Abstract
Beneficial effects of estrogen have been attributed to improved lipid profiles and to direct effects on the arterial wall. Macrophage-derived matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where they may contribute to plaque disruption. We have shown that oxidized low-density lipoprotein (Ox-LDL) increases matrix metalloproteinase-9 (MMP-9) expression in macrophages (Mφ). In this study, we tested the hypothesis that 17β-estradiol regulates basal and Ox-LDL-induced expression of MMPs and their tissue inhibitor (TIMPs) in human Mφ. Peripheral blood mononuclear cells isolated from normal human subjects were cultured for 7 days to transform into Mφ. On day 7, Mφ were starved with serum-free medium for 16 hours and then treated with 17β-estradiol and/or progesterone (PROG) in the presence or absence Ox-LDL for 24 hours. Levels and activity of MMP-2 and MMP-9 and levels of TIMP-1 and TIMP-2 were determined. After exposure to Ox-LDL, MMP-9 expression increased by 60% and TIMP-1 expression decreased by 29% (P < 0.05 and P < 0.05, respectively, compared to control), whereas TIMP-2 expression was unchanged. 17β-estradiol reduced the levels of Ox-LDL-induced MMP-9 protein as measured by Western blot (P < 0.05; n = 5) and Ox-LDL-induced MMP-9 activity (P < 0.05; n = 5) as measured by gelatin zymography. Conclusively, estradiol abolished Ox-LDL-stimulated increase in the levels of macrophage-derived MMP-9 protein and activity in human Mφ. This effect was reversed by TAM but not by PROG. These data suggest that at least part of the protective effect of estrogen occurs by attenuation of Ox-LDL alterations in MMP-9 expression.
Estrogen produces favorable effects on circulating lipids, improves endothelial function, and reduces vascular smooth muscle cell proliferation.1,2Although these diverse biological effects may account at least in part for the atheroprotective effects of estrogens, the molecular basis for the protective effects of estrogen is not fully understood, and other mechanisms may be involved. Most acute cardiovascular events such as myocardial infarction and stroke result from plaque disruption and thrombosis.3,4Plaque disruption is associated with enhanced matrix-degrading activity of macrophages in atherosclerotic plaques attributable to increased expression or activity of matrix metalloproteinases (MMPs) and/or decreased expression of their endogenous inhibitors-tissue inhibitors of matrix metalloproteinases (TIMPs).5-7Oxidized low-density lipoprotein (Ox-LDL) has been postulated to play an important role in atherogenesis, but the molecular mechanisms of its effects on plaque composition and stability have not been fully elucidated. Recently, we reported that Ox-LDL up-regulates MMP-9 and down-regulates TIMP-1 in monocytes-derived macrophages.5,6,8In this study, we tested the hypothesis that estrogen regulates basal and Ox-LDL-induced MMP and TIMP expression and activity in human macrophages (Mφ).
MATERIALS AND METHODS
Materials
Purified mouse monoclonal antibodies to human MMP-9 were purchased from Oncogene Research Products (La Jolla, CA). Goat antimouse IgG horseradish peroxidase secondary antibody for Western blot was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Purified human Ox-LDL was kindly provided by Dr. Judith Berliner, UCLA School of Medicine, Los Angeles, CA. 17β-estradiol (EST), tamoxifen (TAM), and progesterone (PROG; 4-pregnene-3, 20-dione) were purchased from Sigma (St Louis, MO). All tissue culture media and supplements were purchased from Invitrogen (Carlsbad, CA). Fetal calf serum (FCS) was from Hyclone Laboratories (Logan, UT). Enzyme-linked immunosorbent assay (ELISA) kits for measuring MMP-2, MMP-9, TIMP-1, and TIMP-2 were purchased from Amersham (Piscataway, NJ).
Cell Culture
Peripheral blood monocytes were isolated as previously described.9,10Monocyte-derived Mφ were cultured in Iscove Modified Dulbecco Medium (IMDM) containing 10% FCS, 100-U/mL penicillin, 100-μg/mL streptomycin, and 0.25-μg/mL amphotericin B for 7 days and then starved in the culture medium without FCS but with 0.1% low-endotoxin bovine serum albumin (Sigma Chemical Co, St Louis, MO). Experiments were performed in the starvation medium with or without varying amounts of reagents. Briefly, Mφ were incubated with EST in the presence or absence of 50 μg/mL of Ox-LDL for 24 hours. This dose of Ox-LDL was chosen based on our previously published results.10In additional experiments, the estrogen receptor antagonist TAM (10 nmol/L) and PROG (10 nmol/L) were also added to determine their interaction with EST. Culture medium was harvested for MMP and TIMP assays.
Immunoblot Analysis
Lysates of Mφ treated with Ox-LDL in the presence or absence of EST, PRG and/or TAM were isolated as described.9Macrophage cell lysates (50 μg) and known molecular weight markers were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinyl difluoride membranes, and incubated overnight at 4°C with blocking solution (5% skimmed milk in phosphate-buffered saline). Purified mouse monoclonal antibodies (10 μg of IgG per milliliter) to human MMP-9 were incubated with the blots overnight at 4°C in phosphate-buffered saline containing 0.1% Tween 20. Blots were washed and treated with goat antimouse antibody (1:10,000 dilution) coupled to horseradish peroxidase. Immunodetection was accomplished with an enhanced chemiluminescence kit as previously described.9,10
Gelatin Zymography
Equal amounts of the supernatants of Mφ untreated or treated with Ox-LDL in the presence or absence of EST, PROG, and/or TAM were assayed for gelatinolytic activity essentially as described.9,10Briefly, proteins with gelatinolytic activity were analyzed by electrophoresis in discontinuous 10% SDS-PAGE containing 1-mg/mL gelatin. Gels were processed to renature the protein by exchanging SDS to Triton X-100 (2 changes of 2.5% Triton X-100 for a total of 30 minutes). Gels were subsequently incubated for 18 hours at 37°C in 50-mmol/L Tris-HCl (pH 7.4) containing 10-mmol/L CaCl2 and 0.05% Brij 35, and stained with colloidal brilliant blue R (Sigma Chemical Co, St Louis, MO) followed by destaining in 5% methanol and 7% acetic acid.
Enzyme-Linked Immunosorbent Assays for MMP and TIMP
Levels of secreted MMP-2, MMP-9, TIMP-1, and TIMP-2 in the culture media of Mφ were measured with specific ELISAs using commercial kits (Amersham, Piscataway, NJ) according to the manufacturer's instructions. Duplicate evaluations were performed for each sample. Absorbance at 450 nm was measured with an automated reader.
Statistical Analysis
Data on MMPs and TIMPs are expressed as mean ± SEM. Statistical analyses were performed using the Student t test to determine the significance of change in the different treatments. A significant difference was considered present for P < 0.05.
RESULTS
MMPs and TIMPs Quantification by ELISA
Oxidized LDL significantly increased MMP-9 and reduced TIMP-1 levels (Table 1). The increase in the levels of MMP-9 protein and the activity by Ox-LDL were both inhibited by EST. Western blotting and gelatin zymography confirmed these findings (Figs. 1 and 2). 17β-estradiol also blunted the effect of Ox-LDL on TIMP-1 production but had no significant effect on MMP-2 and TIMP-2 levels under basal conditions or in the presence of Ox-LDL. TAM had no effect on basal and Ox-LDL induced increases in MMP-9, but it abolished the inhibitory effects of EST on the Ox-LDL-mediated increase in MMP-9. Progesterone did not reverse the effects of EST on increased expression of MMP-9 induced by Ox-LDL.
Effect of EST, TAM, and PROG on MMP-9 Production
Immunoblotting of cell lysates from Mφ showed constitutive expression of MMP-9 protein (Fig. 1). Oxidized LDL increased the steady-state levels of MMP-9 (P < 0.05). This increased production of MMP-9 in response to Ox-LDL was inhibited by EST. However, EST had no significant effect on MMP-9 levels in the absence of Ox-LDL. TAM also had no effect on basal levels of MMP-9 in the absence of Ox-LDL. Unlike EST, TAM was unable to prevent the increase in MMP-9 expression associated with exposure of Mφ to Ox-LDL. However, TAM did abolish the inhibitory effect of EST on increased levels of MMP-9 induced by Ox-LDL. Like TAM, PROG also had no effect on either basal or Ox-LDL-dependent augmentation of MMP-9 expression.
Effects of EST on MMP-2 and MMP-9 Gelatinolytic Activity
To examine whether levels of secreted MMP-9 protein corresponded to enzymatic activity, we performed SDS-PAGE gelatin zymography on conditioned media of Mφ grown in the presence or absence of Ox-LDL with or without EST, PROG, and/or TAM. Gelatinolytic activity at 92 kd attributable to MMP-9 was increased in the media after exposure to Ox-LDL. The augmentation of MMP-9 activity induced by Ox-LDL was abrogated by EST (P < 0.05). TAM had no effect on MMP-9 activity in the presence of Ox-LDL but reversed the effects of EST in the presence of Ox-LDL. Progesterone also had no effect on basal and Ox-LDL-induced increase in MMP-9 activity. Thus, changes in measurements of MMP-9 activity using ELISA closely paralleled the changes in MMP-9 protein expression. We conclude from these experiments that the effects of EST on Ox-LDL-induced MMP-9 expression were accompanied by parallel changes in MMP-9 activity.
DISCUSSION
Premenopausal women are at decreased risk for cardiovascular events compared to men with similar risk factor profiles.1,2It is thought that this is largely due to the protective effects of estrogen, but a detailed understanding of the molecular mechanisms of the vascular effects of estrogen is lacking. A number of studies suggest that structural destabilization of atherosclerotic plaque leads to increased likelihood of plaque rupture and consequent arterial thrombosis.3A localized imbalance between MMP proteolytic activity and the inhibition of that activity in plaque microenvironments is likely to be a major determinant of plaque vulnerability.11However, little is known about how estrogen might affect the balance between MMP degradative potential and its inhibition by TIMPs.
In this study, we demonstrate that EST abrogates the Ox-LDL-induced increase in MMP-9 level and activity without changing basal MMP-9 levels in human Mφ. The effect of EST was not changed by progesterone but was reversed by the selective estrogen receptor-modulating agent, TAM. 17β-estradiol had no significant effect on either MMP-2 or TIMP-2 expression either under basal conditions or in response to Ox-LDL but tended to attenuate the effect of ox-LDL on TIMP-1 expression.
Matrix metalloproteinases are a family of neutral zinc-requiring proteases produced by Mφ and a variety of other cells.4-6,9-12Matrix metalloproteinases are tightly regulated at the transcriptional level by their requirement for extracellular activation and by endogenous inhibitors such as TIMP-1 and TIMP-2.12Matrix metalloproteinase-1, MMP-2, MMP-3, and MMP-9 as well as TIMP-1 and TIMP-2 are expressed by Mφ and, to a lesser extent, by smooth muscle cells and endothelial cells in rupture-prone shoulder regions of atherosclerotic plaques.13-15These and other results have led to the concept that an imbalance in MMP-mediated extracellular matrix degradation relative to proteolytic inhibition of MMP activity by endogenous inhibitors such as TIMPs is a key factor contributing to plaque disruption leading to thrombosis and acute coronary events.5,6,11A variety of factors have been shown to regulate the ability of Mφ to produce MMPs, and Ox-LDL is one such agent that is present in atherosclerotic lesions.9Recently, we have demonstrated that Ox-LDL significantly increases MMP-9 messenger RNA and protein as well as MMP-9-mediated gelatinolytic activity in cultured human Mφ.9Our findings are consistent with those of Vegeto et al.,16who recently reported that endotoxin-induced activation of MMP-9 gene was inhibited by EST in microglial cells. This study, together with results reported here, hints at the possibility of a general mechanism whereby inhibition of MMP-9 expression by estrogens produce protective effects. However, further investigations in diverse pathologies involving dysregulated MMP expression and/or activity will be required to determine the generalizability of this possibility.
The precise mechanism by which EST reverses the effects of Ox-LDL on MMP-9 expression remains to be defined. Because TAM is a selective estrogen receptor antagonist, these data are consistent with the conclusion that the effects of EST were mediated directly by EST signaling through its receptor and were not nonspecific or secondary effects. Matrix metalloproteinase and TIMP genes are regulated by the transcription factors nuclear factor-κB and activator protein-1, and we speculate that Ox-LDL-induced changes in MMPs and TIMPs are most likely due to increasing nuclear binding of these transcription factors to nuclear gene targets in macrophages.17-20Estrogen has been shown to interact with these transcription factors, suggesting this genomic pathway as a potential mechanism of the effects observed in our study.21Further studies will be necessary to more fully evaluate these potential mechanisms.
In a small crossover-design study of 10 postmenopausal women with known coronary artery disease receiving hormone replacement therapy as well as statins and other drugs, Zanger et al.22reported that serum levels of MMP-9 were significantly increased compared to women not taking EST. This raises the possibility that at least under certain conditions, EST might increase MMP-9 expression. However, this study did not directly measure expression of MMP-9. Furthermore, serum MMP-9 levels to our results may not be compared for a variety of other reasons. For example, the relationship between serum MMP-9 and arterial expression may not be linear, and the increased serum levels could reflect not only increased tissue synthesis of MMP-9 but may also be a manifestation of decreased serum clearance, increased efflux from tissues, or some combination of these. Nevertheless, our study was an in vitro investigation. In addition, only acute effects of EST were evaluated. Whether chronic therapy would have different effects remains to be determined, and the in vivo relevance of our results will also require further investigation.
CONCLUSION
These studies, performed in vitro using cultured human monocyte-derived Mφ, demonstrate that EST abrogates Ox-LDL-mediated induction of MMP-9 levels in human Mφ without affecting basal expression. These effects of EST are reversed by TAM but not by PROG. Our findings suggest that the ability of EST to favorably affect proteases and their inhibitors in Mφ may contribute to the protective effects of EST in atherosclerotic pathologies.