdensity lipoprotein cholesterol level. On the other hand, the existence of functional estrogen receptors in vascular endothelial cells [11] and vascular smooth muscle cells (VSMCs) [12–14] suggests that estrogen acts directly on the arterial wall. Estrogen appears to have both endothelium-independent [15,16] and endothelium-dependent [17–19] vasodilator actions. A calcium-antagonistic effect [15,16], regulation of prostacyclin [20,21] and nitric oxide production [22] may be involved in these effects.
0021-9150/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 2 1 - 9 1 5 0 ( 9 6 ) 0 6 0 2 3 - 6
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M. Akishita et al. / Atherosclerosis 130 (1997) 1–10
Migration and proliferation of VSMCs are fundamental steps in the development of atherosclerosis [23] and neointimal formation after endothelial injury [24]. However, the effect of estrogen on migration of cultured VSMCs is not known. Moreover, the effect of estrogen on proliferation of VSMCs is controversial. Fischer-Dzoga et al. [25] and Vargas et al. [26] reported an inhibitory effect of estrogen on VSMC proliferation, while Farhat et al. [27] demonstrated a stimulatory effect of estrogen. We first aimed at investigating the effect of estrogen on cuff-induced intimal thickening of the rat femoral artery. The reason we used this model was that lipid metabolism was not involved in the development of intimal thickening by cuff placement. We further examined the effect of estrogen on migration and proliferation of cultured VSMCs to investigate the mechanism underlying the effect on intimal thickening.
2. Materials and methods
2.1. Chemicals Estradiol dipropionate was obtained from Teikoku Hormone (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO BRL (Gaithersburg, MD) and fetal bovine serum (FBS) from ICN Biomedicals (Osaka, Japan). Bovine serum albumin (BSA), trichloroacetic acid (TCA) and Triton X100 were purchased from Wako (Osaka, Japan). Phenol red-free DMEM, 17b-estradiol (E2), 17a-estradiol, progesterone, testosterone, endothelin-1, human basic fibroblast growth factor (bFGF), indomethacin, N Gmonomethyl-L-arginine (L-NMMA) and methylene blue were purchased from Sigma. Human plateletderived growth factor-BB (PDGF-BB) was from Genzyme (Cambridge, MA) and type V collagen solution from Koken (Tokyo, Japan). [3H]thymidine and [3H]2deoxy-D-glucose (DOG) were from NEN Research (Boston, MA).
2.2. Animals Eight-week-old female and male Wistar rats (Nippon Bio-Supply Center, Tokyo, Japan) were used in this study. They were kept individually in stainless steel cages in a room where lighting was controlled (12 h on, 12 h off) and room temperature was kept at around 22°C. They were given standard diet (CE-2, Japan Clea, Tokyo, Japan) and water ad libitum. All the surgical procedures were performed under ether anesthesia. All of the experimental protocols were approved by the Animal Research Committee of the University of Tokyo.
2.3. Cuff-induced intimal thickening The surgical procedures of cuff placement was according to the method described by Hirosumi et al. [28] with some modification. The left femoral artery of rats was isolated from the surrounding tissues. A polyethylene tube (5 mm long PE-160; inner diameter, 1.14 mm; outer diameter, 1.57 mm; Becton Dickinson, Parsippany, NJ) was cut longitudinally to open the tube and loosely placed around the artery, then the wounds were sutured. The control rats underwent isolation of the femoral artery without cuff placement. After the experimental period, the rats were killed and the femoral artery was excised together with the cuff. The artery was then fixed in 10% neutral formalin and embedded in paraffin. The middle segment of the artery was cut into cross-sectional pieces with 5 mm thickness and stained by Elastica van Gieson staining. Cross-sectional areas of the intima and the media were measured using a digitizer connected to a Macintosh Computer and the ratio of the intimal area to the medial area (I:M ratio) was calculated. In order to check the time course of intimal thickening, male rats were sacrificed 1–4 weeks (n=7–9 for each week) after cuff placement and the magnitude of intimal thickening was evaluated. Control rats without cuff placement were added (n= 2–3 for each week). To examine the effect of estrogen on cuff-induced intimal thickening, female rats were randomly divided into three groups. Two groups of rats were ovariectomized and the other group of rats were sham operated. After a one-week recovery period, one group of ovariectomized rats received subcutaneous injection of estradiol dipropionate (20 mg/kg) suspended in corn oil as vehicle once a week (OVX + E group, n = 7). In the preliminary experiments, administration of this dose of estradiol dipropionate exhibited a change in plasma E2 concentrations as follows: day 0, 42.7 9 6.1; day 1, 109.0912.5; day 3, 95.89 15.6; day 7, 57.894.7; day 8, 162.0920.3; day 10, 140.09 8.3 and day 14, 78.7 9 8.0 pmol/l (n= 4). The other group of ovariectomized rats (OVX group, n= 7) and sham-operated rats (sham group, n= 7) received the same amount of corn oil once a week. Age-matched male rats (male group, n=7) were also given corn oil injection. The three female groups underwent cuff placement one week after ovariectomy or sham operation and the male group also underwent cuff placement at the same time. Histological analysis was performed 2 weeks after cuff placement. At that time, systolic blood pressure was measured by tail-cuff method (UR-1000/UR-5000, Ueda, Tokyo, Japan) and a blood sample was collected from the abdominal aorta after 16 h fasting to measure serum lipids and plasma estradiol concentration. Serum total cholesterol and triglyceride were measured enzymatically, and high-density lipoprotein cholesterol was
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measured by heparin – Ca2 + Ni2 + precipitation method [29]. Plasma estradiol concentration was measured by sensitive radioimmunoassay.
2.4. Immunohistochemistry To examine the involvement of VSMC proliferation on the effects of estrogen on cuff-induced intimal thickening, the immunohistochemical staining of the sections was carried out by streptavidin – biotin – peroxidase method [30]. We used anti-muscle actin antibody (HHF35, 10 mg/ml; Enzo Diagnostics, New York, NY), anti-proliferating cell nuclear antigen (PCNA) antibody (PC10, 10 mg/ml; Boehringer Mannheim Biochemica, Mannheim, Germany) and normal mouse IgG (10 mg/ ml) as the first antibody. Specifically bound antibodies were disclosed by immersing the sections in a substrate solution of 3,3%-diaminobenzine (Vector Laboratories, Burlingame, CA).
2.5. Cell culture In the present study, we used three types of rat VSMCs in culture. Two of them were isolated from the medial layer of the thoracic aorta by enzymatic digestion, according to the method described previously [31], from 8-week-old male and female Wistar rats (Nippon Bio-Supply Center). These cells were identified as VSMCs by their ‘hill-and-valley’ appearance and by immunohistochemical staining using anti-muscle actin antibody (HHF35). Subcultured VSMCs (5 –9th passage) were used in the experiments. The other type was A10 cells, a cell-line derived from fetal rat aorta [32]. We also used MCF-7 cells, a cell-line derived from human breast cancer [33]. All of the cells were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. At the time of experiments, we used dextran-coated charcoal-stripped FBS (DCC-FBS) and phenol red-free DMEM to avoid contamination of steroids and estrogen receptor agonist. E2 concentration of non-treated FBS was 109 pmol/l, while that of DCC-FBS was 5.3 pmol/l.
2.6. Migration assay Migration of VSMCs was assayed by a modified Boyden’s chamber method [34] using a 96-well microchemotaxis chamber (Neuro Probe, Cabin John, MD) and polycarbonate filters (Neuro Probe) with pore size of 5.0 mm diameter. The filters were treated with 0.5 mol/l acetic acid overnight and then incubated in type V collagen solution (100 mg/ml) for 72 h. The coated filters were air-dried. Cultured VSMCs derived from male rats were trypsinized and suspended at a concentration of 5 × 105 cells/ml in phenol red-free DMEM containing 0.1% DCC-FBS. VSMC suspension
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(225 ml) was placed in the upper chamber and 35 ml phenol red-free DMEM containing 10% DCC-FBS with E2 (0.1–100 nmol/l) or vehicle (0.1% ethanol) was placed in the lower chamber. The chamber was incubated for 4 h. The filter was removed and VSMCs on the upper side of the filter were scraped off. Then, the filter was fixed in methanol and stained with Diff-Quick staining solution (Green Cross Co., Osaka, Japan). The number of VSMCs migrated to the lower side of the filter was counted under a microscope and the migration activity was expressed as the mean number of cells per 5 high power fields (HPF; magnification ×400).
2.7. Measurement of DNA synthesis DNA synthesis was assayed by measuring [3H]thymidine incorporation [35]. The cells were seeded in 24-well multiplates (Corning, NY) at an initial concentration of 5×104 cells/ml in 0.5 ml of DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The medium was then replaced with phenol red-free DMEM to arrest the growth. After 24 h, the medium was replaced again with phenol red-free DMEM containing [3H]thymidine (1 mCi/ml) and various agents. After incubation for another 24 h, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and subsequently incubated with ice-cold 5% TCA for 20 min at 4°C. The cells were washed twice with ice-cold 5% TCA and then with ice-cold PBS. The cells were lysed with 0.5 mol/l NaOH. The radioactivity of the cell lysate was counted using a liquid scintillation counter (LSC-1000, Aloka, Tokyo, Japan).
2.8. Growth of VSMCs A10 cells were seeded in 12-well multiplates (Corning) at an initial concentration of 5× 104 cells/ml in 1 ml of DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The medium was then replaced with phenol red-free DMEM to arrest the growth. After 24 h, the medium was replaced again with phenol red-free DMEM containing 5% DCC-FBS with E2 (1–100 nmol/l) or vehicle (0.1% ethanol). After incubation for 24–48 h, the cells were trypsinized and suspended. Then the number of cells was determined using a Coulter Counter (model ZM, Coulter Electronics, Hialeah, FL).
2.9. E6aluation of VSMC injury Injury of VSMCs was evaluated based on the release of incorporated [3H]2-DOG from VSMCs as described previously [35]. In brief, VSMCs (A10 cells and male VSMCs) were seeded in 12-well multiplates (Corning) at an initial concentration of 5 × 104 cells/ml in 1 ml of
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Fig. 1. Sex-related difference in cuff-induced intimal thickening of the rat femoral artery. A, representative high magnification photomicrographs (Elastica van Gieson staining; original magnification ×200) of the indicated groups are shown: arrow head, internal elastic lamina; arrow, external elastic lamina. B, bar graph showing the ratios of the intimal area to the medial area (I:M ratio) of each group. n = 7 For each group. * P B 0.05 vs. sham and OVX+E.
DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The cells were incubated with [3H]2-DOG (1 mCi/ml) for 18 h at 37°C and were washed twice with Hanks’ balanced salt solution containing 0.5% BSA. The cells were incubated in Hanks’ balanced salt solution containing 0.5% BSA with E2 (1, 100 nmol/l), vehicle (0.1% ethanol) or 0.2% Triton X-100 for 2 h. The radioactivity in the medium and the cell lysate obtained by treatment with 0.5 mol/l NaOH was counted using a liquid scintillation counter (LSC-1000). The specific release of [3H]2-DOG was determined as 100 ×(A − C)/(B −C) (%), where A is the radioactivity in the medium treated with E2 or Triton X-100, B is the total radioactivity in the medium and the cell lysate treated with E2 or Triton X-100 and C is the radioactivity in the medium treated with vehicle.
2.10. Data analysis All of the migration, [3H]thymidine incorporation, cell count and cell injury assays were repeated three times or more and the representative results are shown. The values in the text, figures and table are expressed as mean 9S.E.M. The data were analyzed using one-factor ANOVA. If a statistically significant effect was
found, Newman-Keuls’ test was performed to isolate the difference between the groups. PB 0.05 was considered to be significant.
3. Results
3.1. Sex-related difference in intimal thickening Cuff placement induced diffuse intimal thickening of the rat femoral artery, while no intimal thickening was observed in the control rats without cuff placement. We checked the time-related change in the development of intimal thickening. The I:M ratio was significantly higher 2 weeks after than 1 week after cuff placement (PB0.05) and then tended to decrease until 4 weeks after cuff placement: 1 week, 0.119 0.01 (n=8); 2 weeks, 0.339 0.07 (n= 9); 3 weeks, 0.2590.05 (n=8); 4 weeks, 0.219 0.02 (n= 7). Based on this result, we decided to examine sex difference and the effect of estrogen on intimal thickening 2 weeks after cuff placement. As shown in Fig. 1A and B, intimal thickening was greater in the male than in the sham group. Intimal thickening was enhanced in the OVX compared with those in the sham and OVX+ E groups. All of the
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shown in Table 1, no differences were found in systolic blood pressure and serum total cholesterol, high-density lipoprotein cholesterol and triglyceride levels among the four groups of rats. Plasma estradiol concentration was significantly lower in the OVX and in the male than in the sham group. Plasma estradiol concentration in the OVX+ E was comparable to that in the sham group.
3.2. Effects of E2 on migration and proliferation of cultured VSMCs
Fig. 2. Hematoxylin staining (A) and immunohistochemical staining of cuff-induced intimal thickening using anti-muscle actin antibody (B), anti-proliferating cell nuclear antigen (C) and normal mouse IgG (D). Representative high magnification photomicrographs (original magnification × 200) of ovariectomized rats are shown. arrow head, internal elastic lamina; arrow, external elastic lamina.
neointimal cells as well as medial cells were positively stained with anti-muscle actin antibody, indicating that these cells were VSMCs (The representative photomicrograph is shown in Fig. 2B). The nuclei positive for PCNA staining were observed in the neointima (Fig. 2C). As shown in Fig. 3, PCNA-positive cells in the neointima were increased in the male and OVX compared with those in the sham and OVX + E groups. As
Fig. 3. Sex-related difference in the number of PCNA-positive cells in the neointima. n = 7 For each group. * PB 0.05 vs. sham and OVX +E.
As shown in Fig. 4A, 1–100 nmol/l E2 significantly inhibited migration of male VSMCs in a concentrationdependent manner. Similar concentration of progesterone or testosterone did not show the inhibitory effect on migration (data not shown). As shown in Fig. 4B, 0.01–100 nmol/l E2 also inhibited DNA synthesis in male VSMCs in a concentration-dependent manner. To examine the effect of E2 on DNA synthesis in other cell types, we used three different types of rat VSMCs (male VSMCs, female VSMCs and A10 cells) and MCF-7 cells. As shown in Fig. 5A, E2 (1–100 nmol/l) inhibited DNA synthesis in all of the three types of VSMCs, while E2 did not inhibit DNA synthesis in MCF-7 cells and 100 nmol/l E2 significantly stimulated DNA synthesis in MCF-7 cells as previously reported [33]. In the following experiments, we investigated the mechanism underlying the inhibitory effect of E2 on DNA synthesis. For this purpose, we used A10 cells because they are well characterized [32]. We compared the effect of E2 with those of other sex steroids (Fig. 5B). Unlike 17b-estradiol (E2), 17a-estradiol had no effect on DNA synthesis. No significant change in DNA synthesis was observed when 1–100 nmol/l progesterone or testosterone was applied. To check whether the inhibition by E2 was specific to a certain growth factor, we used various stimuli including 5% DCC-FBS, 10 ng/ml PDGF-BB, 10 ng/ml bFGF and 1 mmol/l endothelin-1 in the presence of 0.2% BSA. As shown in Fig. 5C, E2 (1 nmol/l) completely inhibited the increase in DNA synthesis stimulated by these substances. No inhibition was observed when any of these stimuli was not applied. We examined the possible involvement of prostacyclin and nitric oxide because they are known to inhibit VSMC proliferation [36,37]. E2 has been reported to stimulate the production of these substances [20–22]. As shown in Fig. 5D, simultaneous addition of indomethacin (10 mmol/l), a cyclooxygenase inhibitor, with E2 (1 nmol/l) did not affect the inhibition of DNA synthesis by E2. As shown in Fig. 5E, L-NMMA (10 mmol/l), an inhibitor of nitric oxide production, or methylene blue (10 mmol/l), an inhibitor of cyclic GMP production, did not affect the inhibition of DNA synthesis by E2 as well.
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Table 1 Blood pressure, serum lipids and plasma estradiol concentration in rats with cuff placement
Sham OVX OVX+E Male
SBP (mmHg)
T.chol (mg/dl)
HDL-C (mg/dl)
TG (mg/dl)
E2 (pmol/l)
13893 13593 13993 14092
72.39 2.3 79.4 9 4.2 80.3 9 3.7 68.49 2.3
52.0 9 3.6 56.8 9 3.2 55.3 9 4.6 56.9 9 2.9
27.6 9 2.6 28.0 9 3.0 22.7 9 1.7 26.4 9 1.5
133 927 41 96* 82 918 28 93*
Values are expressed as mean9S.E.M. (n= 7). *, PB0.05 vs sham. SBP, systolic blood pressure; T.chol, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride.
We also checked the change in cell numbers induced by 5% DCC-FBS. As shown in Fig. 6, the increase in cell numbers was significantly inhibited by 40% by incubation with 100 nmol/l E2 for 24 h compared with
vehicle. The inhibition was more marked with incubation for 48 h, and 1, 10 and 100 nmol/l E2 significantly inhibited the increase in cell numbers compared with vehicle by 16, 49 and 68% respectively.
3.3. VSMC injury by E2 As shown in Fig. 7, the specific release of [3H]2-DOG from A10 cells by incubation with E2 (1, 100 nmol/l) was not significantly different from the control, while that by incubation with 0.2% Triton X-100 was markedly elevated. Similar results were obtained when male VSMCs were used (data not shown).
4. Discussion
Fig. 4. Effect of E2 on migration (A) and DNA synthesis (B) of male vascular smooth muscle cells. A, Migration activity was assayed using a chemotaxis chamber as described in Section 2, n= 3. * PB 0.05 vs. vehicle. Cells/HPF, migrated cells per high power field (magnification × 400). B, DNA synthesis was assayed as [3H]thymidine incorporation induced by 5% dextran-coated charcoal-stripped fetal bovine serum for 24 h, n =8. *, ** PB 0.05, 0.01 vs. vehicle.
In the present study, we showed that intimal thickening of the rat femoral artery induced by cuff placement was greater in males than that in control females. Intimal thickening of females was increased by ovariectomy and this increase was reversed by estrogen replacement. Arterial intimal thickening induced by a polyethylene cuff has been reported in the rabbit femoral artery [38] and in the rabbit carotid artery [28]. Histological observations in these studies elucidate that cuff placement causes polymorphonuclear leukocyte infiltration and endothelial injury in the initial step by inflammatory responses. The next important processes for the development of intimal thickening are the migration of medial VSMCs to the intima and the proliferation of migrated VSMCs with deposition of extracellular matrix in the neointima. These steps are similar to those observed in the development of atherosclerosis in man [23]. Therefore, intimal thickening induced by cuff placement might be an adequate model to facilitate understanding atherosclerosis. We checked the time course of polyethylene cuff-induced intimal thickening of the rat femoral artery and the peak of intimal thickening was observed 2 weeks after cuff placement. Therefore, we examined sex-related difference at 2 weeks. The physiological concentration of plasma E2 in female rats was 0.139 0.03 nmol/l according to the
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Fig. 5. (A) Effect of E2 on DNA synthesis in MCF-7 cells, male vascular smooth muscle cells (VSMCs), female VSMCs and A10 cells induced by 5% dextran-coated charcoal-stripped fetal bovine serum (DCC-FBS) n =6. * P B0.05 vs. vehicle. (B) Effect of E2, 17a-estradiol, progesterone and testosterone on DNA synthesis in A10 cells induced by 5% DCC-FBS n =6. * PB 0.05 vs. vehicle. (C) DNA synthesis in A10 cells induced by 5% DCC-FBS, PDGF-BB (10 ng/ml), bFGF (10 ng/ml), endothelin-1 (1 mmol/l) or vehicle (0.2% BSA) with (closed columns) or without (open columns) E2 (1 nmol/l) n =6. * PB 0.05 vs. vehicle; n.s., not significant. (D) Effect of E2 on DNA synthesis in A10 cells induced by 5% DCC-FBS in the absence or presence of indomethacin (10 mmol/l). closed column, E2 1 nmol/l; open column, vehicle n =6. (E) Effect of E2 on DNA synthesis in A10 cells induced by 5% DCC-FBS in the absence or presence of L-NMMA (10 mmol/l) or methylene blue (10 mmol/l). closed column, E2 1 nmol/l; open column, vehicle, n = 6.
results of this study. This is comparable to the previously reported value [39]. Plasma E2 concentrations in estrogen-replaced rats were at the same level as those in control females throughout the experiment. These results indicate that a physiological concentration of estrogen inhibits intimal thickening in female rats. Recently, Sullivan et al. [40] reported that estrogen replacement inhibited the response of the mouse carotid artery to wire injury and Chen et al. [41] reported that estrogen reduced myointimal proliferation after balloon injury of the rat carotid artery. Even though the animals used [40] and the method of vascular injury [40,41]
are different from ours, their studies are consistent with our results. We did not administer estrogen in males, thus it is unclear whether the lack of endogenous estrogen in males augmented intimal thickening. Testosterone might have influenced intimal thickening because exogenous [42] and endogenous [43] testosterone is reported to accelerate atherosclerosis. However, Chen et al. [41] reported that endogenous or exogenous testosterone did not alter, but estrogen attenuated, neointimal formation in male and female rats. Although their data indicate that the sex difference in neointimal for-
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Fig. 6. Effect of E2 on vascular smooth muscle cell growth. Growtharrested A10 cells were incubated in phenol red-free Dulbecco’s modified Eagle’s medium containing 5% dextran-coated charcoalstripped fetal bovine serum with E2 (1–100 nmol/l) or vehicle (0.1% ethanol) for 24 – 48 h, then the number of cells was counted, n = 4. *, **; P B0.05, 0.01 vs. vehicle
mation is estrogen dependent, further study is needed to clarify whether testosterone contributes to the sex difference in cuff-induced intimal thickening. It is suggested that estrogen exhibits anti-atherogenic action by improving several risk factors, especially serum lipid concentration [5,6,8 – 10] and blood pressure
Fig. 7. VSMC injury incubated with 1 or 100 nmol/l E2, vehicle (0.1% ethanol) or 0.2% Triton X-100 for 2 h. VSMC injury was assayed as the specific release of incorporated [3H]2-DOG, n =6. *** P B 0.001 vs. vehicle.
[44–46]. In our experiment, however, no differences were found in serum lipid levels and systolic blood pressure among the groups. Serum lipids were considered to be measured accurately because they were comparable to the previously reported values in male [47] and female [48] Wistar rats. Thus, the inhibitory effect of estrogen on intimal thickening was not due to an effect on these risk factors. Since the arterial wall is reported to contain functional estrogen receptors [11– 14], it is possible that estrogen may act directly on the arterial wall to inhibit intimal thickening. We further performed an immunohistochemical study to evaluate which components contributed to the effects of estrogen on intimal thickening. We used anti-muscle actin antibody (HHF35) as a marker of VSMCs and anti-PCNA antibody (PC10) as a marker of cell proliferation [49]. Immunostaining with antimuscle actin antibody showed that neointimal cells were composed of VSMCs. Furthermore, PCNA-positive cells in the neointima were increased in males and ovariectomized females compared with those in control females and estrogen-replaced females. This data indicates that proliferation of VSMCs contributed to the effects of estrogen on intimal thickening. Sullivan et al. [40] and Chen et al. [41] also demonstrated that inhibition of VSMC proliferation was involved in the effects of estrogen using bromodeoxyuridine uptake [40] or c-myc expression [41]. In order to further investigate the mechanisms involved in the inhibitory action of estrogen on the development of cuff-induced intimal thickening, we examined the effects of this hormone on migration and proliferation of VSMCs in vitro. We found that estrogen inhibited both migration and proliferation of rat VSMCs in a concentration-dependent manner. This inhibition was not due to a toxic effect of estrogen, because sex steroids other than E2 had no effect, and VSMC injury incubated with 1 or 100 nmol/l E2 was the same as that with vehicle. To our knowledge, this is the first report demonstrating the inhibitory effect of E2 on migration of cultured VSMCs. The effect of estrogen on proliferation of VSMCs is controversial. Fischer-Dzoga et al. [25] reported that E2 (73 nmol/l) inhibited [3H]thymidine incorporation and growth of cultured VSMCs from rabbit aorta induced by hyperlipemic serum, and Vargas et al. [26] reported that E2 (180–360 nmol/l) inhibited [3H]thymidine incorporation in pig coronary artery segments. These results are in agreement with ours, although they used much higher concentration of E2 than in our study. On the other hand, Farhat et al. [27] reported that E2 (3–300 nmol/l) potentiated [3H]thymidine incorporation both in canine pulmonary artery segments and in cultured VSMCs from the rat pulmonary artery. The reason why the responses to E2 differ in these experiments may be attributed to the origin of the arteries which were investigated.
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The effective concentration of E2 in the thymidine incorporation study (0.01 nmol/l) was within the physiological range, while those in the cell count study (1 nmol/l) and the migration study (1 nmol/l) somewhat exceeded the physiological range. The concentrations of E2 used in our experiments, however, were significantly lower than those previously reported [25,26]. It is not known why the effective dose of E2 differed among the thymidine incorporation study, the cell count study and the migration study. The difference may depend on the conditions of the experiments or the sensitivities of the assays. Additionally, other factors might have influenced intimal thickening. Estrogen is reported to inhibit adhesion of leukocytes to endothelial cells in vitro [50]. Estrogen replacement improves endothelium-dependent vasomotor responses [17 – 19] suggesting that estrogen could protect endothelial cells from injury. Moreover, estrogen decreases collagen synthesis in cultured VSMCs [51] and in atherosclerotic aortas [52]. Leukocyte infiltration, endothelial injury and synthesis of extracellular matrix are important steps in the development of intimal thickening as described above. Therefore, it is possible that the results of the cuff experiment might be synergistic effects of E2 on these factors as well as migration and proliferation of VSMCs and thus, the discrepancy of the effective doses of E2 could be explained. In thymidine incorporation studies we showed that E2 generally inhibited DNA synthesis in rat VSMCs but not in MCF-7 cells. The question arises as to why the response of VSMCs to E2 is different from that of MCF-7 cells. It is conceivable that the product stimulated by E2 may differ between cell types. Estrogen is reported to stimulate synthesis of specific proteins (not characterized) which may regulate the growth of MCF7 cells [53–55], whereas the existence of these proteins is not known in VSMCs. Production of some substance that inhibits cell growth may be induced in VSMCs by E2. Both prostacyclin and nitric oxide are candidates [20–22]. However, in the present study, prostacyclin or nitric oxide was not involved in the growth-inhibitory effect of E2, because indomethacin, L-NMMA or methylene blue did not affect the action of E2. In the present study, we also showed that the inhibitory effect of E2 on DNA synthesis was not specific to a certain growth factor. Therefore, it is possible that E2 inhibits VSMC proliferation by blocking some common pathway of growth stimulation, although the underlying mechanism still remains to be explored. In conclusion, estrogen inhibited cuff-induced intimal thickening of rat femoral artery. Estrogen also inhibited migration and proliferation of rat VSMCs in culture. Inhibition of VSMC migration and proliferation by estrogen may contribute to the anti-atherogenic effect of estrogen.
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Acknowledgements We would like to thank Masae Watanabe and Hitomi Yamaguchi for their excellent technical assistance. This work was supported by grants from the Uehara Memorial Foundation and the Sankyo Life Science Research Foundation. This work was presented in part at the 10th International Symposium on Atherosclerosis in Montre´al, October 9–14, 1994. References [1] Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and risk of cardiovascular disease: The Framingham Study. Ann Intern Med 1976;85:447 – 452. [2] Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med 1987;316:1105 –1110. [3] Bush TL, Barrett-Conner E, Cowan LD et al. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation 1987;75:1102 – 1109. [4] Stampfer MJ, Colditz GA, Willett WC et al. Postmenopausal estrogen therapy and cardiovascular disease: ten-year follow-up from the Nurses’ Health Study. N Engl J Med 1991;325:756– 762. [5] Kushwaha RS, Hazzard WR. Exogenous estrogens attenuate dietary hypercholesterolemia and atherosclerosis in the rabbit. Metabolism 1981;30:359 – 366. [6] Adams MR, Clarkson TB, Kaplan JR, Koritnik DR. Experimental evidence in monkeys for beneficial effects of estrogen on coronary artery atherosclerosis. Transplant Proc 1989;21:3662– 3664. [7] Cheng LP, Kuwahara M, Jacobsson J, Foegh ML. Inhibition of myointimal hyperplasia and macrophage infiltration by estradiol in aorta allografts. Transplantation 1991;52:967 – 972. [8] Rijpkema AHM, van der Sanden AA, Ruijs AHC. Effects of post-menopausal oestrogen-progestogen replacement therapy on serum lipids and lipoproteins: a review. Maturitas 1990;12:259– 285. [9] Lobo RA. Clinical review 27: effects of hormonal replacement on lipids and lipoproteins in postmenopausal women. J Clin Endocrinol Metab 1991;73:925 – 930. [10] Walsh BW, Schiff I, Rosner B, Greenberg L, Ravnikar V, Sacks FM. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med 1991;325:1196 – 1204. [11] Colburn P, Buonassisi V. Estrogen-binding sites in endothelial cell cultures. Science 1978;201:817 – 819. [12] Nakao J, Chang W-C, Murota S-I, Orimo H. Estradiol-binding sites in rat aortic smooth muscle cells in culture. Atherosclerosis 1981;38:75 – 80. [13] Orimo A, Inoue S, Ikegami A et al. Vascular smooth muscle cells as target for estrogen. Biochem Biophys Res Commun 1993;195:730 – 736. [14] Karas RH, Patterson BL, Mendelsohn ME. Human vascular smooth muscle cells contain functional estrogen receptor. Circulation 1994;89:1943 – 1950. [15] Jiang C, Sarrel PM, Lindsay DC, Poole-Wilson PA, Collins P. Endothelium-independent relaxation of rabbit coronary artery by 17b-oestradiol in vitro. Br J Pharmacol 1991;104:1033–1037. [16] Jiang C, Sarrel PM, Poole-Wilson PA, Collins P. Acute effect of 17b-estradiol on rabbit coronary artery contractile response to endothelin-1. Am J Physiol 1992;263:H271 – 275.
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[36] Graf H. Endothelial control of cell migration and proliferation. Eur Heart J 1993;14:183 – 186. [37] Soyombo AA, Thurston VJ, Newby AC. Endothelial control of vascular smooth muscle proliferation in an organ culture of human saphenous vein. Eur Heart J 1993;14:201 –206. [38] Gebrane J, Roland J, Orcel L. Experimental diffuse intimal thickening of the femoral arteries in the rabbit. Virchows Arch Pathol Anat 1982;396:41 – 59. [39] Smith MS, Freeman ME, Neill JD. The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 1975;96:219 – 226. [40] Sullivan TR, Karas RH, Aronovitz M et al. Estrogen inhibits the response-to-injury in a mouse carotid artery model. J Clin Invest 1995;96:2482 – 2488. [41] Chen S-J, Li H, Durand J, Oparil S, Chen Y-F. Estrogen reduces myointimal proliferation after balloon injury of rat carotid artery. Circulation 1996;93:577 – 584. [42] Fischer GM, Bashey RI, Rosenbaum H, Lyttle CR. A possible mechanism in arterial wall for mediation of sex difference in atherosclerosis. Exp Mol Pathol 1985;43:288 – 296. [43] Clarkson TB, Adams MR, Kaplan JR, Koritnik DR. Gonadal steroids in cardiac atherosclerosis in primates. Transplant Proc 1987;19:85 – 89. [44] Hoeg JM, Willis LR, Weinberger MH. Estrogen attenuation of the development of hypertension in spontaneously hypertensive rats. Am J Physiol 1977;233:369 – 373. [45] Iams SG, Wexler BC. Inhibition of the development of spontaneous hypertension in SH rats by gonadectomy or estradiol. J Lab Clin Med 1979;94:608 – 616. [46] Ouchi Y, Share L, Crofton JT, Iitake K, Brooks D. Sex differences in the development of deoxycorticosterone-salt hypertension in the rat. Hypertension 1987;9:172 – 177. [47] Patel ST, Newman HAI, Yates AJ, Thibert P, Falko JM. Serum lipids and lipoprotein composition in spontaneously diabetic BB Wistar rats. J Lipid Res 1984;25:1072 – 1083. [48] Deshaies Y, LeBlanc J, Richard D. Influence of a palatable, high-fat diet, and exercise training on the high-density lipoprotein to total cholesterol ratio in the rat. Metabolism 1983;32:62 – 65. [49] Zeymer U, Fishbein MC, Forrester JS, Cercek B. Proliferating cell nuclear antigen immunohistochemistry in rat aorta after balloon denudation. Comparison with thymidine and bromodeoxyuridine labeling. Am J Pathol 1992;141:685 – 690. [50] Caulin-Glaser TL, Watson C, Bender JR. The effects of 17bestradiol on leukocyte-endothelial cell interactions. Circulation 1993;88:I – 78. [51] Beldekas JC, Smith B, Gerstenfeld LC, Sonenshein GE, Franzblau C. Effects of 17b-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells. Biochemistry 1981;20:2162 – 2167. [52] Fischer GM, Swain ML. Effects of estradiol and progesterone on the increased synthesis of collagen in atherosclerotic rabbit aortas. Atherosclerosis 1985;54:177 – 185. [53] Edwards DP, Adams DJ, McGuire WL. Estrogen regulation of growth and specific protein synthesis in human breast cancer cells in tissue culture. Adv Exp Med Biol 1981;138:133–149. [54] Vignon F, Capony F, Chambon M, Freiss G, Garcia M, Rochefort H. Autocrine growth stimulation of the MCF-7 breast cancer cells by the estrogen-regulated 52 K protein. Endocrinology 1986;118:1537 – 1545. [55] Dickson RB, Huff KK, Spencer EM, Lippman ME. Induction of epidermal growth factor-related polypeptides by 17b-estradiol in MCF-7 human breast cancer cells. Endocrinology 1986;118:138 – 142.
Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells Masahiro Akishita, Yasuyoshi Ouchi *, Hideyuki Miyoshi, Koichi Kozaki, Satoshi Inoue, Michiro Ishikawa, Masato Eto, Kenji Toba, Hajime Orimo Department of Geriatrics, Faculty of Medicine, Uni6ersity of Tokyo, 7 -3 -1, Hongo, Bunkyo-ku, Tokyo 113, Japan Received 16 April 1996; received in revised form 9 September 1996; accepted 11 November 1996
Abstract The present study was performed to elucidate the mechanism underlying the anti-atherogenic action of estrogen. We investigated the effect of estrogen on intimal thickening of the rat femoral artery induced by cuff placement and further examined the effect of estrogen on migration and proliferation of vascular smooth muscle cells (VSMCs) in culture. Intimal thickening was significantly greater in males than in control females. Intimal thickening in females was increased to the level in males by ovariectomy. Estrogen replacement to ovariectomized rats reversed this effect. Proliferating cell nuclear antigen immunohistochemistry showed that in vivo proliferation of VSMCs contributed to the difference in intimal thickening. There was no difference in blood pressure and serum lipids, suggesting that estrogen directly acted on artery and inhibited intimal thickening. 17b-Estradiol (E2, 1–100 nmol/l) inhibited migration of cultured rat VSMCs, assayed using a microchemotaxis chamber, in a concentration-dependent manner. E2 (0.01–100 nmol/l), but not progesterone or testosterone, also inhibited [3H]thymidine incorporation in rat VSMCs in a concentration-dependent manner. Indomethacin, N G-monomethyl-L-arginine and methylene blue did not influence the inhibitory action of E2 on [3H]thymidine incorporation, suggesting that prostanoids and nitric oxide are not involved in the action of E2. E2 did not provoke VSMC injury, as measured by the release of incorporated [3H]2-deoxy-D-glucose. These results suggest that the inhibition of migration and proliferation of VSMCs contributes to the inhibitory effect of estrogen on intimal thickening. © 1997 Elsevier Science Ireland Ltd. Keywords: Estrogen; Smooth muscle cell; Migration; Proliferation; Atherosclerosis
1. Introduction It is an established epidemiological observation that the incidence of atherosclerosis is lower in premenopausal women than in men and increases after menopause [1,2]. Estrogen replacement therapy is effective to protect women from coronary heart disease [2 – 4]. Estrogen has also been reported to inhibit the development of atherosclerosis in animal experiments [5 – 7]. These epidemiological and experimental findings suggest that estrogen has an anti-atherogenic action. The mechanism, however, is not fully understood. * Corresponding author. Tel.: +81 3 38155411 ext. 3223; fax: +81 3 38122097.
A number of investigators have reported that lipid metabolism is improved by estrogen administration [5,6,8–10]. Estrogen increases serum high-density lipoprotein cholesterol level and decreases serum lowdensity lipoprotein cholesterol level. On the other hand, the existence of functional estrogen receptors in vascular endothelial cells [11] and vascular smooth muscle cells (VSMCs) [12–14] suggests that estrogen acts directly on the arterial wall. Estrogen appears to have both endothelium-independent [15,16] and endothelium-dependent [17–19] vasodilator actions. A calcium-antagonistic effect [15,16], regulation of prostacyclin [20,21] and nitric oxide production [22] may be involved in these effects.
0021-9150/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 2 1 - 9 1 5 0 ( 9 6 ) 0 6 0 2 3 - 6
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Migration and proliferation of VSMCs are fundamental steps in the development of atherosclerosis [23] and neointimal formation after endothelial injury [24]. However, the effect of estrogen on migration of cultured VSMCs is not known. Moreover, the effect of estrogen on proliferation of VSMCs is controversial. Fischer-Dzoga et al. [25] and Vargas et al. [26] reported an inhibitory effect of estrogen on VSMC proliferation, while Farhat et al. [27] demonstrated a stimulatory effect of estrogen. We first aimed at investigating the effect of estrogen on cuff-induced intimal thickening of the rat femoral artery. The reason we used this model was that lipid metabolism was not involved in the development of intimal thickening by cuff placement. We further examined the effect of estrogen on migration and proliferation of cultured VSMCs to investigate the mechanism underlying the effect on intimal thickening.
2. Materials and methods
2.1. Chemicals Estradiol dipropionate was obtained from Teikoku Hormone (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO BRL (Gaithersburg, MD) and fetal bovine serum (FBS) from ICN Biomedicals (Osaka, Japan). Bovine serum albumin (BSA), trichloroacetic acid (TCA) and Triton X100 were purchased from Wako (Osaka, Japan). Phenol red-free DMEM, 17b-estradiol (E2), 17a-estradiol, progesterone, testosterone, endothelin-1, human basic fibroblast growth factor (bFGF), indomethacin, N Gmonomethyl-L-arginine (L-NMMA) and methylene blue were purchased from Sigma. Human plateletderived growth factor-BB (PDGF-BB) was from Genzyme (Cambridge, MA) and type V collagen solution from Koken (Tokyo, Japan). [3H]thymidine and [3H]2deoxy-D-glucose (DOG) were from NEN Research (Boston, MA).
2.2. Animals Eight-week-old female and male Wistar rats (Nippon Bio-Supply Center, Tokyo, Japan) were used in this study. They were kept individually in stainless steel cages in a room where lighting was controlled (12 h on, 12 h off) and room temperature was kept at around 22°C. They were given standard diet (CE-2, Japan Clea, Tokyo, Japan) and water ad libitum. All the surgical procedures were performed under ether anesthesia. All of the experimental protocols were approved by the Animal Research Committee of the University of Tokyo.
2.3. Cuff-induced intimal thickening The surgical procedures of cuff placement was according to the method described by Hirosumi et al. [28] with some modification. The left femoral artery of rats was isolated from the surrounding tissues. A polyethylene tube (5 mm long PE-160; inner diameter, 1.14 mm; outer diameter, 1.57 mm; Becton Dickinson, Parsippany, NJ) was cut longitudinally to open the tube and loosely placed around the artery, then the wounds were sutured. The control rats underwent isolation of the femoral artery without cuff placement. After the experimental period, the rats were killed and the femoral artery was excised together with the cuff. The artery was then fixed in 10% neutral formalin and embedded in paraffin. The middle segment of the artery was cut into cross-sectional pieces with 5 mm thickness and stained by Elastica van Gieson staining. Cross-sectional areas of the intima and the media were measured using a digitizer connected to a Macintosh Computer and the ratio of the intimal area to the medial area (I:M ratio) was calculated. In order to check the time course of intimal thickening, male rats were sacrificed 1–4 weeks (n=7–9 for each week) after cuff placement and the magnitude of intimal thickening was evaluated. Control rats without cuff placement were added (n= 2–3 for each week). To examine the effect of estrogen on cuff-induced intimal thickening, female rats were randomly divided into three groups. Two groups of rats were ovariectomized and the other group of rats were sham operated. After a one-week recovery period, one group of ovariectomized rats received subcutaneous injection of estradiol dipropionate (20 mg/kg) suspended in corn oil as vehicle once a week (OVX + E group, n = 7). In the preliminary experiments, administration of this dose of estradiol dipropionate exhibited a change in plasma E2 concentrations as follows: day 0, 42.7 9 6.1; day 1, 109.0912.5; day 3, 95.89 15.6; day 7, 57.894.7; day 8, 162.0920.3; day 10, 140.09 8.3 and day 14, 78.7 9 8.0 pmol/l (n= 4). The other group of ovariectomized rats (OVX group, n= 7) and sham-operated rats (sham group, n= 7) received the same amount of corn oil once a week. Age-matched male rats (male group, n=7) were also given corn oil injection. The three female groups underwent cuff placement one week after ovariectomy or sham operation and the male group also underwent cuff placement at the same time. Histological analysis was performed 2 weeks after cuff placement. At that time, systolic blood pressure was measured by tail-cuff method (UR-1000/UR-5000, Ueda, Tokyo, Japan) and a blood sample was collected from the abdominal aorta after 16 h fasting to measure serum lipids and plasma estradiol concentration. Serum total cholesterol and triglyceride were measured enzymatically, and high-density lipoprotein cholesterol was
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measured by heparin – Ca2 + Ni2 + precipitation method [29]. Plasma estradiol concentration was measured by sensitive radioimmunoassay.
2.4. Immunohistochemistry To examine the involvement of VSMC proliferation on the effects of estrogen on cuff-induced intimal thickening, the immunohistochemical staining of the sections was carried out by streptavidin – biotin – peroxidase method [30]. We used anti-muscle actin antibody (HHF35, 10 mg/ml; Enzo Diagnostics, New York, NY), anti-proliferating cell nuclear antigen (PCNA) antibody (PC10, 10 mg/ml; Boehringer Mannheim Biochemica, Mannheim, Germany) and normal mouse IgG (10 mg/ ml) as the first antibody. Specifically bound antibodies were disclosed by immersing the sections in a substrate solution of 3,3%-diaminobenzine (Vector Laboratories, Burlingame, CA).
2.5. Cell culture In the present study, we used three types of rat VSMCs in culture. Two of them were isolated from the medial layer of the thoracic aorta by enzymatic digestion, according to the method described previously [31], from 8-week-old male and female Wistar rats (Nippon Bio-Supply Center). These cells were identified as VSMCs by their ‘hill-and-valley’ appearance and by immunohistochemical staining using anti-muscle actin antibody (HHF35). Subcultured VSMCs (5 –9th passage) were used in the experiments. The other type was A10 cells, a cell-line derived from fetal rat aorta [32]. We also used MCF-7 cells, a cell-line derived from human breast cancer [33]. All of the cells were maintained in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2. At the time of experiments, we used dextran-coated charcoal-stripped FBS (DCC-FBS) and phenol red-free DMEM to avoid contamination of steroids and estrogen receptor agonist. E2 concentration of non-treated FBS was 109 pmol/l, while that of DCC-FBS was 5.3 pmol/l.
2.6. Migration assay Migration of VSMCs was assayed by a modified Boyden’s chamber method [34] using a 96-well microchemotaxis chamber (Neuro Probe, Cabin John, MD) and polycarbonate filters (Neuro Probe) with pore size of 5.0 mm diameter. The filters were treated with 0.5 mol/l acetic acid overnight and then incubated in type V collagen solution (100 mg/ml) for 72 h. The coated filters were air-dried. Cultured VSMCs derived from male rats were trypsinized and suspended at a concentration of 5 × 105 cells/ml in phenol red-free DMEM containing 0.1% DCC-FBS. VSMC suspension
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(225 ml) was placed in the upper chamber and 35 ml phenol red-free DMEM containing 10% DCC-FBS with E2 (0.1–100 nmol/l) or vehicle (0.1% ethanol) was placed in the lower chamber. The chamber was incubated for 4 h. The filter was removed and VSMCs on the upper side of the filter were scraped off. Then, the filter was fixed in methanol and stained with Diff-Quick staining solution (Green Cross Co., Osaka, Japan). The number of VSMCs migrated to the lower side of the filter was counted under a microscope and the migration activity was expressed as the mean number of cells per 5 high power fields (HPF; magnification ×400).
2.7. Measurement of DNA synthesis DNA synthesis was assayed by measuring [3H]thymidine incorporation [35]. The cells were seeded in 24-well multiplates (Corning, NY) at an initial concentration of 5×104 cells/ml in 0.5 ml of DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The medium was then replaced with phenol red-free DMEM to arrest the growth. After 24 h, the medium was replaced again with phenol red-free DMEM containing [3H]thymidine (1 mCi/ml) and various agents. After incubation for another 24 h, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and subsequently incubated with ice-cold 5% TCA for 20 min at 4°C. The cells were washed twice with ice-cold 5% TCA and then with ice-cold PBS. The cells were lysed with 0.5 mol/l NaOH. The radioactivity of the cell lysate was counted using a liquid scintillation counter (LSC-1000, Aloka, Tokyo, Japan).
2.8. Growth of VSMCs A10 cells were seeded in 12-well multiplates (Corning) at an initial concentration of 5× 104 cells/ml in 1 ml of DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The medium was then replaced with phenol red-free DMEM to arrest the growth. After 24 h, the medium was replaced again with phenol red-free DMEM containing 5% DCC-FBS with E2 (1–100 nmol/l) or vehicle (0.1% ethanol). After incubation for 24–48 h, the cells were trypsinized and suspended. Then the number of cells was determined using a Coulter Counter (model ZM, Coulter Electronics, Hialeah, FL).
2.9. E6aluation of VSMC injury Injury of VSMCs was evaluated based on the release of incorporated [3H]2-DOG from VSMCs as described previously [35]. In brief, VSMCs (A10 cells and male VSMCs) were seeded in 12-well multiplates (Corning) at an initial concentration of 5 × 104 cells/ml in 1 ml of
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Fig. 1. Sex-related difference in cuff-induced intimal thickening of the rat femoral artery. A, representative high magnification photomicrographs (Elastica van Gieson staining; original magnification ×200) of the indicated groups are shown: arrow head, internal elastic lamina; arrow, external elastic lamina. B, bar graph showing the ratios of the intimal area to the medial area (I:M ratio) of each group. n = 7 For each group. * P B 0.05 vs. sham and OVX+E.
DMEM containing 10% FBS per well and cultured until a confluent state was obtained. The cells were incubated with [3H]2-DOG (1 mCi/ml) for 18 h at 37°C and were washed twice with Hanks’ balanced salt solution containing 0.5% BSA. The cells were incubated in Hanks’ balanced salt solution containing 0.5% BSA with E2 (1, 100 nmol/l), vehicle (0.1% ethanol) or 0.2% Triton X-100 for 2 h. The radioactivity in the medium and the cell lysate obtained by treatment with 0.5 mol/l NaOH was counted using a liquid scintillation counter (LSC-1000). The specific release of [3H]2-DOG was determined as 100 ×(A − C)/(B −C) (%), where A is the radioactivity in the medium treated with E2 or Triton X-100, B is the total radioactivity in the medium and the cell lysate treated with E2 or Triton X-100 and C is the radioactivity in the medium treated with vehicle.
2.10. Data analysis All of the migration, [3H]thymidine incorporation, cell count and cell injury assays were repeated three times or more and the representative results are shown. The values in the text, figures and table are expressed as mean 9S.E.M. The data were analyzed using one-factor ANOVA. If a statistically significant effect was
found, Newman-Keuls’ test was performed to isolate the difference between the groups. PB 0.05 was considered to be significant.
3. Results
3.1. Sex-related difference in intimal thickening Cuff placement induced diffuse intimal thickening of the rat femoral artery, while no intimal thickening was observed in the control rats without cuff placement. We checked the time-related change in the development of intimal thickening. The I:M ratio was significantly higher 2 weeks after than 1 week after cuff placement (PB0.05) and then tended to decrease until 4 weeks after cuff placement: 1 week, 0.119 0.01 (n=8); 2 weeks, 0.339 0.07 (n= 9); 3 weeks, 0.2590.05 (n=8); 4 weeks, 0.219 0.02 (n= 7). Based on this result, we decided to examine sex difference and the effect of estrogen on intimal thickening 2 weeks after cuff placement. As shown in Fig. 1A and B, intimal thickening was greater in the male than in the sham group. Intimal thickening was enhanced in the OVX compared with those in the sham and OVX+ E groups. All of the
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shown in Table 1, no differences were found in systolic blood pressure and serum total cholesterol, high-density lipoprotein cholesterol and triglyceride levels among the four groups of rats. Plasma estradiol concentration was significantly lower in the OVX and in the male than in the sham group. Plasma estradiol concentration in the OVX+ E was comparable to that in the sham group.
3.2. Effects of E2 on migration and proliferation of cultured VSMCs
Fig. 2. Hematoxylin staining (A) and immunohistochemical staining of cuff-induced intimal thickening using anti-muscle actin antibody (B), anti-proliferating cell nuclear antigen (C) and normal mouse IgG (D). Representative high magnification photomicrographs (original magnification × 200) of ovariectomized rats are shown. arrow head, internal elastic lamina; arrow, external elastic lamina.
neointimal cells as well as medial cells were positively stained with anti-muscle actin antibody, indicating that these cells were VSMCs (The representative photomicrograph is shown in Fig. 2B). The nuclei positive for PCNA staining were observed in the neointima (Fig. 2C). As shown in Fig. 3, PCNA-positive cells in the neointima were increased in the male and OVX compared with those in the sham and OVX + E groups. As
Fig. 3. Sex-related difference in the number of PCNA-positive cells in the neointima. n = 7 For each group. * PB 0.05 vs. sham and OVX +E.
As shown in Fig. 4A, 1–100 nmol/l E2 significantly inhibited migration of male VSMCs in a concentrationdependent manner. Similar concentration of progesterone or testosterone did not show the inhibitory effect on migration (data not shown). As shown in Fig. 4B, 0.01–100 nmol/l E2 also inhibited DNA synthesis in male VSMCs in a concentration-dependent manner. To examine the effect of E2 on DNA synthesis in other cell types, we used three different types of rat VSMCs (male VSMCs, female VSMCs and A10 cells) and MCF-7 cells. As shown in Fig. 5A, E2 (1–100 nmol/l) inhibited DNA synthesis in all of the three types of VSMCs, while E2 did not inhibit DNA synthesis in MCF-7 cells and 100 nmol/l E2 significantly stimulated DNA synthesis in MCF-7 cells as previously reported [33]. In the following experiments, we investigated the mechanism underlying the inhibitory effect of E2 on DNA synthesis. For this purpose, we used A10 cells because they are well characterized [32]. We compared the effect of E2 with those of other sex steroids (Fig. 5B). Unlike 17b-estradiol (E2), 17a-estradiol had no effect on DNA synthesis. No significant change in DNA synthesis was observed when 1–100 nmol/l progesterone or testosterone was applied. To check whether the inhibition by E2 was specific to a certain growth factor, we used various stimuli including 5% DCC-FBS, 10 ng/ml PDGF-BB, 10 ng/ml bFGF and 1 mmol/l endothelin-1 in the presence of 0.2% BSA. As shown in Fig. 5C, E2 (1 nmol/l) completely inhibited the increase in DNA synthesis stimulated by these substances. No inhibition was observed when any of these stimuli was not applied. We examined the possible involvement of prostacyclin and nitric oxide because they are known to inhibit VSMC proliferation [36,37]. E2 has been reported to stimulate the production of these substances [20–22]. As shown in Fig. 5D, simultaneous addition of indomethacin (10 mmol/l), a cyclooxygenase inhibitor, with E2 (1 nmol/l) did not affect the inhibition of DNA synthesis by E2. As shown in Fig. 5E, L-NMMA (10 mmol/l), an inhibitor of nitric oxide production, or methylene blue (10 mmol/l), an inhibitor of cyclic GMP production, did not affect the inhibition of DNA synthesis by E2 as well.
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Table 1 Blood pressure, serum lipids and plasma estradiol concentration in rats with cuff placement
Sham OVX OVX+E Male
SBP (mmHg)
T.chol (mg/dl)
HDL-C (mg/dl)
TG (mg/dl)
E2 (pmol/l)
13893 13593 13993 14092
72.39 2.3 79.4 9 4.2 80.3 9 3.7 68.49 2.3
52.0 9 3.6 56.8 9 3.2 55.3 9 4.6 56.9 9 2.9
27.6 9 2.6 28.0 9 3.0 22.7 9 1.7 26.4 9 1.5
133 927 41 96* 82 918 28 93*
Values are expressed as mean9S.E.M. (n= 7). *, PB0.05 vs sham. SBP, systolic blood pressure; T.chol, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglyceride.
We also checked the change in cell numbers induced by 5% DCC-FBS. As shown in Fig. 6, the increase in cell numbers was significantly inhibited by 40% by incubation with 100 nmol/l E2 for 24 h compared with
vehicle. The inhibition was more marked with incubation for 48 h, and 1, 10 and 100 nmol/l E2 significantly inhibited the increase in cell numbers compared with vehicle by 16, 49 and 68% respectively.
3.3. VSMC injury by E2 As shown in Fig. 7, the specific release of [3H]2-DOG from A10 cells by incubation with E2 (1, 100 nmol/l) was not significantly different from the control, while that by incubation with 0.2% Triton X-100 was markedly elevated. Similar results were obtained when male VSMCs were used (data not shown).
4. Discussion
Fig. 4. Effect of E2 on migration (A) and DNA synthesis (B) of male vascular smooth muscle cells. A, Migration activity was assayed using a chemotaxis chamber as described in Section 2, n= 3. * PB 0.05 vs. vehicle. Cells/HPF, migrated cells per high power field (magnification × 400). B, DNA synthesis was assayed as [3H]thymidine incorporation induced by 5% dextran-coated charcoal-stripped fetal bovine serum for 24 h, n =8. *, ** PB 0.05, 0.01 vs. vehicle.
In the present study, we showed that intimal thickening of the rat femoral artery induced by cuff placement was greater in males than that in control females. Intimal thickening of females was increased by ovariectomy and this increase was reversed by estrogen replacement. Arterial intimal thickening induced by a polyethylene cuff has been reported in the rabbit femoral artery [38] and in the rabbit carotid artery [28]. Histological observations in these studies elucidate that cuff placement causes polymorphonuclear leukocyte infiltration and endothelial injury in the initial step by inflammatory responses. The next important processes for the development of intimal thickening are the migration of medial VSMCs to the intima and the proliferation of migrated VSMCs with deposition of extracellular matrix in the neointima. These steps are similar to those observed in the development of atherosclerosis in man [23]. Therefore, intimal thickening induced by cuff placement might be an adequate model to facilitate understanding atherosclerosis. We checked the time course of polyethylene cuff-induced intimal thickening of the rat femoral artery and the peak of intimal thickening was observed 2 weeks after cuff placement. Therefore, we examined sex-related difference at 2 weeks. The physiological concentration of plasma E2 in female rats was 0.139 0.03 nmol/l according to the
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Fig. 5. (A) Effect of E2 on DNA synthesis in MCF-7 cells, male vascular smooth muscle cells (VSMCs), female VSMCs and A10 cells induced by 5% dextran-coated charcoal-stripped fetal bovine serum (DCC-FBS) n =6. * P B0.05 vs. vehicle. (B) Effect of E2, 17a-estradiol, progesterone and testosterone on DNA synthesis in A10 cells induced by 5% DCC-FBS n =6. * PB 0.05 vs. vehicle. (C) DNA synthesis in A10 cells induced by 5% DCC-FBS, PDGF-BB (10 ng/ml), bFGF (10 ng/ml), endothelin-1 (1 mmol/l) or vehicle (0.2% BSA) with (closed columns) or without (open columns) E2 (1 nmol/l) n =6. * PB 0.05 vs. vehicle; n.s., not significant. (D) Effect of E2 on DNA synthesis in A10 cells induced by 5% DCC-FBS in the absence or presence of indomethacin (10 mmol/l). closed column, E2 1 nmol/l; open column, vehicle n =6. (E) Effect of E2 on DNA synthesis in A10 cells induced by 5% DCC-FBS in the absence or presence of L-NMMA (10 mmol/l) or methylene blue (10 mmol/l). closed column, E2 1 nmol/l; open column, vehicle, n = 6.
results of this study. This is comparable to the previously reported value [39]. Plasma E2 concentrations in estrogen-replaced rats were at the same level as those in control females throughout the experiment. These results indicate that a physiological concentration of estrogen inhibits intimal thickening in female rats. Recently, Sullivan et al. [40] reported that estrogen replacement inhibited the response of the mouse carotid artery to wire injury and Chen et al. [41] reported that estrogen reduced myointimal proliferation after balloon injury of the rat carotid artery. Even though the animals used [40] and the method of vascular injury [40,41]
are different from ours, their studies are consistent with our results. We did not administer estrogen in males, thus it is unclear whether the lack of endogenous estrogen in males augmented intimal thickening. Testosterone might have influenced intimal thickening because exogenous [42] and endogenous [43] testosterone is reported to accelerate atherosclerosis. However, Chen et al. [41] reported that endogenous or exogenous testosterone did not alter, but estrogen attenuated, neointimal formation in male and female rats. Although their data indicate that the sex difference in neointimal for-
8
M. Akishita et al. / Atherosclerosis 130 (1997) 1–10
Fig. 6. Effect of E2 on vascular smooth muscle cell growth. Growtharrested A10 cells were incubated in phenol red-free Dulbecco’s modified Eagle’s medium containing 5% dextran-coated charcoalstripped fetal bovine serum with E2 (1–100 nmol/l) or vehicle (0.1% ethanol) for 24 – 48 h, then the number of cells was counted, n = 4. *, **; P B0.05, 0.01 vs. vehicle
mation is estrogen dependent, further study is needed to clarify whether testosterone contributes to the sex difference in cuff-induced intimal thickening. It is suggested that estrogen exhibits anti-atherogenic action by improving several risk factors, especially serum lipid concentration [5,6,8 – 10] and blood pressure
Fig. 7. VSMC injury incubated with 1 or 100 nmol/l E2, vehicle (0.1% ethanol) or 0.2% Triton X-100 for 2 h. VSMC injury was assayed as the specific release of incorporated [3H]2-DOG, n =6. *** P B 0.001 vs. vehicle.
[44–46]. In our experiment, however, no differences were found in serum lipid levels and systolic blood pressure among the groups. Serum lipids were considered to be measured accurately because they were comparable to the previously reported values in male [47] and female [48] Wistar rats. Thus, the inhibitory effect of estrogen on intimal thickening was not due to an effect on these risk factors. Since the arterial wall is reported to contain functional estrogen receptors [11– 14], it is possible that estrogen may act directly on the arterial wall to inhibit intimal thickening. We further performed an immunohistochemical study to evaluate which components contributed to the effects of estrogen on intimal thickening. We used anti-muscle actin antibody (HHF35) as a marker of VSMCs and anti-PCNA antibody (PC10) as a marker of cell proliferation [49]. Immunostaining with antimuscle actin antibody showed that neointimal cells were composed of VSMCs. Furthermore, PCNA-positive cells in the neointima were increased in males and ovariectomized females compared with those in control females and estrogen-replaced females. This data indicates that proliferation of VSMCs contributed to the effects of estrogen on intimal thickening. Sullivan et al. [40] and Chen et al. [41] also demonstrated that inhibition of VSMC proliferation was involved in the effects of estrogen using bromodeoxyuridine uptake [40] or c-myc expression [41]. In order to further investigate the mechanisms involved in the inhibitory action of estrogen on the development of cuff-induced intimal thickening, we examined the effects of this hormone on migration and proliferation of VSMCs in vitro. We found that estrogen inhibited both migration and proliferation of rat VSMCs in a concentration-dependent manner. This inhibition was not due to a toxic effect of estrogen, because sex steroids other than E2 had no effect, and VSMC injury incubated with 1 or 100 nmol/l E2 was the same as that with vehicle. To our knowledge, this is the first report demonstrating the inhibitory effect of E2 on migration of cultured VSMCs. The effect of estrogen on proliferation of VSMCs is controversial. Fischer-Dzoga et al. [25] reported that E2 (73 nmol/l) inhibited [3H]thymidine incorporation and growth of cultured VSMCs from rabbit aorta induced by hyperlipemic serum, and Vargas et al. [26] reported that E2 (180–360 nmol/l) inhibited [3H]thymidine incorporation in pig coronary artery segments. These results are in agreement with ours, although they used much higher concentration of E2 than in our study. On the other hand, Farhat et al. [27] reported that E2 (3–300 nmol/l) potentiated [3H]thymidine incorporation both in canine pulmonary artery segments and in cultured VSMCs from the rat pulmonary artery. The reason why the responses to E2 differ in these experiments may be attributed to the origin of the arteries which were investigated.
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The effective concentration of E2 in the thymidine incorporation study (0.01 nmol/l) was within the physiological range, while those in the cell count study (1 nmol/l) and the migration study (1 nmol/l) somewhat exceeded the physiological range. The concentrations of E2 used in our experiments, however, were significantly lower than those previously reported [25,26]. It is not known why the effective dose of E2 differed among the thymidine incorporation study, the cell count study and the migration study. The difference may depend on the conditions of the experiments or the sensitivities of the assays. Additionally, other factors might have influenced intimal thickening. Estrogen is reported to inhibit adhesion of leukocytes to endothelial cells in vitro [50]. Estrogen replacement improves endothelium-dependent vasomotor responses [17 – 19] suggesting that estrogen could protect endothelial cells from injury. Moreover, estrogen decreases collagen synthesis in cultured VSMCs [51] and in atherosclerotic aortas [52]. Leukocyte infiltration, endothelial injury and synthesis of extracellular matrix are important steps in the development of intimal thickening as described above. Therefore, it is possible that the results of the cuff experiment might be synergistic effects of E2 on these factors as well as migration and proliferation of VSMCs and thus, the discrepancy of the effective doses of E2 could be explained. In thymidine incorporation studies we showed that E2 generally inhibited DNA synthesis in rat VSMCs but not in MCF-7 cells. The question arises as to why the response of VSMCs to E2 is different from that of MCF-7 cells. It is conceivable that the product stimulated by E2 may differ between cell types. Estrogen is reported to stimulate synthesis of specific proteins (not characterized) which may regulate the growth of MCF7 cells [53–55], whereas the existence of these proteins is not known in VSMCs. Production of some substance that inhibits cell growth may be induced in VSMCs by E2. Both prostacyclin and nitric oxide are candidates [20–22]. However, in the present study, prostacyclin or nitric oxide was not involved in the growth-inhibitory effect of E2, because indomethacin, L-NMMA or methylene blue did not affect the action of E2. In the present study, we also showed that the inhibitory effect of E2 on DNA synthesis was not specific to a certain growth factor. Therefore, it is possible that E2 inhibits VSMC proliferation by blocking some common pathway of growth stimulation, although the underlying mechanism still remains to be explored. In conclusion, estrogen inhibited cuff-induced intimal thickening of rat femoral artery. Estrogen also inhibited migration and proliferation of rat VSMCs in culture. Inhibition of VSMC migration and proliferation by estrogen may contribute to the anti-atherogenic effect of estrogen.
9
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