Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1691-1700

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by R.W. St Clair, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by R.W. St Clair, S.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1691-1700.)
© 1997 American Heart Association, Inc.

Articles

Effect of 17ß-Estradiol on Metabolism of Acetylated Low-Density Lipoprotein by THP-1 Macrophages in Culture

Sulistiyani R.W. St Clair

From the Department of Pathology, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC.

Correspondence to Richard W. St Clair, PhD, The Bowman Gray School of Medicine of Wake Forest University, Department of Pathology, Medical Center Blvd, Winston-Salem, NC 27157-1072.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Evidence from numerous epidemiological and animal studies has shown a protective effect of estrogens on the development of atherosclerosis. Since not all of the beneficial effects of estrogen can be explained by alterations in plasma lipoprotein profiles, estrogens may have a direct effect on the arterial wall on one or more of the key steps in the pathogenesis of atherosclerosis. In the present study we tested the hypothesis that estrogens decrease macrophage foam cell formation by reducing lipoprotein uptake via the scavenger receptor pathway. Incubation of the human THP-1 macrophage cell line with 17ß-estradiol reduced the uptake and metabolism of 125I-labeled human acetylated LDL (acLDL) in a concentration-dependent manner (from 10-9 to 10-5 mol/L) by 30% to 40% at the highest concentrations used. This decrease was accompanied by a reduction in cholesterol accumulation and esterification. When chloroquine was used to block lysosomal degradation, 17ß-estradiol retained its ability to decrease accumulation of acLDL. This finding suggested that the effect of estrogen occurs before degradation of acLDL by lysosomes. 17ß-Estradiol had no effect on binding of 125I-acLDL at 4°C. When 125I-acLDL was bound at 4°C and warmed to 37°C, less acLDL was internalized and degraded in cells treated with 17ß-estradiol, due to greater dissociation of the bound acLDL from the surface of estrogen-treated cells during internalization. We conclude that as a result of the estrogen-induced increase in dissociation of acLDL, less lipoprotein cholesterol is delivered to macrophages, resulting in a reduced rate of foam cell formation. This may be one mechanism by which estrogens reduce the development of atherosclerosis.


Key Words: estrogen • acLDL metabolism • macrophage scavenger receptor • foam cells • atherosclerosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Evidence from numerous studies indicates that estrogen replacement therapy has a protective effect against the development of coronary heart disease in postmenopausal women.1 Only a part of this beneficial effect, however, can be explained by alterations in plasma lipoprotein profile.2 This observation suggests that estrogens may also influence the development of atherosclerosis by acting directly on the arterial wall.

Recently we reported a significant reduction in atherosclerosis development by ethynylestradiol and 17{alpha}-dihydroequilin sulfate, a water-soluble estrogen of Premarin, in ovariectomized cholesterol-fed rabbits.3 In this study the reduction in atherosclerosis development was independent of changes in plasma cholesterol or lipoprotein concentrations. Other studies in ovariectomized nonhuman primates4 5 and rabbits6 7 have shown similar results; ie, 17ß-estradiol reduced the amount of atherosclerosis without affecting plasma lipoprotein concentrations. The mechanism by which estrogens exert this protective effect against atherosclerosis is not known; however, a direct effect of estrogens on one or more aspects of lipoprotein metabolism in the arterial wall is consistent with other data in the literature. Wagner et al8 reported that administration of 17ß-estradiol and cyclic progesterone reduced accumulation and degradation of LDL in monkey coronary arteries in the early stages of atherosclerosis development. Similarly, Hough and Zilversmit6 showed a reduction of lipoprotein cholesteryl ester accumulation in aortas of rabbits treated with 17ß-estradiol. Since estrogens do not appear to affect aortic permeability to LDL,9 it is likely that estrogens have their greatest effect on the metabolism or accumulation of lipoproteins after they have entered the arterial wall.

In both nonhuman primates10 and rabbits,11 the most prominent initial pathological feature of atherosclerosis is the accumulation of macrophage foam cells. Thus, estrogens could influence the development of atherosclerosis by reducing the formation of macrophage-derived foam cells by altering cellular processes that are involved in the accumulation of cholesterol esters within these cells. Consistent with this hypothesis is the observation that accelerated cardiac transplant atherosclerosis in the rabbit aorta showed a decrease in the number of macrophage-derived foam cells in the lesions of estrogen-treated animals.12 In the present study, we tested the hypothesis that estrogens inhibit macrophage foam cell formation by reducing lipoprotein uptake by the scavenger receptor pathway. For this purpose, we studied the effect of 17ß-estradiol on the uptake and metabolism of acLDL by the THP-1 human macrophage cell line.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Sodium [125I]iodide (carrier free in NaOH solution, pH 7 to 11) was purchased from Amersham Corp; [14C]oleic acid and [3H]cholesterol were from New England Nuclear. 17ß-Estradiol and stigmasterol were purchased from Steraloids; RPMI-1640 medium, penicillin, streptomycin, vitamins, and L-glutamine were from JRH Biosciences; FBS, from Atlanta Biologicals, was heat inactivated at 56°C for 1 hour; chloroquine and N-ethyl maleimide were obtained from Sigma Chemical Co. Tissue-culture dishes were purchased from Corning Glass Works. Silica gel–coated glass plates for TLC were purchased from Curtis Matheson Scientific, Inc. Other reagents and solvents were purchased from Scientific Products.

Cell Culture
The THP-1 macrophage cell line13 was obtained from American Type Culture Collection. The cells were grown in suspension in T-75 flasks in RPMI-1640 medium containing 10% heat-inactivated FBS and 2-ß-mercaptoethanol (5x10-5 mol/L, Fisher Scientific) supplemented with L-glutamine (200 mmol/L), penicillin (100 IU/mL), streptomycin (100 µg/mL), and vitamins. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. To convert the THP-1 cells to a macrophage phenotype, at least 1x106 cells were seeded into 35-mm culture dishes in RPMI-1640 medium buffered with HEPES and containing phorbol myristate acetate (Sigma Chemical Co) at a final concentration of 10-7 mol/L. The RPMI-1640 did not contain phenol red, since it has been reported to have estrogenic activity.14 The cells were incubated for 72 hours before initiation of experiments.

Thioglycollate-elicited mouse peritoneal macrophages were obtained from female CD-1 mice (18 to 20 g; Charles River Labs, Raleigh, North Carolina). Macrophages were elicited with injection of a solution of 10% thioglycollate (Fisher Scientific) into the peritoneal cavity 3 days before isolation of cells. Mice were killed by cervical dislocation and cells were harvested by peritoneal lavage as described15 and plated into plastic tissue-culture dishes. This procedure was approved by the Wake Forest University Animal Care and Use Committee. Nonadherent cells were washed off after 4 hours, and the adherent macrophages were incubated overnight before their use in experiments.

Lipoprotein Isolation, Characterization, Modification, and Labeling
Human LDL was isolated from plasma obtained from the Red Cross. Rabbit ß-VLDL was isolated from plasma of New Zealand White rabbits (Franklin Rabbitry, Wake Forest, North Carolina) that had been fed a commercial pelleted rabbit diet (Agway PROLAB rabbit formula) containing 0.5% cholesterol and 5% corn oil for at least 4 weeks. Human LDL was isolated by sequential ultracentrifugation.16 Briefly, VLDL (d<1.006 g/mL) was removed by ultracentrifugation for 24 hours at 36 000 rpm in an SW-40 rotor. LDL was isolated at d<1.063 g/mL from the d>1.006 g/mL fraction by adjusting this fraction to 1.080 g/mL with solid KBr and overlayering it with a 1.063-g/mL solution followed by ultracentrifugation for 44 hours at 50 000 rpm using a 50.2 Ti rotor. Rabbit ß-VLDL was isolated at d<1.006 g/mL after ultracentrifugation of plasma for 20 hours at 36 000 rpm in an SW-40 rotor. Plasma and isolated lipoproteins contained EDTA (1 mg/mL) and were kept at 4°C during all procedures. Acetylation of human LDL was carried out using acetic anhydride as described by Basu et al.17 Verification of acetylation was determined by analyzing lipoprotein electrophoretic mobility in 1% agarose gels. Lipoproteins were labeled with 125I, using the iodine monochloride method of MacFarlane as modified by Bilheimer.18 Free iodine was removed by passing the iodination mixture over a Sephadex G-25 M column (Pharmacia column PD-10) followed by exhaustive dialysis against a saline-EDTA solution at pH 7.4. All lipoprotein preparations were sterilized by filtration through a 0.45-µm filter (Millipore Corp), stored at 4°C, and used within 4 weeks of isolation.

Experimental Design
To test the effect of estrogen on macrophage scavenger receptor activity, cells were incubated in the presence or absence of 17ß-estradiol in RPMI-1640 medium containing 5% steroid-free FBS. Steroids were extracted from FBS with charcoal.19 17ß-Estradiol was dissolved in ethanol and added to the culture medium so that the maximum ethanol concentration did not exceed 0.5%. The same concentration of ethanol alone was added to control cells. For most experiments, cells were incubated in the presence or absence of 17ß-estradiol for 48 hours before addition of acLDL. Depending on the experiment, 17ß-estradiol was either present or absent during incubation with acLDL. Cells were incubated with acLDL for the times indicated in the tables and figures.

Uptake and Degradation of 125I-acLDL
Lipoprotein uptake and degradation were determined after incubation with 125I-acLDL at 37°C for 5 hours, unless indicated otherwise. Cell surface–bound acLDL was determined after incubation for 4 hours at 4°C. Following incubation, macrophages were washed five times with PBS containing 2 mg/mL BSA, then three times with PBS as described by Goldstein et al.18 Cell-associated radioactivity (surface-bound at 4°C and surface-bound plus internalized at 37°C) was measured after digestion of the cells with 1N NaOH overnight at room temperature. Lipoprotein degradation was determined as TCA-soluble, noniodide 125I in the postincubation medium.18 To determine specific uptake and degradation via the acLDL receptor, incubations with 125I-acLDL were carried out in the absence and presence of a 20-fold excess of unlabeled acLDL. The specific binding and metabolism were calculated by subtracting the results in the presence of unlabeled acLDL from that in its absence.18 Specific binding was evaluated by the LIGAND computer program (BIOSOFT).

Cellular Cholesterol Accumulation and Esterification
Cellular cholesterol content was determined essentially as described previously.15 After cells were washed, the cellular lipids were extracted directly from the tissue-culture dishes by isopropanol following the method of McCloskey20 as modified by Bernard et al.21 Stigmasterol was added volumetrically to the isopropanol extract to serve as an internal standard. Aliquots of the isopropanol extract were used to determine free and total cholesterol mass by gas-liquid chromatography according to the method of Ishikawa et al.22 The conditions of gas-liquid chromatography were as described previously.15 Esterified cholesterol was calculated as the difference between total and free cholesterol. Results are expressed as milligrams cell protein.

Cholesterol esterification was determined by the incorporation of [14C]oleate into cholesteryl esters as described previously.23 Two hours before the end of incubation, cells were washed with PBS and incubated with 0.17 mmol/L [14C]oleate-albumin substrate for 2 hours at 37°C. [3H]Cholesterol was added as an internal standard before extraction of cell lipids with isopropanol. Individual lipids were separated by TLC on silica gel plates in a solvent system of hexane/ethyl ether/glacial acetic acid (146:50:4, vol/vol/vol). The cholesterol and cholesteryl ester fractions were identified by comparison with cholesterol and cholesteryl oleate standards. The free and esterified cholesterol bands were scraped from the TLC plates and counted directly for 3H and 14C radioactivity in a Beckman LS 230 liquid scintillation counter. All counts were corrected for quenching and for 14C spill into the 3H channel. Samples were counted to a 2 sigma error of <5%.

Other Analytical Methods
Lipoprotein and cell protein content were measured by the method of Lowry et al24 using BSA as the standard. Turbid lipoprotein samples were cleared by extraction of the Lowry reaction mixture with chloroform before measurement of absorbance. After extraction of cellular lipids, the cells were dissolved in 1N NaOH before protein determination.

Unless indicated otherwise, results are expressed as means of triplicate dishes at each point±SEM. Statistical significance was analyzed with Student's t test, except, where indicated, a one-way ANOVA was used. Most experiments were repeated at least twice, with similar results.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Fig 1Down shows the effect of different concentrations of 17ß-estradiol on the uptake and metabolism of 125I-acLDL by THP-1 cells. There was a dose-dependent reduction in specific degradation of 125I-acLDL over the range of 17ß-estradiol used (10-9 to 10-5 mol/L). At 10-5 mol/L 17ß-estradiol, degradation of acLDL was 30% to 40% less than in the control cells (P<.05). A similar reduction in cell-associated 125I-acLDL was seen, although when considering all experiments, the effect of 17ß-estradiol on acLDL metabolism was most consistently seen by the reduction in degradation. The reduction in acLDL metabolism by 17ß-estradiol was secondary to a reduction in specific uptake and degradation, since there was no effect on nonspecific processes. Over the range of 17ß-estradiol used, there was no effect on cell protein per dish (data not shown). An effect of estrogen on acLDL degradation could be demonstrated as early as 18 to 24 hours after preincubation with estrogen (data not shown). To maximize the effects of estrogens on acLDL metabolism, most of the subsequent studies were done with 10-5 mol/L 17ß-estradiol.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Effects of 17ß-estradiol on 125I-acLDL metabolism in THP-1 macrophages. After treatment with phorbol myristate acetate as described in "Methods," THP-1 cells were incubated in RPMI-1640 medium (without phenol red) containing 5% steroid-free FBS plus ethanol alone (none; 0.5% final concentration) or 17ß-estradiol dissolved in ethanol (0.5% final concentration) at concentrations ranging from 10-9 to 10-5 mol/L. After 48 hours, the cells were washed with PBS and incubated in serum-free RPMI-1640 medium (without phenol red) containing the indicated concentration of 17ß-estradiol and 5 µg/mL 125I-labeled human acLDL (256 cpm/ng protein) for 5 hours at 37°C in the absence or presence of a 20-fold excess of unlabeled human acLDL. 17ß-Estradiol at the indicated concentrations was present during incubation with 125I-acLDL. At the end of the incubation period, cell-associated and degraded 125I were measured. Specific metabolism of 125I-acLDL is shown and was calculated as described in "Methods." Nonspecific cell-associated and degraded 125I-acLDL were not different, regardless of the concentration of 17ß-estradiol, ranging from 46 to 47 and 25 to 35 µg/mg cell protein for cell-associated and degraded 125I, respectively. Results are the average of triplicate dishes (±SEM). This experiment was repeated, with similar results.

The effect of 17ß-estradiol on the time course of metabolism of 125I-acLDL is shown in Fig 2Down. Control cells were preincubated for 48 hours in the absence of 17ß-estradiol, while the two estrogen groups were preincubated for 48 hours with 10-5 mol/L 17ß-estradiol before incubation with 125I-acLDL for the indicated times. In one of the estrogen groups, 17ß-estradiol at 10-5 mol/L also was present during the time-course incubation with 125I-acLDL, while in the other group the estrogen was omitted during the time-course incubation with 125I-acLDL. At 1 hour, cell-associated radioactivity was similar for all groups. Thereafter, at all time points, the estrogen groups had lower amounts of cell-associated acLDL than control cells (P<.05). This difference was greater when 17ß-estradiol also was present during the 24 hours in which 125I-acLDL metabolism was being measured. A similar relationship was seen in degraded 125I- acLDL. The suppression in acLDL degradation, however, was similar with or without the estrogen in the medium during the first 12 hours over which 125I-acLDL metabolism was being measured. In the second 12 hours, there was a trend for acLDL degradation to be suppressed to a greater extent when 17ß-estradiol was kept in the medium during the 24 hours of incubation with 125I-acLDL. This finding implies that the effect of a 48-hour preincubation of THP-1 cells with 10-5 mol/L 17ß-estradiol retains its full effect on the reduction of acLDL degradation for at least 12 hours after removal of the estrogen. In preliminary studies with thioglycollate-elicited mouse peritoneal macrophages and human monocyte macrophages, a similar reduction in the uptake and degradation of 125I-acLDL was seen (data not shown).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Time course of specific 125I-acLDL metabolism by THP-1 macrophages treated with 17ß-estradiol. Cells were preincubated with 10-5 mol/L 17ß-estradiol or ethanol alone as described in the legend to Fig 1Up. Cells were then incubated at 37°C in serum-free RPMI-1640 medium with 4 µg/mL 125I-acLDL (55 cpm/ng) ±20-fold excess of unlabeled acLDL for the indicated times up to 24 hours. In one batch of cells, 17ß-estradiol (10-5 mol/L) was kept in the RPMI-1640 medium during the time course of incubation with 125I-acLDL, while in the other group, only the ethanol vehicle was present. Specific cell-associated and degraded 125I-acLDL are presented and were determined as described in "Methods." Results are the mean±SEM of triplicate dishes at each point and are representative of two similar experiments. Where error bars are not seen, they fall within the data points.

The effect of 17ß-estradiol on the concentration-dependent metabolism of 125I-acLDL at 37°C for 5 hours is shown in Fig 3Down. Estrogen-treated macrophages showed a 30% to 40% reduction in uptake and degradation of 125I-acLDL compared with control cells (P<.05). This reduction was seen at all concentrations of 125I-acLDL used. Cell-associated and degraded acLDL plateaued at concentrations above 20 µg/mL.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 3. Concentration dependence of the metabolism of 125I-acLDL by THP-1 macrophages treated with 17ß-estradiol. Cells were preincubated with or without 17ß-estradiol (5x10-6 mol/L) for 48 hours followed by incubation for 5 hours with medium containing 125I-labeled human acLDL (161 cpm/ng) in the presence or absence of a 20-fold excess of unlabeled human acLDL. 17ß-Estradiol (5x10-6 mol/L) was present in the medium during incubation with 125I-acLDL. Data points are the average of triplicate dishes (±SEM) and represent specific metabolism of 125I-acLDL. Where error bars are not seen, they fall within the data points.

Fig 4Down shows the effect of 17ß-estradiol on the time course of cellular cholesterol accumulation when incubated with acLDL. As shown in Fig 4ADown, both estrogen-treated and control THP-1 macrophages increased their cholesterol content with time on incubation with 100 µg/mL acLDL for up to 24 hours. At all time points, the accumulation of cholesterol was less in cells treated with 17ß-estradiol. In THP-1 macrophages, there was an increase in both free and esterified cholesterol, and both were reduced with estrogen (Fig 4BDown). In data not shown, 17ß-estradiol also decreased the accumulation of esterified cholesterol in thioglycollate-elicited mouse peritoneal macrophages. In these cells, incubated for 24 hours with 100 µg/mL acLDL, 10-5 mol/L 17ß-estradiol significantly (P<.05) reduced cellular esterified cholesterol content from 40.0±0.47 to 35.5±1.17 µg/mg cell protein (mean±SEM, n=3). The effect of 17ß-estradiol on cholesterol esterification in THP-1 macrophages incubated with acLDL is shown in Fig 4CDown. 17ß-Estradiol had no significant effect on ACAT activity in THP-1 macrophages, except perhaps for the 24-hour time point (P<.05). On the other hand, there was a 30% (P<.05) reduction with 17ß-estradiol treatment in the rate of cholesterol esterification in mouse peritoneal macrophages incubated for 24 hours with 100 µg/mL acLDL from 33.2±3.4 to 23.1±1.0 nmol cholesteryl oleate formed per milligram cell protein after a 2-hour incubation with [14C]oleate (n=3).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Effects of 17ß-estradiol on cellular cholesterol accumulation (A and B) and esterification in THP-1 macrophages (C). Cells were preincubated with or without 17ß-estradiol at a concentration of 10-5 mol/L for 48 hours followed by incubation with 100 µg/mL acLDL at 37°C for the indicated times. During incubation with acLDL, 17ß-estradiol was either present or absent as indicated. For the last 2 hours of incubation, [14C]oleate was added to each dish (12 780 dpm/nmol, 0.17 mmol/L) to measure cholesterol esterification. After 2 hours of incubation, cells were washed and lipids extracted in isopropanol. Total cellular cholesterol (A), free cholesterol, and esterified cholesterol (B) in control and estrogen-treated cells were determined as described in "Methods." Cholesterol esterification is shown in C. Each data point is the mean of triplicate dishes (±SEM). Where error bars are not seen, they are contained within the data points.

To determine whether the reduction in acLDL uptake by 17ß-estradiol was due to an effect of 17ß-estradiol on endocytosis in general or whether it was specific to scavenger receptor–mediated endocytosis, we carried out experiments using other ligands that were taken up by receptor-mediated endocytosis. Data in Table 1Down show that the uptake and degradation of 125I-labeled native human LDL and rabbit ß-VLDL by THP-1 macrophages, which occur through the LDL receptor pathway,25 were either unchanged or increased by 17ß-estradiol treatment.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of 17ß-Estradiol on Metabolism of 125I–Human LDL and 125I–Rabbit ß-VLDL by THP-1 Macrophages

The effect of other estrogens and progesterone on degradation of 125I-acLDL is shown in Table 2Down. At a concentration of 10-5 mol/L, estrone and 17{alpha}-estradiol had a similar effect to 17ß-estradiol in reducing the degradation of 125I-acLDL by THP-1 macrophages. At similar concentrations, ethynylestradiol, 17{alpha}-dihydroequilin (an equine estrogen component of Premarin), and progesterone had little effect.


View this table:
[in this window]
[in a new window]
  		Table 2. Effet of Various Estrogens and Progesterone on the Degradation of 125I-acLDL by THP-1 Macrophages

The results described thus far suggest that 17ß-estradiol inhibits cholesterol influx into macrophages by reducing acLDL uptake and metabolism via the scavenger receptor pathway. To determine whether estrogens act by reducing the number of scavenger receptors or their affinity for acLDL, 4°C binding experiments were conducted using 125I-acLDL (Fig 5Down). 17ß-Estradiol had no effect on specific binding of 125I-acLDL to THP-1 cells at 4°C.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. Binding of 125I-acLDL at 4°C to THP-1 macrophages treated with 17ß-estradiol. THP-1 macrophages were preincubated with or without estrogen (10-5 mol/L) as described previously in the legend to Fig 2Up. The cells were then chilled to 4°C and ice-cold RPMI-1640 medium was added with the indicated concentrations of 125I-acLDL (136 cpm/ng) plus or minus a 50-fold excess of unlabeled acLDL. The cells were incubated at 4°C for 4 hours. The data represent the specific binding to control and estrogen-treated cells. Results are the mean±SEM of triplicate dishes and are representative of two similar experiments. Where error bars are not present, they are contained within the data points.

Since there was no difference in binding of 125I-acLDL to estrogen-treated THP-1 cells, we tested the possibility that 17ß-estradiol reduced the uptake of bound acLDL. If this hypothesis is correct, then 17ß-estradiol should continue to reduce acLDL uptake even if degradation is blocked. This possibility was tested in Fig 6Down by using chloroquine to inhibit lysosomal degradation of 125I-acLDL. As shown in Fig 6BDown, specific degradation of 125I-acLDL was abolished on treatment with chloroquine, which was reflected by an enhanced accumulation of undegraded cell-associated 125I. Nevertheless, chloroquine did not eliminate the effect of estrogen on diminishing the accumulation of cell-associated 125I-acLDL (Fig 6ADown). This finding suggests that estrogen acts at the level of internalization of acLDL and not degradation.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 6. Effects of chloroquine (75 µmol/L) on the uptake and metabolism of 125I-acLDL by THP-1 macrophages treated with 17ß-estradiol. Cells were preincubated with 10-5 mol/L 17ß-estradiol for 48 hours as described in the legend to Fig 2Up. The culture medium was changed and cells were incubated with or without 75 µmol/L chloroquine for 45 minutes at 37°C. RPMI-1640 medium containing 5 µg/mL 125I-acLDL (177 cpm/ng) was then added in the presence or absence of a 20-fold excess of unlabeled acLDL, and the cells were incubated together for the indicated times at 37°C. 17ß-Estradiol (10-5 mol/L) also was present during incubation with 125I-acLDL. Cell-associated and degraded 125I-acLDL were determined as described in "Methods." Results are the mean±SEM of triplicate dishes. Where error bars are not shown, they fall within the data points.

To directly test the hypothesis that 17ß-estradiol reduces the internalization of acLDL, we carried out the pulse-chase experiment shown in Fig 7Down. THP-1 cells were pulse labeled for 3 hours at 4°C with 125I-acLDL and the cells were washed extensively. This procedure was followed by a chase incubation at 37°C with unlabeled acLDL for the times indicated in the figure. Cell-associated and degraded 125I-acLDL were measured at each time point. The data are expressed as a percent of total 125I-acLDL bound at 4°C at zero time. Cell-associated 125I-acLDL decreased significantly (P<.02) more rapidly in the estrogen-treated cells during the first 40 minutes of the chase period and appeared as degraded products (TCA-soluble counts) in the media at a slower rate (first seen at 40 minutes) in estrogen-treated cells than controls. This is consistent with the conclusion that for the same amount of acLDL bound to the surface of macrophages, a smaller percentage is internalized with estrogen treatment.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 7. Effect of 17ß-estradiol on the internalization and degradation of 125I-acLDL after pulse labeling at 4°C. Cells were preincubated with or without 17ß-estradiol (10-5 mol/L) for 48 hours as indicated previously. Cells were then cooled to 4°C and incubated with RPMI-1640 medium containing 15 µg/mL 125I-acLDL (254 cpm/ng) for 3 hours at 4°C. At the end of this incubation, the cells were washed extensively with BSA and PBS, the medium was replaced with fresh medium containing unlabeled acLDL (30 µg/mL), and the cells were incubated at 37°C for up to 180 minutes. At the indicated times, cells were quickly cooled on ice, and cell-associated and degraded 125I-acLDL from control and estrogen-treated cells were determined as described in "Methods." Values are expressed as a percentage of total 125I-acLDL bound at 4°C before the chase with unlabeled acLDL (100%). The 100% control values are 108±8 ng/mg cell protein for control and 139±21 ng/mg cell protein for estrogen-treated cells. Results are the mean of triplicate dishes at each point.

Although estrogen treatment had little effect on binding of 125I-acLDL (Fig 5Up), a reduction in internalization leading to an eventual reduction in degradation could have resulted if estrogen treatment caused a greater proportion of the bound 125I-acLDL to detach from the cell during the process of internalization at 37°C. To test this possibility, we carried out another pulse-chase experiment similar to that shown in Fig 7Up, except that the amount of TCA-precipitable 125I-acLDL released into the chase medium was measured. As shown in Fig 8Down, by 40 minutes into the chase period, the amount of undegraded 125I-acLDL released from the surface of the cell plateaued, with nearly 75% of that found at zero time released into the medium in estrogen-treated cells compared with 50% for control cells.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 8. Effect of 17ß-estradiol on the release of bound 125I-acLDL from THP-1 cells into medium after rewarming to 37°C. Cells were preincubated with or without 17ß-estradiol for 48 hours, followed by incubation at 4°C with 125I-acLDL, as described in the legend to Fig 7Up. Cells were washed and then rewarmed to 37°C for the indicated times. At these times, cells were immediately cooled on ice and the amount of TCA-precipitable 125I-acLDL in the chase media from control and estrogen-treated cells was determined. Values are expressed as the percentage of total 125I-acLDL bound at zero time. The 100% control values are 36±4 ng/mg cell protein for control and 20±2 ng/mg cell protein for estrogen-treated cells. Results are the mean of triplicate dishes at each point.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous studies by us8 and others7 have shown a beneficial effect of estrogens on atherosclerosis development that is greater than can be accounted for by changes in plasma lipoproteins alone. This finding suggests that estrogens may act directly on the arterial wall on one or more critical components in the pathogenesis of atherosclerosis. There are a number of potential modes of action. Perhaps foremost among these is an effect on some aspect of lipoprotein metabolism, ranging from influx of plasma lipoproteins to their metabolism by cells of the arterial wall. A recent study by Haarbo et al9 showed that estrogens do not alter the initial rate of entry of LDL into the arterial wall, implying that estrogens probably act at a later stage in the pathogenesis. This is consistent with our studies on the effects of estrogens on the uptake and metabolism of LDL.8 The macrophage foam cell plays a critical role in the early pathogenesis of atherosclerosis in humans and all animal models that have been studied. In cholesterol-fed rabbits in particular, the macrophage plays a predominant role in foam cell development.11 Since estrogens reduce atherosclerosis in cholesterol-fed rabbits,3 a likely site of action of estrogens is on some aspect of macrophage foam cell formation. The results of the present study are consistent with the conclusion that one way estrogens may mediate this effect is by a reduction in the uptake of lipoprotein via the macrophage scavenger receptor pathway.

In the human THP-1 monocyte-macrophage cell line used in these studies, 17ß-estradiol reduced uptake of acLDL by scavenger receptors in a concentration-dependent manner (from 10-9 to 10-5 mol/L) by as much as 40% at the highest concentrations used. This action was an effect on specific uptake and degradation of acLDL, as nonspecific uptake and degradation were unaffected. This observation implies that the effect of estrogens on acLDL metabolism was not secondary to changes in overall endocytotic processes. The reduction in cell uptake and degradation of 125I-acLDL by 17ß-estradiol was accompanied by a reduction in cell cholesterol content. In THP-1 macrophages, this reduction was seen for both free and esterified cholesterol, while in thioglycollate-elicited mouse peritoneal macrophages, the reduction was mainly in cholesteryl esters. There was also a reduction in the rate of cholesterol esterification in both cell types. The reduction in cholesterol esterification with 17ß-estradiol was less in THP-1 cells than mouse peritoneal macrophages, perhaps due to the fact that phorbol ester treatment of THP-1 cells to convert them to their macrophage phenotype may have already suppressed ACAT activity.26 Since it has been shown that 17ß-estradiol does not directly inhibit ACAT activity in macrophages,27 it is likely that the reduction in cholesterol esterification by estrogens was secondary to a reduced delivery of acLDL-derived cholesterol to the cells.

Several studies were carried out to determine the site of action of estrogens on reduction in acLDL metabolism. Estrogens could act by reducing the number of scavenger receptors, their affinity, or the postreceptor processing of acLDL. Since the effect of 17ß-estradiol was most consistently seen by a reduction in degradation, it was possible that the slower rate of degradation was due to a primary effect of estrogen on lysosomal enzyme activity. An effect of estrogens on lysosomal enzymes in uterine endometrial cells has been reported.28 If estrogens reduced only the lysosomal degradation step of the endocytotic pathway, the addition of a lysosomotropic agent such as chloroquine should equalize acLDL accumulation in control and estrogen-treated cells. When this experiment was done, the accumulation of acLDL by macrophages treated with 17ß-estradiol and chloroquine remained significantly less than by macrophages treated with chloroquine alone (Fig 6Up). These data suggest that the effect of estrogen occurs before degradation of acLDL in lysosomes. 17ß-Estradiol, however, had no effect on binding of 125I-acLDL to cells at 4°C, suggesting that differences in binding affinity or capacity of acLDL to scavenger receptors could not account for the reduced uptake of 125I-acLDL. This conclusion must be tempered by the fact that a difference in binding at 4° and 37°C has been described for scavenger receptors and was attributed to the fact that the ligand-binding domain of the scavenger receptor is temperature sensitive.29 30 We have carried out pilot studies to address this question by measuring binding of 125I-acLDL to THP-1 cells at 37°C in cells that had been preincubated at 4°C for 30 minutes with 1 mmol/L N-ethyl maleimide and then warmed to 37°C for the binding studies. Consistent with the works of others,31 this procedure completely inhibited receptor-mediated endocytosis. Although a small reduction in binding was seen with estrogen, it was not sufficiently different from controls to detect a significant difference in binding kinetics using the LIGAND program. Thus, our data suggest that 17ß-estradiol primarily reduces the internalization of acLDL by a mechanism that is not secondary to a reduction in the number of scavenger receptors. Interestingly, a similar observation has been reported for the effect of dexamethasone32 on the uptake and degradation of ß-VLDL by mouse peritoneal macrophages and for hydrocortisone on the uptake and degradation of LDL by cultured human fibroblasts.33

A possible mechanism for the estrogen effect is suggested by the data in Figs 7Up and 8Up. When 125I-acLDL was bound at 4°C and then warmed to 37°C, less 125I-acLDL was internalized and degraded in estrogen-treated cells than in controls (Fig 7Up). This was due to the fact that more of the bound 125I-acLDL was released from the estrogen-treated cells during warm-up than from non–estrogen-treated cells (Fig 8Up), resulting in less acLDL being internalized and degraded.

The mechanism by which 17ß-estradiol mediates the enhanced release of acLDL from the surface of THP-1 macrophages is unclear. One possibility is that the effect is mediated by estrogen receptors. Although human monocyte-macrophages, rat peritoneal macrophages, and some macrophage cell lines34 35 have been reported to have estrogen receptors, we are aware of no reports in the literature demonstrating estrogen receptors on THP-1 cells. In preliminary studies not reported here, we analyzed THP-1 cells that had been treated with phorbol esters for estrogen receptors by using immunohistochemistry with antibodies specific for human estrogen receptors36 and [3H]17ß-estradiol–binding studies for type I and type II receptors.37 Neither method showed any evidence of estrogen receptors. When these results are coupled with the fact that 17{alpha}-estradiol, an isomer of estradiol that does not bind to estrogen receptors,38 was effective in reducing uptake and degradation of 125I-acLDL, the most likely interpretation is that estrogen receptors probably do not mediate the effect on scavenger receptor activity in THP-1 cells. A definitive answer to the role estrogen receptors play in this response will have to await future studies.

Another possibility is that estrogens, particularly at high concentrations, may act simply by virtue of their lipophilic properties by partitioning to hydrophobic domains on plasma membranes and influencing a variety of ligand receptor–mediated endocytotic processes in a nonspecific fashion. Although we cannot completely eliminate this possibility, several factors argue against it. Perhaps the most compelling is the fact that estrogens affected specific receptors differently. While scavenger receptor activity was reduced, LDL/ß-VLDL receptor activity was either increased or unchanged. The fact that estrogens affect specific receptors differently is in agreement with recent findings by Szanto et al,39 who reported that administration of a pharmacological dose of ethynylestradiol increased rat hepatic LDL receptor expression fivefold but decreased {alpha}2MR/LRP expression by 50%. Similarly, Burgess and Stanley40 reported that treatment with ethynylestradiol decreased asialoglycoprotein receptor expression by 50%, while LDL receptor activity was increased. Estrogens are not the only steroids that have been shown to have differential effects on membrane receptor activity. Dexamethasone, for example, increases acLDL receptor activity and reduces LDL receptor activity in human monocyte-macrophages.41 These observations, although arguing against a nonspecific effect, do not rule out the possibility that changes in the physical properties of membranes resulting from the hydrophobic association of estrogens could trigger specific signal-transduction mechanisms (eg, phosphorylation/dephosphorylation), to which various receptors respond differently.

In all of these studies with estrogens and other steroids in macrophages, the activity of scavenger receptors and LDL/ß-VLDL receptors seems to vary inversely. Thus, another potential mechanism by which estrogens could act is by changing the activation or differentiation state of the macrophages. Estrogens have been reported to stimulate macrophage activation, as evidenced by increased phagocytic activity42 and the production of interleukin-1.43 Since scavenger receptor activity is downregulated by macrophage activation,44 45 46 it is possible that the effect of 17ß-estradiol on scavenger receptor activity was secondary to activation of the macrophages. In turn, activated macrophages may secrete products such as interferon-{gamma} that are inhibitory to scavenger receptor functions. Interestingly, Fong et al47 reported that interferon-{gamma} inhibited acLDL degradation in mouse macrophages without any effect on acLDL binding and suggested that the inhibitory effect on degradation was mediated by an alteration in the transport of internalized acLDL. Such a mechanism would be consistent with our studies showing that several estrogens (17ß-estradiol, estrone, and 17{alpha}-estradiol) were effective in inhibiting scavenger receptor activity.

Regardless of the mechanism, the effect of estrogens on scavenger receptor activity was dose dependent over a range of 17ß-estradiol concentrations, from physiological (10-9 mol/L) to pharmacological (10-5 mol/L). Although most of these concentrations are higher than the concentration of 17ß-estradiol thought to be required for estrogen receptor–mediated physiological function in vivo (10-9 to 10-10 mol/L),48 it should be kept in mind that the effect on scavenger receptor activity does not appear to be mediated by estrogen receptors, and other estrogens present in the blood (eg, estrone) are equally potent. Furthermore, it is not possible to know the true concentration of estrogens at the level of the cells of various tissues. For example, Henkin et al49 showed that cortisol concentration in the cat brain was about 30 times higher than in plasma. Thus, it is possible that uptake of estrogen by the arterial wall and its partition into hydrophobic domains may result in a higher concentration of estrogens in arterial tissue than in plasma.

At the highest concentration of 17ß-estradiol (10-5 mol/L) used, there was a maximum reduction in scavenger receptor activity of 30% to 40%. Whether this is sufficient to explain the up to 70% reduction in atherosclerosis previously reported3 6 7 is unclear. Those studies, however, were from 24 to 33 weeks in duration. Thus, it is possible that a relativity small effect on scavenger receptor activity is all that is needed to balance the influx and efflux of lipoprotein-derived macrophage cholesterol, resulting in a significant reduction in the rate of development of atherosclerosis.

Previously we reported that oral administration of ethynylestradiol and 17{alpha}-dihydroequilin sulfate, a conjugated estrogen found in pregnant mares' urine and one of the active ingredients of Premarin, reduced atherosclerosis development in ovariectomized cholesterol-fed rabbits.3 In our present study, neither ethynylestradiol nor 17{alpha}-dihydroequilin had a significant effect on acLDL metabolism in THP-1 macrophages. This could imply that reduction of macrophage scavenger receptor activity is not the mechanism responsible in vivo for reduced atherosclerosis or that estrogens act on the arterial wall in multiple ways to reduce atherosclerosis. Recently, several conjugated equine estrogens, including 17{alpha}-dihydroequilin, have been reported to have antioxidant properties, as have other estrogens.50 Since oxidative modification of LDL has been proposed to play a role in the early pathogenesis of atherosclerosis,51 this finding could provide another mechanism by which estrogens protect against the development of atherosclerosis. Clearly, however, the failure of ethynylestradiol to reduce scavenger receptor uptake of acLDL suggests that structural characteristics different from those required for binding to estrogen receptors are required for reducing uptake by the scavenger receptor.

In conclusion, these studies have shown that incubation of the human THP-1 macrophage cell line and elicited mouse peritoneal macrophages with estrogens in vitro reduces uptake of acLDL, resulting in a decrease in cholesterol accumulation and cholesterol esterification. This may be one mechanism by which estrogens protect against the development of atherosclerosis.


*    Selected Abbreviations and Acronyms
 
ACAT = acyl coenzyme A:cholesterol acyltransferase
acLDL = acetylated human LDL
FBS = fetal bovine serum
TCA = trichloroacetic acid


*    Acknowledgments
 
This study was supported by a grant from the Wyeth-Ayerst Company and grant HL-49211 from the United States Public Health Service. This work was done in partial fulfillment of the requirements for the PhD degree for Sulistiyani from the Bowman Gray School of Medicine of Wake Forest University. The authors thank Susie Hester and Linda Glass for technical assistance, Dr Tim Kute for performing estrogen receptor assays, Dr Steve Adelman for the 17{alpha}-dihydroequilin, and Joyce Stafford and Janet Powers for preparation of the manuscript.

Received August 8, 1996; accepted January 21, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Barrett-Connor E. Estrogen and estrogen-progestogen replacement: therapy and cardiovascular diseases. Am J Med. 1993;95:40S–43S.[Medline] [Order article via Infotrieve]
  2. Bush TL, Barrett-Connor E, Cowan LD, Criqui MH, Wallace RB, Suchindran CM, Tyroler HA, Rifkind BM. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from the Lipid Research Clinics Program Follow-up Study. Circulation. 1987;75:1102-1109.[Abstract/Free Full Text]
  3. Sulistiyani, Adelman SJ, Chandrasekaran A, Jayo J, St Clair RW. Effect of 17{alpha}- dihydroequilin sulfate, a conjugated equine estrogen, and ethynylestradiol on atherosclerosis in cholesterol-fed rabbits. Arterioscler Thromb Vasc Biol. 1995;15:837-846.[Abstract/Free Full Text]
  4. Adams MR, Kaplan JR, Manuck SB, Koritnik DR, Parks JS, Wolfe MS, Clarkson TB. Inhibition of coronary artery atherosclerosis by 17ß-estradiol in ovariectomized monkeys. Arteriosclerosis. 1990;10:1051-1057.[Abstract/Free Full Text]
  5. Kushwaha RS, Lewis DS, Carey KD, McGill HC Jr. Effects of estrogen and progesterone on plasma lipoproteins and experimental atherosclerosis in the baboon (Papio sp.). Arterioscler Thromb. 1991;11:23-31.[Abstract/Free Full Text]
  6. Hough JL, Zilversmit DB. Effect of 17ß-estradiol on aortic cholesterol content and metabolism in cholesterol-fed rabbits. Arteriosclerosis. 1986;6:57-63.[Abstract/Free Full Text]
  7. Haarbo J, Leth-Espensen P, Stender S, Christiansen C. Estrogen monotherapy and combined estrogen-progestogen replacement therapy attenuate aortic accumulation of cholesterol in ovariectomized cholesterol-fed rabbits. J Clin Invest. 1991;87:1274-1279.
  8. Wagner JD, Clarkson TB, St Clair RW, Schwenke DC, Shively CA, Adams MR. Estrogen and progesterone replacement therapy reduces LDL accumulation in the coronary arteries of surgically postmenopausal cynomolgus monkeys. J Clin Invest. 1991;88:1995-2002.
  9. Haarbo J, Nielsen LB, Stender S, Christiansen C. Aortic permeability to LDL during estrogen therapy: a study in normocholesterolemic rabbits. Arterioscler Thromb. 1994;14:243-247.[Abstract/Free Full Text]
  10. Masuda J, Ross R. Atherogenesis during low level hypercholesterolemia in the nonhuman primate, I: fatty streak formation. Arteriosclerosis. 1990;10:164-177.[Abstract/Free Full Text]
  11. Rosenfeld ME, Khoo JC, Miller E, Parthasarathy S, Palinski W, Witztum JL. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts. J Clin Invest. 1991;87:90-99.
  12. 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.[Medline] [Order article via Infotrieve]
  13. Hara H, Tanishita H, Yokoyama S, Tajima S, Yamamoto A. Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of human monocytic leukemia cell line (THP-1 cell). Biochem Biophys Res Commun. 1987;146:802-808.[Medline] [Order article via Infotrieve]
  14. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS. Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci U S A. 1986;83:2496-2500.[Abstract/Free Full Text]
  15. Yancey PG, St Clair RW. Cholesterol efflux is defective in macrophages from atherosclerosis-susceptible White Carneau pigeons relative to resistant Show Racer pigeons. Arterioscler Thromb. 1991;12:1291-1304.[Abstract/Free Full Text]
  16. Sulistiyani, St Clair RW. The method of isolation of primary cells and their subculture influences the expression of LDL receptors on pigeon and chicken embryo cells in culture. Atherosclerosis. 1991;91:123-135.[Medline] [Order article via Infotrieve]
  17. Basu SK, Goldstein JL, Anderson GW, Brown MS. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178-3182.[Abstract/Free Full Text]
  18. Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241-260.[Medline] [Order article via Infotrieve]
  19. Eckert RL, Katzenellenbogen BS. Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells: differences with anti-estrogen and estrogen. J Biol Chem. 1982;257:8840-8846.[Free Full Text]
  20. McCloskey HM, Rothblat GH, Glick JM. Incubation of acetylated low-density lipoprotein with cholesterol-rich dispersions enhances cholesterol uptake by macrophages. Biochim Biophys Acta. 1987;921:320-332.[Medline] [Order article via Infotrieve]
  21. Bernard DW, Rodriguez A, Rothblat GH, Glick JM. Influence of high density lipoprotein on esterified cholesterol stores in macrophages and hepatoma cells. Atherosclerosis. 1990;10:135-144.
  22. Ishikawa TT, MacGee J, Morrison JA, Glueck CJ. Quantitative analysis of cholesterol in 5 to 20 µL of plasma. J Lipid Res. 1974;15:286-291.[Abstract]
  23. Adelman SJ, St Clair RW. ß-VLDL metabolism by pigeon macrophages: evidence for two binding sites with different potentials for promoting cholesterol accumulation. Arteriosclerosis. 1989;9:673-683.[Abstract/Free Full Text]
  24. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]
  25. Ellsworth JL, Kraemer FB, Cooper AD. Transport of ß-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway. J Biol Chem. 1987;262:2316-2325.[Abstract/Free Full Text]
  26. Via DP, Pons L, Dennison DK, Fanslow AE, Bernini F. Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1. J Lipid Res. 1989;30:1515-1524.[Abstract]
  27. Goldstein JL, Faust JR, Dygos JH, Chorvat RJ, Brown MS. Inhibition of cholesteryl ester formation in human fibroblasts by an analogue of 7-ketocholesterol and by progesterone. Proc Natl Acad Sci U S A. 1978;75:1877-1881.[Abstract/Free Full Text]
  28. Duval D, Durant S, Homo-Delarche F. Non-genomic effects of steroids: interactions of steroid molecules with membrane structures and functions. Biochim Biophys Acta. 1983;737:409-442.[Medline] [Order article via Infotrieve]
  29. Zhang H, Yang Y, Steinbrecher UP. Structural requirements for the binding of modified proteins to the scavenger receptor of macrophages. J Biol Chem. 1993;268:5535-5542.[Abstract/Free Full Text]
  30. Doi T, Higashino K, Kurihara Y, Wada Y, Miyazaki T, Nakamura H, Uesugi S, Imanishi T, Kawabe Y, Itakura H, Yazaki Y, Matsumoto A, Kodama T. Charged collagen structure mediates the recognition of negatively charged macromolecules by macrophage scavenger receptors. J Biol Chem. 1993;268:2126-2133.[Abstract/Free Full Text]
  31. Brown MS, Goldstein JL. Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptor-bound low density lipoprotein. Cell. 1976;9:663-674.[Medline] [Order article via Infotrieve]
  32. Asai K, Funaki C, Hayashi T, Yamada K, Naito M, Kuzuya M, Yoshida F, Yoshimine N, Kuzuya F. Dexamethasone-induced suppression of aortic atherosclerosis in cholesterol-fed rabbits: possible mechanisms. Arterioscler Thromb. 1993;13:892-899.[Abstract/Free Full Text]
  33. Henze K, Chait A, Albers JJ, Bierman EL. Hydrocortisone decreases the internalization of low density lipoprotein in cultured human fibroblasts and arterial smooth muscle cells. Eur J Clin Invest. 1983;13:171-177.[Medline] [Order article via Infotrieve]
  34. Gulshan S, McCruden AB, Stimson WH. Oestrogen receptors in macrophages. Scand J Immunol. 1990;31:691-697.[Medline] [Order article via Infotrieve]
  35. Danel L, Souweine G, Monier JC, Saez S. Specific estrogen binding sites in human lymphoid cells and thymic cells. J Steroid Biochem. 1983;18:559-563.[Medline] [Order article via Infotrieve]
  36. Greene GL, Press MF. Immunochemical evaluation of estrogen receptor and progesterone receptor in breast cancer. In: Ceriani RL, ed. Immunological Approaches to the Diagnosis and Therapy of Breast Cancer. New York, NY: Plenum Press; 1987:119.
  37. Kute TE, Huske MS, Shore A, Rhyne AL. Improvements in steroid receptor assays including rapid computer analysis of data. Anal Biochem. 1980;103:272-279.[Medline] [Order article via Infotrieve]
  38. Stern MD. Pharmacology of conjugated oestrogens. Maturitas. 1982;4:333-339.[Medline] [Order article via Infotrieve]
  39. Szanto A, Balasubramaniam S, Roach PD, Nestel PJ. Modulation of the low-density-lipoprotein-receptor–related protein and its relevance to chylomicron-remnant metabolism. Biochem J. 1992;288:791-794.
  40. Burgess JW, Stanley KK. 17{alpha}-Ethynylestradiol increases transcytosis of asialoglycoproteins in rat liver. J Biol Chem. 1994;269:3482-3488.[Abstract/Free Full Text]
  41. Hirsch LJ, Mazzone T. Dexamethasone modulates lipoprotein me-tabolism in cultured human monocyte-derived macrophages: stimulation of scavenger receptor activity. J Clin Invest. 1986;77:485-490.
  42. Baranao RI, Tenenbaum A, Rumi LS. Effects of sexual steroid hormones on the functionality of murine peritoneal macrophages. Steroids. 1991;56:481-485.[Medline] [Order article via Infotrieve]
  43. Hu S-K, Mitcho YL, Rath NC. Effect of estradiol on interleukin 1 synthesis by macrophages. Int J Immunopharmacol. 1988;10:247-252.[Medline] [Order article via Infotrieve]
  44. Kraemer FB, Tavangar K, Gandjei RK, Kirlew K, Behr SR. Effects of activation on lipid and lipoprotein metabolism in murine macrophages. Arteriosclerosis. 1990;10:8-16.[Abstract/Free Full Text]
  45. Lopes-Virella MF, Klein RL, Stevenson HC. Low-density lipoprotein metabolism in human macrophages stimulated with microbial or microbial-related products. Arteriosclerosis. 1987;7:176-184.[Abstract/Free Full Text]
  46. De Whalley CV, Riches DW. Influence of the cytocidal macrophage phenotype on the degradation of acetylated low density lipoproteins: dual regulation of scavenger receptor activity and of intracellular degradation of endocytosed ligand. Exp Cell Res. 1991;192:460-468.[Medline] [Order article via Infotrieve]
  47. Fong LG, Fong AT, Cooper AD. Inhibition of mouse macrophage degradation of acetyl-low density lipoprotein by interferon-gamma. J Biol Chem. 1990;265:11751-11760.[Abstract/Free Full Text]
  48. Barnes RB, Lobo RA. Pharmacology of estrogens. In: Mishell DR Jr, ed. Menopause: Physiology and Pharmacology. Chicago, Ill: Year Book Medical Publishers; 1987:310.
  49. Henkin RI, Casper AGT, Brown R, Harlan AB, Bartter FC. Presence of corticosterone and cortisol in the central and peripheral nervous system of the cat. Endocrinology. 1968;82:1058-1061.[Medline] [Order article via Infotrieve]
  50. Subbiah MTR, Kessel B, Agrawal M, Rajan R, Abplanalp W, Rymaszewski Z. Antioxidant potential of specific estrogens on lipid peroxidation. J Clin Endocrinol Metab. 1993;77:1095-1097.[Abstract]
  51. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785-1792.



This article has been cited by other articles:


Home page
 Am. J. Physiol. Renal Physiol.Home page
R. K. Dubey and E. K. Jackson
Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms
Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. G. Fong and D. Le
The Processing of Ligands by the Class A Scavenger Receptor Is Dependent on Signal Information Located in the Cytoplasmic Domain
J. Biol. Chem., December 17, 1999; 274(51): 36808 - 36816.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
J. A. McCrohon, S. Nakhla, W. Jessup, K. K. Stanley, and D. S. Celermajer
Estrogen and Progesterone Reduce Lipid Accumulation in Human Monocyte-Derived Macrophages : A Sex-Specific Effect
Circulation, December 7, 1999; 100(23): 2319 - 2325.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
W. Cheng, O. D. Lau, and N. A. Abumrad
Two Antiatherogenic Effects of Progesterone on Human Macrophages; Inhibition of Cholesteryl Ester Synthesis and Block of Its Enhancement by Glucocorticoids
J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 265 - 271.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend