Role of Activin-A and Follistatin in Foam Cell Formation of THP-1 Macrophages
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Abstract
Abstract Macrophage (Mφ) foam cell formation is a characteristic event that occurs in the early stage of atherosclerosis. To examine the roles of activin-A, a member of the transforming growth factor-β superfamily, and follistatin, the binding protein for activin-A, in Mφ function, we investigated their effects on foam cell formation of THP-1 Mφs. When THP-1 Mφs were treated with activin-A (5 nmol/L), foam cell formation and cellular cholesteryl ester accumulation were decreased. This downregulation was paralleled by a reduction in cell association and degradation of acetylated LDL. The inhibitory effect of activin-A on cell association and degradation was dose dependent, and the effect was blocked by concomitant addition of follistatin. Activin-A (5 nmol/L) also decreased the Bmax for acetylated LDL and scavenger receptor mRNA expression. Follistatin showed an effect opposite to that of activin-A and promoted Mφ foam cell formation and cellular cholesteryl ester accumulation. It increased binding, cell association, and degradation of acetylated LDL and upregulated scavenger receptor mRNA expression. Because follistatin is the binding protein for activin-A, follistatin’s effect is considered to be mediated by blocking the inhibitory effect of intrinsic activin-A. These results indicate that activin-A inhibits and follistatin promotes Mφ foam cell formation by regulating scavenger receptor mRNA expression. We conclude that activin-A and follistatin play important roles in the process of atherosclerosis by regulating Mφ foam cell formation.
- Received November 8, 1996.
- Accepted April 28, 1997.
Foam cell formation of macrophages is observed in the early stage of atherosclerosis.1 Mφs scavenge lipoproteins that have undergone oxidative modification in the vascular wall. Mφs take up modified LDLs via the SR pathway. Unlike the LDL receptor, the SR is not downregulated by cellular cholesterol content; consequently, Mφs become foam cells when they take up excessive amounts of modified lipoproteins via the SR.2 It has been shown that several cytokines and factors regulate SR expression and/or activity. M-CSF is a positive regulator that increases the activity of the SR.3 4 5 Lipopolysaccharide,6 tumor necrosis factor-α,6 7 TGF-β,8 interferon-γ,9 10 and GM-CSF11 are negative regulators.
Activin-A, a member of the TGF-β superfamily,12 13 has a variety of important biological functions: it promotes secretion of follicle-stimulating hormone from anterior pituitary cells,14 induces differentiation of erythroid cells,12 promotes survival of neuronal cells,15 and induces mesoderm in Xenopus during development.16 Follistatin is a 35-kD glycoprotein that binds to activin and neutralizes its effect.17 18 19 Follistatin is widely expressed in the body20 21 and is considered to be an important modulator of activin. We previously demonstrated that activin-A is expressed in arteriosclerotic lesions of the Watanabe heritable hyperlipidemic rabbit.22 We also showed that follistatin is expressed in the neointima and media of the rat denuded carotid artery and is produced by vascular SMCs.23 Although it has been reported that activin-A inhibits vascular endothelial cell growth24 and promotes SMC growth,25 nothing has been reported about the effect of activin-A and follistatin on the function of Mφ, which are key regulators of atherogenesis. In the present study, we examined the effects of activin-A and follistatin on Mφ function and found that the transformation of THP-1 Mφ into foam cells was inhibited by activin-A and promoted by follistatin. We also revealed that this regulation may take place at the mRNA level of the SR. Together with the existence of these factors in atherosclerotic lesions, our results suggest that activin-A and follistatin may play important roles in the development of atherosclerosis.
Methods
Materials
Recombinant human activin-A was provided by Dr Y. Eto of the Central Research Laboratory of Ajinomoto Co, Ltd. Activin-A was purified from the media of Chinese hamster ovary cells bearing an expression vector for the cDNA of the βA subunit of activin.26 It was confirmed that purified activin-A was free of endotoxin. Recombinant human follistatin was also provided by Dr Y. Eto. Recombinant human M-CSF was obtained from Morinaga Milk Industry Co, Ltd. The following materials were purchased from various sources: human platelet-derived growth factor-BB from Genzyme; [α-32P]dCTP and [125I]NaI from NEN Research Products; RPMI 1640 from GIBCO BRL; fetal bovine serum from Bioserum; and TPA from Sigma Chemical Co. All other chemicals were of analytical grade.
Cell Culture
THP-1 cells from the American Type Culture Collection (Rockville, Md) were cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum, 1.5 g/L NaHCO3, 80 mg/mL kanamycin monosulfate, and 15 mmol/L HEPES (pH 7.4). The cells were maintained in a humidified 5% CO2 chamber at a cell density of 2 to 10 ×105/mL. THP-1 cells were differentiated in RPMI 1640–5% fetal bovine serum medium containing 100 ng/mL TPA for 48 hours. After being washed three times with PBS, differentiated THP-1 cells, termed THP-1 Mφ, were cultured in RPMI 1640–5% LPDS medium in the presence of activin-A or follistatin for 24 to 48 hours.
Preparation of Ac-LDL, Lipoprotein-Deficient Serum, and 125I-Labeling of Ac-LDL
Plasma was collected from normolipidemic volunteers in a fasting state. LDL was isolated from the plasma by sequential density ultracentrifugation.27 The fraction of d>1.21 g/mL was collected and dialyzed extensively in sodium phosphate buffer (150 mmol/L NaCl and 2 mmol/L sodium phosphate, pH 7.4). It was sterilized by filtration (0.45 μm) and used as LPDS. LDL was acetylated by repetitive additions of acetic anhydride as described previously.28 Ac-LDL was radioiodinated with [125I]NaI by the iodine monochloride method.29
Oil Red O Staining
After THP-1 Mφs (1.6×106 cells per well in six-well plates) were cultured with activin-A (5 nmol/L) or follistatin (500 ng/mL) for 48 hours, cells were incubated in RPMI 1640–5% LPDS medium containing 50 μg/mL Ac-LDL in the presence of activin-A (5 nmol/L) or follistatin (500 ng/mL) at 37°C for 24 hours. After being washed three times with PBS, cells were fixed with 6% paraformaldehyde–0.1 mol/L sodium phosphate (pH 7.4) and stained with oil red O and hematoxylin. To evaluate the extent of foam cell formation, the red-stained areas were quantified by using NIH Image 1.56. In brief, the red-stained areas were “extracted” from the original image (×400 magnification). Then their sizes were calculated and divided by the size of the entire original area (cell area was almost the same in all observed fields). This analysis was performed in four random fields for control, activin-A–treated, and follistatin-treated cells.
Measurement of CE
After THP-1 Mφs (2.0×106 cells per well in six-well plates) were cultured with activin-A (5 nmol/L), follistatin (500 ng/mL), or fucoidan (100 μg/mL) for 48 hours, cells were incubated in RPMI 1640–5% LPDS medium containing 50 μg/mL Ac-LDL in the presence of activin-A (5 nmol/L), follistatin (500 ng/mL), or fucoidan (100 μg/mL) at 37°C for 36 hours. After the cells were washed three times with TBS (pH 7.4) containing 2 mg/mL bovine serum albumin and another three times with TBS, cellular lipids were extracted in 2 mL hexane/isopropanol (3:2, vol/vol). The extracts were evaporated under a stream of N2 gas and resolved in 150 μL isopropanol. Total and free cholesterol values were quantified by a fluorimetric enzymatic method using Determiner TC 555 and Determiner FC 555 (Kyowa Medics). The difference between the amounts of total and free cholesterol was determined as cellular CE content. Each value was corrected with respect to the amount of cell protein that was measured by the Bio-Rad protein assay (Bio-Rad Labs).
Cell Association, Degradation, and Binding of 125I–Ac-LDL
THP-1 Mφs (1×106 cells per well in 12-well plates) were cultured with activin-A (5 nmol/L) or follistatin (500 ng/mL) for 48 hours. After being washed three times with PBS, cells were incubated in RPMI 1640–5% LPDS medium containing 125I–Ac-LDL at 37°C for 6 hours (to measure cell association and degradation) or at 0°C for 2 hours (to measure binding) as described previously.30 Each value was corrected with respect to the amount of cell protein. Nonspecific cell association, degradation, and binding were determined by adding a 40-fold excess of unlabeled Ac-LDL. Specific values were calculated by subtracting the nonspecific value from the total value.
RNA Extraction and Northern Blot Analysis
Total RNA was extracted by the acid guanidinium thiocyanate/phenol/chloroform method31 from THP-1 Mφs that had been cultured with activin-A (5 nmol/L) or follistatin (500 ng/mL) for 24 hours. Poly(A)+ RNA was prepared by using Oligo-dT30 “Super” (Nippon Roche) according to the manufacturer’s protocol. Then, 4 μg poly(A)+ RNA was subjected to electrophoresis in a 1% formaldehyde/agarose gel and transferred to a nylon membrane (Gene-Screen Plus, NEN Research Products). The membrane was hybridized with a randomly primed, 32P-labeled human Mφ SR cDNA probe32 or with a human β-actin cDNA probe (Wako Pure Chemicals). The membrane was washed in 15 mmol/L NaCl, 1.5 mmol/L sodium citrate, and 0.1% SDS solution at 65°C for 20 minutes and then exposed to x-ray film at −80°C for 2 days. The signal density of SR1 and SR2 was measured by densitometry, and values were corrected by comparison with the value for the density of β-actin.
Statistics
All values in the text and figures are expressed as mean±SEM. The data were analyzed by one-factor ANOVA. If statistical significance was found, the Newman-Keuls test was performed to isolate the difference between groups. A value of P<.05 was considered significant.
Results
Foam Cell Formation of THP-1 Mφs
When THP-1 Mφs were incubated with Ac-LDL (50 μg/mL) for 24 hours, lipid droplets accumulated within the cytoplasm (Fig 1A⇓, red stained area). Compared with control THP-1 Mφs, activin-A suppressed and follistatin enhanced transformation of foam cells (Fig 1B⇓).
Oil red O staining. THP-1 Mφs (1.6×106 cells per well in six-well plates) that had been treated with activin-A (5 nmol/L) or follistatin (500 ng/mL) for 48 hours were incubated in RPMI 1640–5% LPDS containing 50 μg/mL Ac-LDL in the presence of activin-A (5 nmol/L) or follistatin (500 ng/mL) at 37°C for 24 hours. Then cells were fixed and stained with oil red O and hematoxylin (×400 magnification). A, Oil red O staining (photomicrograph). B, Quantitative analysis of red-stained areas. The size of the red-stained areas was quantified by using NIH Image 1.56 and that value was divided by the size of the entire original area and shown as percent. This was performed in four random fields for control, activin-A–treated, and follistatin-treated cells, respectively. All values are expressed as mean±SEM.
CE Content in THP-1 Mφs
Because cellular CE accumulation is known to parallel Mφ foam cell formation,2 we examined the effects of activin-A and follistatin on cellular CE content. CE mass after a 36-hour incubation with 50 μg/mL Ac-LDL was 14.9±1.9 μg/mg cell protein in control THP-1 Mφs (Fig 2⇓). Activin-A decreased CE content to 11.0±0.7 μg/mg cell protein (74% of control). Fucoidan, which is a competitive inhibitor of Ac-LDL, also markedly decreased cellular CE content. On the other hand, follistatin increased cellular CE content to 18.5±0.8 μg/mg cell protein (124% of control). This effect was almost equivalent to that of anti–activin-A antibody (data not shown).
CE content in THP-1 Mφs. THP-1 Mφs (2.0×106 cells per well in six-well plates) that had been treated with activin-A (5 nmol/L), follistatin (500 ng/mL), or fucoidan (100 μg/mL) for 48 hours were incubated with 50 μg/mL Ac-LDL in the presence of activin-A (5 nmol/L), follistatin (500 ng/mL), or fucoidan (100 μg/mL) at 37°C for 48 hours. Then cellular lipid was extracted and CE content measured by fluorimetric enzymatic methods. Each value was corrected with respect to the amount of cell protein. All values are expressed as mean±SEM (n=4). *P<.05, **P<.01 vs control.
Cell Association and Degradation of 125I–Ac-LDL
To clarify the mechanisms of regulation of foam cell formation by activin-A and follistatin, we examined their effects on the activity of the SR. As shown in Fig 3⇓ activin-A decreased cell association and degradation of 125I–Ac-LDL in a dose-dependent manner. Although the inhibitory effect of activin-A was less potent than that of TGF-β, activin-A (>5 nmol/L) significantly decreased SR activity. The inhibitory effect of activin-A (5 nmol/L) was abolished when follistatin (500 ng/mL) was added concomitantly. Because follistatin is the binding protein for activin-A, this result confirms that the inhibition of SR activity was indeed caused by activin-A. Follistatin at concentrations >100 ng/mL significantly increased cell association and degradation of 125I–Ac-LDL (Fig 4⇓). The effect of follistatin was almost as strong as that of M-CSF, which is known to increase activity of the SR.
Dose-dependent effect of activin-A on cell association and degradation of 125I–Ac-LDL. THP-1 Mφs (1×106 cells per well in 12-well plates) that had been treated with 0.05, 0.5, 5, or 50 nmol/L activin-A; 0.5 nmol/L TGF-β; or activin-A (5 nmol/L) together with follistatin (500 ng/mL) were incubated with 15 μg/mL 125I–Ac-LDL at 37°C for 6 hours. Cell association of 125I–Ac-LDL (left) after 6-hour incubation and degradation of 125I–Ac-LDL (right) during the 6-hour incubation were measured. Each value was corrected with respect to the amount of cell protein. Nonspecific values (open columns) were determined in the presence of a 40-fold excess of unlabeled Ac-LDL. Specific values (designated columns) were calculated by subtracting nonspecific values from total values. All values are expressed as mean±SEM (n=3). *P<.05, **P<.01 vs control.
Effects of follistatin (dose dependence) and M-CSF on cell association and degradation of 125I–Ac-LDL. THP-1 Mφs (1×106 cells per well in 12-well plates) that had been treated with 1, 10, 100, or 1000 ng/mL follistatin or with M-CSF (100 ng/mL) were incubated with 15 μg/mL 125I–Ac-LDL at 37°C for 6 hours. Cell association (left) and degradation (right) of 125I–Ac-LDL were measured. Each value was corrected with respect to the amount of cell protein. Specific values are shown (mean±SEM, n=4). *P<.05 vs control.
Binding of 125I–Ac-LDL
Next we investigated the effects of activin-A and follistatin on the binding of Ac-LDL. As shown in Fig 5⇓, all binding curves showed saturation. Activin-A (5 nmol/L) decreased and follistatin (500 ng/mL) increased the binding of 125I–Ac-LDL. Scatchard analysis revealed that Bmax was decreased by activin-A and increased by follistatin (Bmax=60, 38, and 79 ng/mg cell protein for control, activin-A–treated, and follistatin-treated cells, respectively). On the other hand, the binding affinity for Ac-LDL was not altered (Kd=120 μg/mL).
Binding of 125I–Ac-LDL to THP-1 Mφs. THP-1 Mφs (1×106 cells per well in 12-well plates) that had been treated with 5 nmol/L activin-A or 500 ng/mL follistatin were incubated with 1, 2, 4, 6, 9, or 15 μg/mL 125I–Ac-LDL at 0°C for 2 hours. Cell-bound 125I–Ac-LDL after 2-hour incubation was measured. A, Saturation curve. Each value was corrected with respect to the amount of cell protein. All values are expressed as mean±SEM (n=3). Control, activin-A–treated, and follistatin-treated cells are shown by circles, triangles, and squares, respectively. Normal, dotted, and bold lines indicate total, nonspecific, and specific binding curves, respectively. B, Corresponding Scatchard plot.
Northern Blot Analysis for SR mRNA Expression
To clarify the mechanisms of this downregulation/upregulation of Ac-LDL binding, we examined expression of the SR at the mRNA level. THP-1 Mφs expressed two types of SR mRNA, 5.5-kb SR1 and 4.4-kb SR2. Activin-A slightly decreased SR1 and clearly decreased SR2 mRNA expression to 80% and 40% of control levels, respectively (Fig 6⇓). In contrast, both SR1 and SR2 mRNA expression was markedly increased by follistatin to 310% and 170% of control levels, respectively.
Northern blot analysis for SR mRNA expression. Poly(A)+ RNA was extracted from THP-1 Mφs that had been treated with 5 nmol/L activin-A or 500 ng/mL follistatin for 24 hours. Then 4 μg of each poly(A)+ RNA was subjected to gel electrophoresis, transferred to a nylon membrane, and hybridized with 32P-labeled human SR or β-actin cDNA probe. SR1 and SR2 indicate type I and type II SRs, respectively.
Discussion
Activin-A, first identified as an activating factor for the release of follicle-stimulating hormone from anterior pituitary cells, is now known to be expressed widely in the body and is recognized as a regulator of diverse important cellular functions.33 In vascular wall cells, activin-A has been shown to inhibit endothelial cell growth24 and promote SMC growth.25 In the present study, we have shown that activin-A also had an effect on Mφs, which play a major role in the atherosclerotic process.
Our results revealed that activin-A inhibited foam cell formation of and CE accumulation in THP-1 Mφs. This downregulation was paralleled by a reduction in cell association and degradation of Ac-LDL. Because it is known that CE in foamy Mφs is derived from cholesterol released from degraded lipoprotein particles,2 our results suggest that inhibition of foam cell formation and CE accumulation may be mediated by reductions in the cell association and degradation of Ac-LDL. This reduction is possibly caused by a decrease in the number of SRs, because the binding study showed that activin-A decreased Bmax, whereas it had little influence on affinity.
Which process yields the reduction in SR expression/activity by activin-A? The possibility that activin-A reduces Ac-LDL binding by directly interfering with ligand binding can be excluded, because in the cell association and degradation study, cells were treated with activin-A only before Ac-LDL was added. Furthermore, if activin-A were a competitive inhibitor, it could not suppress SR mRNA expression. Therefore, it is unlikely that activin-A acted as a competitive inhibitor.
Previous studies have established that lipopolysaccharide can inhibit Mφ scavenger function by blocking ligand binding and by decreasing receptor gene expression. To rule out the possibility that the purified activin-A that we used in the present study was contaminated with lipopolysaccharide, we measured the level of endotoxin in activin-A solution by a turbidimetric time assay.34 As a result, endotoxin was undetectable (<10 pg/mL). Therefore, it is unlikely that the reduction in SR expression/activity was caused by the concurrent presence of lipopolysaccharide with activin-A.
With respect to the reduced binding capacity for Ac-LDL, we also found that activin-A downregulated the SR at the mRNA level. However, the precise mechanism of this downregulation (eg, instability, transcriptional downregulation, or the effect of another protein) is unknown. Taken together, our data suggest that the inhibition of foam cell formation by activin-A is regulated, at least in part, by downregulation of the SR at the mRNA level.
The inhibitory effect of activin-A was modest compared with that of TGF-β (Fig 3⇑). Previously, Bottalico et al38 had shown that TGF-β inhibited Ac-LDL degradation by ≈70%. They also showed that 24 pmol/L TGF-β was sufficient to exert the maximum effect for suppression of Ac-LDL degradation. Activin-A and TGF-β bind to heteromeric receptor complexes known as type I and type II receptors. The type II receptor determines ligand-binding specificity, and each receptor interacts with a distinct repertoire of type I receptor.35 Therefore, even though the inhibitory effect of activin-A is less potent than that of TGF-β, the functional roles of activin-A and TGF-β are considered to be different.
Follistatin is the binding protein of activin-A and is known to inhibit its action.17 18 19 In the present study, we have shown that follistatin itself promotes Mφ foam cell formation and CE accumulation. Follistatin also increased the activity and expression of the SR. Because it is already shown that THP-1 Mφs can produce activin-A,12 36 these effects of follistatin may result from blockade of the effect of intrinsic activin-A. Indirectly, the evidence that enhancement of CE accumulation by follistatin was almost equivalent to that by anti–activin-A antibody indicates that follistatin acted as a blocker of intrinsic activin-A. When THP-1 cells and human monocytes are induced to differentiate, both types of cell come to express the SR37 38 39 40 and produce activin-A.12 36 41 42 Because we showed that activin-A decreased SR expression, it is suggested that basal expression of SR is kept suppressed by simultaneously produced activin-A during Mφ differentiation.
Several lines of evidence indicate that activin-A and follistatin are involved in atherogenesis. We found that activin-A is expressed in arteriosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.22 Activin-A is predominantly expressed in the neointima of the diseased artery. In vitro experiments from other groups have shown that activin-A is produced by human monocytes/Mφs41 42 as well as THP-1 Mφs.12 36 On the other hand, follistatin is expressed in the neointima and media of the rat denuded carotid artery.23 Furthermore, follistatin expression is increased in atherosclerotic lesions in comparison with normal vascular tissue. In vitro experiments have suggested that SMCs can produce follistatin.23 Although it is not known how activin-A and follistatin are differently regulated in arteriosclerotic lesions, the balance of these factors may determine the level of SR expression on Mφs.
In conclusion, we demonstrated that activin-A inhibited the expression and activity of the SR in THP-1 Mφ, resulting in suppression of cellular CE accumulation and Mφ foam cell formation. We also revealed that follistatin, probably by blocking the inhibitory effect of intrinsic activin-A, increased expression and activity of the SR, thus promoting accumulation of cellular CE and the transformation of Mφs into foam cells. Activin-A and follistatin, which are expressed in arteriosclerotic lesions, may regulate the Mφ foam cell formation that is a key step in the process of atherosclerosis.
Selected Abbreviations and Acronyms
Ac-LDL | = | acetylated LDL |
CE | = | cholesteryl ester |
(G)M-CSF | = | (granulocyte) macrophage colony stimulating factor |
LPDS | = | lipoprotein-deficient serum |
Mφ | = | macrophage |
SMC | = | smooth muscle cell |
SR | = | scavenger receptor |
TGF-β | = | transforming growth factor-β |
Acknowledgments
This study was supported in part by a grant-in-aid (No. 08670771) for Scientific Research from the Ministry of Education, Science and Culture of Japan. We would like to thank Dr Y. Eto of the Central Research Laboratory of Ajinomoto Co, Ltd, for providing us with activin-A and follistatin and giving us useful advice. We also thank Masae Watanabe and Hitomi Yamaguchi for excellent technical assistance.
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- Role of Activin-A and Follistatin in Foam Cell Formation of THP-1 MacrophagesKoichi Kozaki, Masahiro Akishita, Masato Eto, Masao Yoshizumi, Kenji Toba, Satoshi Inoue, Michiro Ishikawa, Masayoshi Hashimoto, Tatsuhiko Kodama, Nobuhiro Yamada, Hajime Orimo and Yasuyoshi OuchiArteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2389-2394, originally published November 1, 1997https://doi.org/10.1161/01.ATV.17.11.2389
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- Role of Activin-A and Follistatin in Foam Cell Formation of THP-1 MacrophagesKoichi Kozaki, Masahiro Akishita, Masato Eto, Masao Yoshizumi, Kenji Toba, Satoshi Inoue, Michiro Ishikawa, Masayoshi Hashimoto, Tatsuhiko Kodama, Nobuhiro Yamada, Hajime Orimo and Yasuyoshi OuchiArteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2389-2394, originally published November 1, 1997https://doi.org/10.1161/01.ATV.17.11.2389