Estrogen Effects on Insulin-Like Growth Factor-I (IGF-I)–Induced Cell Proliferation and IGF-I Expression in Native and Allograft Vessels
Background Estrogen protects against cardiovascular disease in both patients and animal models and regulates insulin-like growth factor-I (IGF-I), an important cell-cycle progression factor.
Methods and Results Smooth muscle cells and tissues were harvested from male recipient rabbits that 6 weeks earlier had received a cardiac allograft transplant consisting of a donor heart and ascending aorta. Segments of the ascending aorta from the native and allograft hearts from 9 placebo-treated and 8 estradiol-treated recipients were compared by using IGF-I–stimulated [3H]thymidine incorporation. The responses of the native vessel segments were similar (175.3±32% and 166.9±41%, respectively; P>.05) whether or not the recipients had been treated for 6 weeks with estradiol. In the grafts, however, estradiol markedly inhibited vascular cell thymidine incorporation (328.04±56% compared with 67.3±11%; P<.02). Smooth muscle cells were derived from the native aorta of the placebo-treated rabbits to study the effect of estradiol in vitro. IGF-I increased cell counts in a concentration-dependent manner. In serum-starved cells estradiol further decreased cell proliferation; this effect was blocked by the specific estrogen receptor antagonist ZK-119.010. Immunohistochemistry staining for IGF-I protein in the coronary arteries and ascending aorta of the cardiac allograft from the placebo-treated recipients revealed extensive IGF-I expression in the myointima. In contrast, IGF-I protein was not expressed in the coronary arteries and ascending aorta of the cardiac allograft from the estradiol-treated recipients. The IGF-I protein was extensively expressed only in the placebo-treated graft vessels. Myointimal thickening of the coronary arteries was significantly reduced by estradiol treatment (17.9±1.5% versus 44.3±3.7%; P<.02).
Conclusions In vivo estradiol treatment abolishes both IGF-I mitogenic effects and IGF-I protein expression in the vascular wall, which may be causally related to the inhibitory effect of estradiol on transplant arteriosclerosis.
The protective role of estrogen against cardiovascular disease in women is well documented.1 2 We and others find experimentally that estrogen treatment suppresses vascular intimal hyperplasia following balloon injury3 4 5 6 and cardiac transplantation.7 Vascular SMC proliferation is an important component of this process and also of the progression of arteriosclerosis.8 9 IGF-I is one of the potent growth regulators of vascular SMC proliferation. This growth factor is only modestly expressed in normal adult vessels and growth-arrested cells.10 11 However, significantly increased expression of IGF-I is found in SMCs contained in human restenotic coronary atherectomy plaques.12 Further, there is increased IGF-I gene expression after balloon injury13 14 and after induction of hypertension.11 The mitogenic effect of IGF-I has been convincingly demonstrated in SMCs in vitro15 16 and in arterial explants.17
The purpose of this study was to demonstrate that the inhibitory effect of estrogen on SMC proliferation is associated with attenuation of the mitogenic effect of IGF-I and the expression of IGF-I in cardiac allografts.
Male New Zealand White rabbits weighing 3.0 to 3.5 kg received cardiac grafts from male Dutch Belted rabbits weighing 1.8 to 2.3 kg (Hazleton Research Products Inc). The animals were housed separately at constant room temperature with a 12-hour light cycle in a facility approved by the American Association for Accreditation of Laboratory Animal Care. The rabbits were fed a 0.5% cholesterol diet for 7 days prior to transplantation. The cardiac allograft recipients continued this diet until they were killed. Heterotopic cardiac transplantation was performed using right-sided end-to-side aorta–carotid artery and pulmonary artery external jugular vein anastomosis. Cyclosporin A (Sandoz) 10 mg·kg−1·d−1 was given as an immunosuppressant from the time of transplantation until death. Penicillin G 150 000 U IM (Wyeth Laboratories, Inc) was administered before surgery to prevent infection.
The animals were divided into two experimental groups: one group (n=8) received estradiol cypionate (Upjohn) 100 μg·kg−1·d−1 IM, and the other (n=9) received placebo. The recipients were killed 42 days following transplantation.
Cardiac Allograft Tissues
At the time of death the cardiac allografts were isolated and pressure-perfused with tissue fixative (Histochoice, Amresco). Five serial cross sections were obtained from each cardiac allograft and embedded in paraffin blocks. Slides (3 μm) were cut from each block for immunohistochemistry study and stained with hematoxylin-eosin or elastin stain for histology and morphometry analysis.
IGF-I Mitogenic Study in Aorta Explants
The aorta explants from the cardiac allograft and the native hearts of 9 placebo-treated and 8 estradiol-treated recipients were harvested at the time of death, cleaned of adherent tissue, and divided into 5-mm segments under sterile conditions. These segments were incubated overnight at 37°C in serum-free and phenol red–free IMEM and then incubated for 24 hours in fresh IMEM with or without IGF-I (20 ng/mL; UBI). [3H]thymidine (2 μCi; Amersham) was added to the culture medium 6 hours before harvesting. The explant segments were washed with PBS (pH 7.4) and incubated in 1 nmol/L thymidine for 30 minutes to remove nonspecific [3H]thymidine binding. Each segment was subsequently digested in 0.5N NaOH at 60°C overnight, and radioactivity was counted in duplicate in a scintillation counter (TR1600). Protein concentration was measured by using Lowry’s method.18 [3H]thymidine incorporation in the vessels was calculated and expressed as counts per minute per milligram of protein.
Primary vascular SMC cultures were obtained from the native aorta of the placebo-treated recipient rabbits. The vessels were removed, rinsed with ice-cold PBS, and cleaned of adherent tissue. The endothelial cells were gently removed by forceps. Each vessel was dissected into 3- to 5-mm segments and placed in a 24-well plate with the luminal side down. The segments were incubated in a growth medium (DMEM, 10% FCS, 2 mmol/L glutamine, 10 U/mL penicillin, and 10 U/mL streptomycin) at 37°C until one third of the surface was covered by the outgrown cells. The segments were then removed, and the cells were trypsinized and transferred to a flask with growth medium to continue growing. After verifying that the cells displayed a characteristic SMC α-actin immunoreactivity, the second through the sixth passages of SMCs were seeded (104 or 105 cells/well) with growth medium overnight in 96-well plates for measuring [3H]thymidine incorporation or in 6-well plates for cell counting. All experiments described below in SMC cultures were performed in triplicate and repeated three times. The results are expressed as the mean of the three different experiments.
IGF-I Mitogenic Study in SMC Cultures
After >24 hours of incubation, the seeded SMCs remained subconfluent (80%). The cells were washed three times with IMEM and remained quiescent in IMEM for 48 hours. The medium was then substituted with IGF-I in concentrations of 20, 50, and 75 ng/mL for 24, 48, and 75 hours in 5% CCS in IMEM. For the specific IGF-I study, anti–IGF-I monoclonal antibody (10 μg/mL; Upstate Biotechnology, Inc) was added to the medium in the presence or absence of IGF-I (20 ng/mL) in the cell culture for 48 hours. At the end of each time period, cell growth was examined under the microscope to ensure that the cells were intact. The cells were then washed with PBS, trypsinized, and counted (Coulter Electronics). DNA synthesis analysis was performed as follows: 1μCi/mL [3H]thymidine was added to the medium 24 hours before harvesting. At the time of harvest, cells were washed three times with PBS, and cellular DNA was precipitated with ice-cold 10% trichloroacetic acid for 30 minutes. After washing in 95% ethanol and air drying, the DNA was solubilized in 0.5N NaOH. Radioactivity and protein concentration were measured in the same manner as described for the explants, and thymidine incorporation in the cell culture was calculated. Data are expressed as percent changes in comparison with nonstimulated SMC cultures.
Estradiol Effects on IGF-I–Induced SMC Proliferation In Vitro
After SMCs were quiescent for 48 hours, the medium was replaced by 1 μmol/L 17β-estradiol with 5% CCS/IMEM, and incubation was continued for 3 hours. Different concentrations of IGF-I were then added as described above. In a separate experiment, half the cells were exposed to the competitive estrogen receptor antagonist ZK-119.010 (5 μmol/L; Schering AG) in IMEM for 3 hours, after which the medium was replaced with different concentrations of estradiol (10−9 to 10−6 mol/L) in 5% CCS/IMEM. Cell proliferation was measured after 48 hours and expressed as percent changes in cell count.
Immunohistochemistry staining for IGF-I was performed by using a monoclonal IGF-I antibody (3D1/2/3; final dilution, 1:10 000) from the National Institute of Diabetes and Digestive and Kidney Disease. Preimmune mouse IgG (10 μg/mL; Sigma) served as a negative control. A histostain SP kit (Zymed) was used according to the protocol provided by the manufacturer. Briefly, the paraffin-embedded cardiac cross sections were dewaxed in xylene and rehydrated in a series of concentrations of ethanol. Endogenous peroxidase activity was reduced in 3% hydrogen peroxide/methanol. IGF-I protein was detected by incubating with primary antibody for 1 hour, with biotin-labeled secondary antibody for 30 minutes, and then with a streptavidin-peroxidase reaction system.
Semiquantitation of the localization of IGF-I was determined by observing the intensity of the positive staining in the myocardium and coronary arteries. Staining was graded as follows: (1) negative (same as negative control); (2) weak (with single scattered red staining); (3) moderate (patched staining in <25% of the area); and (4) extensive positive (>25% of the area positively stained and color of the staining stronger than moderate staining).
Morphometry was performed on elastin-stained cross sections of the cardiac allograft. The area of intimal thickening was determined in cross sections of the coronary arteries by using computerized morphometric analysis (The Morphometer, Woods Hole Educational Associates). Measurements were taken from a total of 30 coronary arteries in 5 cross sections in each cardiac graft. The intimal thickness was averaged from 8 grafts in each group and expressed as the myointimal area/total vessel area×100%.
Statistical Analysis and Significance
All immunostaining measurements and scoring were performed blindly by two investigators. In the event of disagreement, the sections were reexamined, and a consensus was reached. Unless otherwise noted, comparisons between estradiol- and placebo-treated groups were done by using an ANOVA and unpaired Student’s t test; a probability value of <.05 was considered significant. All in vitro experiments were performed in triplicate and repeated three times, and all values are expressed as mean±SEM.
Estradiol Effects on IGF-I Mitogenicity for Allograft and Native Arterial Explants
IGF-I (20 ng/mL) significantly increased DNA synthesis as measured by [3H]thymidine incorporation in both native (175.3±32%; P<.05) and allograft (328±56%; P<.005) aorta explants from 9 placebo-treated recipients ex vivo (Fig 1⇓). The mitogenic effect of IGF-I was substantially larger in the allografts than in the native vessels. The IGF-I–induced cell proliferation of the allografts was completely abrogated by chronic estradiol treatment of the recipients (n=8) (from 328±56% to 67.3±1%; P<.02). In contrast, the mitogenic effect of IGF-I was unaffected in the native vessel explants from recipients receiving chronic estradiol treatment (166.9±41% versus 175.3±32% in the native vessel explants from the placebo-treated recipients; P>.05).
Estradiol Effects on IGF-I Mitogenicity for Aorta SMCs in Culture
Exogenous IGF-I also induced rabbit aorta SMC proliferation in 5% CCS/IMEM in a dose-dependent manner as measured by cell count at 24 and 48 hours (Fig 2⇓). At 24 hours a significant proliferative effect was seen at the two higher IGF-I concentrations, 50 ng/mL (140.6±16%; P<.05) and 75 ng/mL (148.7±17%; P<.02) (Fig 2A⇓). At 48 hours all three doses induced a significant increase in cell number: 158.9±16% increase at 20 ng/mL, 176.0±20% at 50 ng/mL, and 261.7±33% at 75 ng/mL (Fig 2B⇓). At 72 hours the cell proliferation was still seen, but the dose-dependent effect was no longer apparent (Fig 2C⇓). The specific monoclonal anti–IGF-I antibody (10 μg/mL) abolished the mitogenic effect of 20 ng/mL IGF-I in SMCs in vitro from 258±45% to 117±19% in three separate experiments at 48 hours (P<.05; Fig 3⇓).
Preincubation with estradiol (1 μmol/L) for 3 hours inhibited IGF-I–induced SMC proliferation at 24, 48, and 72 hours. At 24 hours a significant inhibition was seen at the two higher doses of IGF-I, from 140.6±15% to 99.0±8.5% at 50 ng/mL (P<.02) and from 148.7±17% to 111.9±8% at 75 ng/mL (P<.05; Fig 2A⇑). At 48 hours, SMC proliferation was reduced to 116.5±26% at IGF-I 50 ng/mL (P<.05) and to 87.0±16% at 75 ng/mL (P<.02; Fig 2B⇑). At 72 hours the mitogenic effect of IGF-I at all three concentrations was completely abolished by estradiol (Fig 2C⇑).
Estradiol inhibited aorta SMC growth in 5% CCS/IMEM in a dose-dependent manner and was significant at 100 nmol/L (71±8%; P<.05) and 1 μmol/L (61±7%; P<.02). The inhibitory effect of estradiol was reversed by the specific estrogen receptor antagonist ZK-119.010 at 5 μmol/L (Fig 4⇓).
Estradiol Effects on IGF-I Expression in Allograft Vessels
Chronic estradiol treatment inhibited endogenous IGF-I expression in allograft coronary arteries 6 weeks after cardiac transplantation. The entire myointima of the ascending aorta of the cardiac allograft from the placebo-treated recipients exhibited extensive positive staining with the anti–IGF-I antibody (Fig 5A⇓); the media of the vascular wall remained negative. IGF-I was not detected in arteries in the native hearts of the recipients in the placebo-treated group (Fig 5B⇓). Strong IGF-I immunostaining was seen in the myointima and adventitia in the coronary arteries in the grafts from the placebo-treated recipients (Fig 5C⇓). In contrast, negative staining was observed in the allograft coronary arteries from the estradiol-treated recipients (Fig 5D⇓).
Estradiol Effects on IGF-I Expression in Allograft Myocardium and Myointimal Hyperplasia
IGF-I immunostaining was positive in allograft myocardium in the placebo-treated recipients and showed a nonhomogeneous distribution. Chronic estradiol treatment reduced IGF-I protein expression in allograft myocardium (Fig 5F⇑) compared with that of placebo-treated recipients (Fig 5E⇑).
Estradiol treatment significantly reduced the myointimal thickening of the coronary arteries from 44.3±3.7% in control to 17.9±1.5% in the estradiol-treated recipients (P<.02).
We found that inhibition of transplant arteriosclerosis by chronic estradiol treatment is associated with abolition of the mitogenic effect of IGF-I in allografts and with inhibition of the expression of IGF-I in allograft arteries. We showed that the mitogenic effect of IGF-I on SMCs is specific since it was abolished by an IGF-I monoclonal antibody. The inhibitory effect of estradiol on SMC proliferation was also demonstrated to be specific in that a pure estrogen receptor antagonist reversed this inhibitory effect.
The current study also demonstrated that exogenous IGF-I accelerates DNA synthesis in both native and allograft aorta explants ex vivo. The strong proliferative response to IGF-I in the allograft vessel is probably due to proliferating SMCs. IGF-I, as a progression factor, is necessary for cells to enter the S phase of the cell cycle,16 19 but it is thought to be nonessential for the earlier entry from the G0 into the G1 phase of the cell cycle. Platelet-derived growth factor, a competence factor, promotes IGF-I mRNA expression in SMCs10 ; platelet-derived growth factor as well as fibroblast growth factor increase the number of IGF-IRs on vascular SMCs.20 These growth factors, by upregulating IGF-IRs, may sensitize cells to the effects of exogenous IGF-I, leading to accelerated cell-cycle progression.21 22 We obtained a concentration-dependent cell proliferation at 24 and 48 but not 72 hours, at which time cells were confluent. This suggests that the mitogenic effect of IGF-I is influenced by the phase of the cell cycle and confluence of the SMC culture.
The contribution of proliferating macrophages in the aorta allograft may explain some of the increased DNA synthesis in the allograft vessel compared with the native vessel. However, although allografts from recipients treated with estradiol do contain macrophages and adventitial fibroblasts,23 they failed to respond to IGF-I with increased proliferation, supporting the hypothesis that the proliferative response, at least in part, is due to SMC proliferation.
The temporal relationship between the development and progression of myointimal hyperplasia and IGF-I appearance in the allograft arterial wall is not clear. IGF-I is known to be a potent migration factor and mitogen for SMCs.15 16 24 In normal vessels IGF-I expression is weak and is not found in the intima and media but occasionally in the adventitia.11 However, following balloon injury, IGF-I protein and IGF-I mRNA increase in the aorta within 24 hours to reach a maximum at 4 to 5 days after injury.13 14 25 In human coronary atheromatous plaques obtained by atherectomy, an intense IGF-I protein expression has been found and localized to synthetic SMCs.12 The extensive presence of IGF-I in the entire myointimal area of the arteries of the cardiac allografts from all the placebo-treated recipients was associated with substantial myointimal hyperplasia. These findings suggest that the ongoing presence of the mitogenic growth factor IGF-I is important for the development of transplant arteriosclerosis.
The substantial inhibition of IGF-I expression in the graft coronary arteries induced by chronic estradiol treatment of the recipients was associated with decreased transplant arteriosclerosis. To confirm that the increased DNA synthesis in the aorta explants was a result of SMC proliferation, the same study was performed in vitro by using rabbit SMCs derived from the placebo-treated recipient native aortas. Similar significantly increased rates of cell proliferation were obtained. This mitogenic effect of IGF-I was abolished by coincubation with a specific monoclonal anti–IGF-I antibody, confirming the specific mitogenic role of IGF-I in SMC proliferation. Recent studies support the likelihood that blocking IGF-I or IGF-IRs will decrease SMC proliferation. For example, introducing an antisense IGF-IR oligonucleotide,26 an antisense IGF-IR expression cassette,27 and a D-analog of IGF-I25 28 29 have all achieved the same antimitogenic effect. These results further suggest that attenuation of IGF-I expression is linked to the inhibition of transplant arteriosclerosis and that the mechanism for the inhibition of transplant arteriosclerosis by chronic estradiol treatment is by preventing IGF-I expression.
It is interesting that chronic estradiol treatment completely abrogated cell proliferation induced by IGF-I in the allograft but had no effect on the mitogenic effect of IGF-I in the native aorta. This may suggest that estradiol treatment reduces the growth rate in synthetic phenotype cells but not in contractile phenotype cells, as has been shown in osteoblastic (UMR106)29 and breast cancer (MC3T3-E1)30 cell lines. This suggests that the beneficial role of estradiol is specifically targeted to the proliferating rather than the normal cells. We speculate that the allograft SMCs are mostly in the progression phase, where they are targets for estradiol. Epifanova31 has demonstrated in a classic estrogen target cell that estradiol treatment decreases the time the cells spend synthesizing new DNA, ie, shortening both the G1 and S phases, which would increase cell proliferation. This contrasts with our finding of estradiol-induced inhibition of proliferation in SMCs, a nonclassic estrogen target cell. In addition to the effects of estradiol being exerted through transcriptional mechanisms, more recently rapid nongenomic estradiol effects on cell membranes, which block voltage-dependent calcium channels, have been suggested.32 However, such nongenomic, nonreceptor-mediated mechanisms are unlikely in our in vitro system, in which a specific estrogen receptor antagonist reversed the estradiol inhibition of SMC proliferation.
In vitro, estradiol inhibited SMC proliferation in a concentration-dependent manner. The significant inhibition was found at 100 nmol/L (P<.05) and 1 μmol/L (P<.02) estradiol. When adding 1 μmol/L estradiol to the medium 3 hours before introducing IGF-I, the SMC proliferation induced by three different concentrations of IGF-I was abolished after 24 to 72 hours. This inhibition was similar to the effect of chronic estradiol treatment on aorta explants ex vivo. It is also in agreement with the previous observation by our group4 7 and others5 6 33 that estradiol treatment significantly suppresses arterial SMC proliferation after injury.
A nonhomogeneous type of IGF-I staining was also present in the myocardium of the cardiac allografts from placebo-treated recipients, but chronic estradiol treatment decreased IGF-I expression in the myocardium of the cardiac allografts. This difference in IGF-I expression may partially be due to a lesser degree of mononuclear cell infiltration found in the cardiac allografts from the estradiol-treated recipients (2.08±0.27) versus the placebo group (2.72±0.12; P<.05).23 Reiss et al34 report that after acute myocardial infarction both IGF-I and IGF-IR mRNA and protein are unregulated in myocytes and coronary arteries and that this lack of regulation is associated with an increase in proliferating cell nuclear antigen mRNA and mitotic images in myocytes. The IGF-I autocrine system is thought to modulate myocyte repair after ischemia/reperfusion injury following transplantation.35 In our study, chronic estradiol treatment reduced IGF-I expression and still protected the myocytes, which showed no myocyte necrosis at 6 weeks after transplantation.
Little information is available regarding the relationship between estradiol and IGF-I in SMCs. Most studies have focused on the reproductive organs and other classic estrogen-sensitive cells and tissues. In these cells or tissues estradiol is often related to an increase in the effect of IGF-I,36 as shown in breast cancer cells37 and in vivo in the uterus.38 39 However, inhibitory effects of estradiol on serum IGF-I are seen after oral estrogen treatment in postmenopausal women.40 More recently, estrogen has been shown to induce repression of IGF-I gene transcription41 42 43 ; the IGF-I gene promoter is negatively regulated by the ligand-activated nuclear estrogen receptor through inhibition of activator protein 1, a complex of jun/jun and jun/fos dimers required for cell proliferation.43 These studies suggest that estrogen is an important regulator of the IGF-I gene.
In our study, the competitive estrogen receptor antagonist ZK-119.010 abrogated the inhibitory effect of estradiol on SMC proliferation. Ma et al44 report that another antagonist, ICI 182780, blocks the growth-promoting activity of IGF-I and IGF-II on a neuroblastoma cell line (SK-ER3) in which an estrogen receptor is integrated in a stable manner. The rabbit SMCs we used expressed IGF-I, estrogen receptor, and IGF-IR (data not shown). We suggest that SMCs are estrogen target cells. Further studies are necessary to determine whether the antiproliferative effects of estradiol occur directly through regulating IGF-I and IGF-IR expression at the transcriptional or the translational level in the SMC.
In the current study, we have shown that IGF-I promotes rabbit vascular SMC proliferation and estradiol-abrogated cell proliferation induced by IGF-I in vitro and transplant arteriosclerosis in vivo. Although it is not completely understood how estradiol affects these changes, our current findings demonstrate that this beneficial effect is associated with decreased IGF-I expression in the arteries of cardiac allografts in the rabbits. These results suggest that the inhibitory effect of estradiol on the development of graft arteriosclerosis may be due to an inhibition of SMC proliferation through attenuation of IGF-I expression.
Selected Abbreviations and Acronyms
|CCS||=||charcoal-stripped calf serum|
|IGF-I||=||insulin-like growth factor-I|
|IGF-IR||=||insulin-like growth factor-I receptor|
|IMEM||=||improved minimum essential medium|
|SMC||=||smooth muscle cell|
This work was supported by NHLBI program project grants HL 40069, HL 47035, and HL 45317, and by the E. Schering Foundation. P. Delafontaine is an Established Investigator of the American Heart Association. We greatly thank Cecile Heatley for manuscript preparation.
- Received November 5, 1996.
- Revision received January 31, 1997.
- Accepted February 5, 1997.
- Copyright © 1997 by American Heart Association
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