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【摘要】
Background- Macrophage scavenger receptors facilitate the uptake of modified low-density lipoprotein (LDL), formation of foam cells, and development of atherosclerosis. Given that proinflammatory cytokines, including IL-6, can modulate the macrophage foaming process, the aim of the present study was to determine whether the synthetic retinoic acid receptor- /ß-specific agonist Am80, which is also an IL-6 inhibitor, can modulate macrophage lipid accumulation and foam cell formation.
Methods and Results- Am80 suppressed IL-6 production induced by 12-myristate 13-acetate (PMA) or angiotensin II in mouse Raw264 macrophages. It also suppressed expression of the 2 major scavenger receptors (scavenger receptor-A [SR-A] and CD36), in part by inhibiting IL-6, and inhibited macrophage foam cell formation. Systemic administration of Am80 led to reductions in the areas of atherosclerotic lesions and foam cell accumulation in the aortas of apolipoprotein E (apoE) -deficient mice and reduced serum concentrations of IL-6 and IL-1ß without affecting body weights, serum lipid profiles or IL-10 levels.
Conclusions- Am80 suppresses scavenger receptor expression and macrophage foam cell formation in vitro and prevents atherogenesis in apoE -deficient mice in vivo. This suggests Am80 is a novel candidate agent that could be highly useful in the prevention and treatment of atherosclerosis.
Synthetic retinoid Am80 inhibits IL-6 signaling and suppresses scavenger receptor expession in macrophages. Moreover, Am80 prevents macrophage from cell formation in vitro and inhibited atherogenesis in apoE -dificient moce. Am80 is a novel candidate agent that could be highly useful in the prevention and treatment of atherosclerosis.
【关键词】 macrophage IL CD scavenger receptorA retinoid
Introduction
Macrophage foam cell formation is a hallmark of both early and late atherosclerotic lesions. 1 During that process, macrophages take up modified low-density lipoprotein (LDL) via scavenger receptors in the vessel wall and release a variety of immune mediators, reactive oxygen species and proteases, thereby playing a pivotal role in atherogenesis. Two macrophage scavenger receptors, scavenger receptor-A (SR-A) and CD36, mediate the majority of modified LDL uptake and promote the development of atherosclerosis. 2 Indeed, mice lacking these 2 receptors do not accumulate esterified cholesterol derived from modified LDL. 3 Thus, development of drugs that modulate scavenger receptor function could potentially provide a strong basis for a novel antiatherogenic therapy. 2
Proinflammatory cytokines are known to affect both the expression of scavenger receptors and the formation of macrophage foam cells. 2 IL-6, for example, is expressed within atherosclerotic lesions in macrophage-rich areas 4 and may stimulate inflammatory responses in macrophages, as well as proliferation of smooth muscle cells (SMCs) and pro-thrombotic activity. 5 Recent studies have also shown that IL-6 plays a key role in angiotensin II (Ang II)-mediated CD36 expression and uptake of oxidized LDL in mouse peritoneal macrophages, and promotes atherogenesis. 6,7 That mouse peritoneal macrophages obtained from IL-6 -deficient mice do not show upregulation of CD36 expression in response to Ang II stimulation 6 suggests inhibition of IL-6 is potentially promising therapeutic strategy for the treatment of atherosclerosis.
Am80 is a retinoic acid receptor (RAR) /ß-specific synthetic retinoid, and it neither binds to nor transactivates the retinoid X receptors (RXRs). 8 Am80 is also known to inhibit the IL-6 signaling. 9-11 Am80 suppresses IL-6 production in splenic mononuclear cells and reduces the severity and progression of inflammatory disease models, including 2,4-dinitrofluorobenzene-induced contact dermatitis, 10 collagen-induced arthritis, 11 and allergic encephalomyelitis. 9 Recently, we reported that Am80 inhibits neointima formation in a mouse vascular injury model, suggesting it also modulates inflammatory and remodeling processes in the vessel wall. 12 However, it is not yet known whether Am80 has the capacity to modulate macrophage function.
The aims of the present study were to determine 1 whether Am80 can inhibit macrophage IL-6 production and foam cell formation in vitro, and 2 whether treatment with Am80 can affect the development of atherosclerotic lesions in apolipoprotein E (apoE) -deficient mice in vivo. Our findings indicate that Am80?s ability to inhibit IL-6 expression enables it to suppress both macrophage foam cell formation and atherogenesis.
Materials and Methods
Plasmids
The IL-6 promoter reporter constructs pIL6-luc651, pIL6-luc651 NF- B, and pIL6-luc651 C/EBPß were a generous gift from Dr O. Eickelberg. 13 The CD36 promoter reporter constructs pGL-CD36 (-273/luc), was a generous gift from Dr R.M. Evans. 14 The RXRa expression vector CMX-hRXR was a general gift from Dr R. Schule. 15 The PPAR expression vector pCAG-PPAR was previously described. 16
For enhanced Materials and Methods used in this article, please see http://atvb.ahajournals.org.
Results
Am80 Inhibits IL-6 Expression
We first analyzed the effect of Am80 on production of IL-6 in macrophages. Raw264 cells were cultured with or without various concentrations of Am80 in the presence of 100 ng/mL PMA, a model agonist that induces scavenger receptor expression 14 or 1 µmol/L Ang II. As shown in Figure 1 A, PMA and Ang II induced significant IL-6 production and secretion in Raw264 cells, and this effect was dose-dependently inhibited by Am80. Likewise, Am80 dose-dependently inhibited PMA and Ang II-induced IL-6 mRNA expression ( Figure 1 B).
Figure 1. Am80 suppresses IL-6 production in macrophages. Raw264 cells were pretreated with the indicated concentration of Am80 for 12 hours and then stimulated with 100 ng/mL PMA or 1 µmol/L Ang II for 24 hours (A) or 12 hours (B). IL-6 released into the medium was analyzed by enzyme-linked immunosorbent assay (ELISA) (A). IL-6 mRNA expression was assessed by real-time polymerase chain reaction (PCR; B). Data are expressed as means±SD of triplicate wells. # P <0.01 vs untreated control; * P <0.05, ** P <0.01 vs PMA alone. C, Am80 suppresses IL-6 promoter activity; Raw264 or Ang II cells were transfected with pIL6-luc651, pIL6-luc651 NF- B or pIL6-luc651 C/EBPß reporter plasmid. Am80 (10 -7 mol/L) was added 18 hours after transfection, and after an additional 6 hours the cells were treated with PMA (100 ng/mL). Luciferase activity was measured 48 hours after transfection. To correct for variation in transfection efficiency, we cotransfected pCMV-ßgal in all experiments. The ratio of the luciferase activity to the ß-galactosidase activity in each sample served as a measure of the normalized luciferase activity. Data are expressed as means±SD of triplicate wells and are representative of three independent experiments. # P <0.05, ## P <0.01 vs control of each construct; * P <0.05, ** P <0.01 vs PMA alone of each construct.
Am80 Inhibits the IL-6 Promoter Through C/EBPß
Its inhibition of IL-6 mRNA expression suggested that Am80 may inhibit IL-6 gene expression at the level of transcription. To test that idea, we analyzed the effect of Am80 on IL-6 promoter activity. Raw264 cells were transfected with an IL-6 promoter-reporter construct (pIL6-luc651), after which the transfected cells were incubated with or without Am80 (10 -7 mol/L) for 6 hour and then treated with PMA for 24 hour. As expected, PMA stimulated IL-6 promoter activity ( Figure 1 C). This effect was inhibited by Am80, further confirming that the retinoid suppresses IL-6 production at least in part by inhibiting IL-6 transcription.
The IL-6 promoter is known to be controlled by C/EBPß (NF-IL6) and NF- B. 17,18 Because earlier studies have shown that ligand-bound RAR inhibits transactivation by C/EBPß, 18 we hypothesized that Am80 might suppress IL-6 transcription by inhibiting C/EBPß. To test that idea, we transfected cells with mutant IL-6 promoter constructs in which either the C/EBPß or NF- B binding site was mutated. Mutation of the NF- B binding site resulted in a significant (49%) reduction in IL-6 promoter activity, as compared with the wild-type construct under the basal culture conditions; mutation of the C/EBPß binding site reduced activity to a slightly lesser degree (24%). PMA significantly increased the activity of both mutant promoter constructs. Am80, however, significantly reduced the reporter activity of the NF- B site mutant (pIL6-luc651 NF- B) but had no effect on that of the C/EBPß site mutant (pIL6-luc651 C/EBPß) ( Figure 1 C). Apparently, an intact C/EBPß binding site is required for Am80 to exert an effect on IL-6 promoter activity.
We also tested whether Am80 treatment would influence C/EBPß expression and found that it had no effect on basal expression of C/EBPß, nor did it affect the ability of PMA to stimulate C/EBPß expression 19 (Figure I, see http://atvb.ahajournals.org). This suggests that Am80 may inhibit IL-6 promoter activity by interfering with the function of C/EBPß.
Am80 Reduces the Cholesterol Content and the Size and Number of Lipid Droplets in Mouse Peritoneal Macrophages
We next tested whether Am80?s ability to inhibit IL-6 production in macrophages might affect foam cell formation. When peritoneal macrophages were incubated with acetylated or oxidized LDL in the presence or absence of Am80, the number and size of intracellular lipid droplets were markedly smaller in the Am80-treated cells ( Figure 2 A). Consistent with those results, Am80 significantly reduced intracellular levels of both cholesterol ester and free cholesterol ( Figure 2 B). Taken together, these findings indicate that Am80 can indeed suppress macrophage-to-foam cell transformation.
Figure 2. Am80 reduces the cholesterol content and the size and number of lipid droplets in mouse peritoneal macrophages. A, Oil Red O staining. Mouse peritoneal macrophages were treated with 10 -7 mol/L Am80 for 12 hours and then stimulated with 50 µg/mL acetylated LDL (upper) or 25 µg/mL oxidized LDL (lower) for 48 hour. The macrophages were then fixed and stained with Oil Red O. The scale bar indicates 20 µm. B, Cellular cholesterol content. Mouse peritoneal macrophages were treated with the indicated concentration of Am80 for 12 hours and then stimulated with 50 µg/mL acetylated LDL (left) or 25 µg/mL oxidized LDL (right) for 48 hours, after which intracellular total cholesterol (TC) and free cholesterol (FC) were measured using an enzymatic fluorometric microassay. The amount of esterified cholesterol was calculated by subtracting FC from TC. Data are means±SD of 3 independent experiments. # P <0.01 vs each control; * P <0.05, ** P <0.01 vs acetylated or oxidized LDL alone.
Am80 Suppresses Expression of SR-A and CD36 in Mouse Peritoneal and Raw264 Macrophages
Modified LDL promotes its own uptake into macrophages by upregulating the scavenger receptors SR-A and CD36. 20 We therefore hypothesized that Am80 might affect expression of these receptors. To test that idea, we treated Raw264 macrophages with PMA, which is known to upregulate expression of both SR-A and CD36. 21,22 As expected, PMA treatment increased mRNA expression of both of those genes in peritoneal macrophages, Raw264 cells (Abelson virus-transformed, murine macrophage-derived cell line 23 ) and THP-1 cells (human acute monocytic leukemia cell line 24 ) ( Figure 3 A). Am80 dose-dependently inhibited the PMA-induced expression of the 2 scavenger receptors in peritoneal macrophages and Raw264 cells and reduced expression of SR-A in THP-1 cells ( Figure 3 A). However, CD36 expression was somewhat upregulated by Am80 in THP-1 cells. Although the exact mechanism is unknown, the differential effect of Am80 on CD36 expression in macrophages (Raw264 and peritoneal macrophages) and monocytic THP-1 cells might reflect differences in species and in the differentiation state of the cells (see Discussion). Because the patterns of regulation of both SR-A and CD36 were similar in mouse peritoneal macrophages and Raw264 cells, we deemed Raw264 cells to be an appropriate model for use in the following experiments.
Figure 3. Effect of Am80 on scavenger receptor expression in macrophages. A, Peritoneal macrophages, Raw264 cells, and THP-1 cells were pretreated with Am80 (10 -6 mol/L), atRA(10 -6 mol/L) or vehicle for 12 hours, and then treated with 50 ng/mL of PMA or vehicle for additional 48 hours. Expression of SR-A and CD36 mRNA was analyzed by real-time PCR. B, Raw264 cells were treated with the indicated concentrations of Am80 for 12 hour and then stimulated with 25 µg/mL of oxidized LDL for 48 hours. C, IL-6 restores scavenger receptors in PMA/Am80-treated cells. Raw264 cells were treated with Am80 (10 -6 mol/L) for 12 hours and then stimulated with the indicated concentrations of mouse IL-6 and 50 ng/mL PMA for 48 hours. Data are means±SD of 3 independent experiments. # P <0.05, ## P <0.01 vs untreated control (A,B); * P <0.05, ** P <0.01 vs PMA (A) or oxidized LDL (B) alone. * P <0.05, ** P <0.01 vs PMA/Am80 alone (C). D, Flow cytometry; Raw264 cells were treated with Am80 (10 -7 mol/L) or vehicle for 12 hours, and then stimulated with 50 ng/mL PMA with or without 30 ng/mL mouse IL-6 for 30 hours. SR-A (A) and CD36 (B) expression were detected by flow cytometry. Black line histogram represents the control (untreated) cells; blue is PMA treated; red is PMA/Am80; and green is PMA/Am80 plus IL-6, respectively.
Like PMA, oxidized LDL upregulated SR-A and CD36 in Raw264 cells, and that upregulation was dose-dependently inhibited by Am80 ( Figure 3 B), which is consistent with the Am80-induced inhibition of cholesterol uptake by peritoneal macrophages seen in Figure 2. When we considered whether its inhibitory effect on IL-6 signaling might be involved in Am80?s inhibition of scavenger receptor expression, we found that addition of exogenous IL-6 partially restored expression of CD36 and SR-A mRNA, which was otherwise inhibited by Am80 ( Figure 3 C). Similarly, induction of the surface CD36 and SR-A proteins in Raw264 cells was inhibited by Am80 by flow cytometric analysis ( Figure 3 D), and this inhibition was partially restored by IL-6. These data demonstrate that Am80 acts to suppress expression of scavenger receptors at least in part via effects on IL-6 expression.
Am80 Inhibits the IL-6 Signaling
We then analyzed if Am80 might affect the IL-6-induced CD36 and SR-A expression. As expected, IL-6 upregulated expression of the scavenger receptor genes in Raw264 cells ( Figure 4 A). Am80 treatment resulted in decreases in the levels of the gene expression, suggesting Am80 might modulate the signaling mechanism that is elicited by IL-6 and leads to upregulation of CD-36 and SR-A.
Figure 4. Effect of Am80 on IL-6 induced signaling. A, Raw264 cells were pretreated with 10 ng/mL IL-6 or vehicle for 12 hours, and then treated with the indicated concentrations of Am80 or vehicle for additional 48 hours. Expression of SR-A and CD36 mRNA was analyzed by real-time PCR. Data are means±SD of 3 independent experiments. B, CD36 promoter activity; Raw264 cells were transfected with human CD36 promoter, pCAG-PPAR and CMX-hRXR. Am80 (10 -7 mol/L) and IL-6 (20 ng/mL) was added 24 hours after transfection. All luciferase activity was measured 36 hour after transfection. Data were expressed as means±SD of triplicate wells and are representative of three independent experiments. # P <0.05, ## P <0.01 vs untreated control; * P <0.05 vs IL-6 alone.
To further analyze effects of Am80 on the signaling mechanism elicited by IL-6, we analyzed its effects on the CD36 promoter in Raw264 cells. As with the endogenous CD36 expression, CD36 promoter activity was augmented by IL-6 treatment ( Figure 4 B). This activation of the promoter was suppressed by Am80. These results suggest that Am80 affects both expression of IL-6 and the signaling elicited by IL-6.
Effect of Am80 on Atherosclerosis in ApoE -Deficient Mice
Given our observations that it inhibits IL-6 production and signaling elicited by IL-6, scavenger receptor expression and foam-cell formation in mouse macrophages, we hypothesized that Am80 might be able to modulate atherogenesis in vivo. To test that idea, 8-week-old apoE -deficient mice were fed a western diet for 2 months, during which they were orally administered Am80 (1.0 mg/kg body weight) or vehicle daily. There were no significant differences in the body weights or serum lipid profiles in the 2 groups ( Table ). Serum IL-6 levels were significantly reduced in the Am80-treated group, as expected. Levels of the proinflammatory cytokine IL-1ß were also reduced in the Am80 group, but levels of the anti-inflammatory cytokine IL-10 were not. Fatty atherosclerotic lesions, measured as the percentage of the entire aorta affected or as the affected area of the aortic roots, were significantly smaller in the Am80-treated mice than in those receiving only vehicle (entire aorta: vehicle-treated, 7.3±0.8%; Am80-treated, 0.5±0.3%; P <0.01; n=8 in each group; aortic roots: vehicle-treated, 206 429±12 352 µm 2; Am80-treated, 149 021±19 282 µm 2; P <0.01; n=8 in each group) ( Figure 5A to 5 E). Immunohistochemical analysis showed that expression of IL-6 was decreased in plaques in the Am80-treated animals ( Figure 5 F). In addition, accumulation of extracellular matrix components around the aortic sinus and coronary artery was also reduced in Am80-treated mice ( Figure 5 G). Thus, Am80 does appear capable of inhibiting macrophage foam cell formation and atherogenesis in vivo.
Characteristics of ApoE-Deficient Mice Treated With Am80 on Western Diet for 2 Months
Figure 5. Am80 reduces plaque formation in apoE -deficient mice. Representative photographs showing the appearance of the aortic arch (A) and en face atherosclerotic lesions over the entire aorta stained with Sudan IV (B). C, Percentage of aortic area affected by en face atherosclerotic lesions (n=8). Representative photomicrographs of the aortic sinus stained with Oil Red O (D), IL-6 antibody (F), and Masson trichrome stains (G). E, Area affected by atherosclerotic lesions in the aortic sinus. The scale bar indicates 100 µm (F). Data are means±SD. * P <0.05, ** P <0.01 vs vehicle-treated group.
Discussion
A hallmark of atherosclerotic lesions is the accumulation of macrophage foam cells, which play a central role in the development and progression of atherosclerosis. During that process, modified LDL is taken up primarily via 2 scavenger receptors, SR-A and CD36. 2 Macrophages taken from mice lacking these 2 receptors fail to accumulate esterified cholesterol, 3 suggesting these scavenger receptors represent potential therapeutic targets for inhibition of atherogenesis. In that regard, we have shown here that Am80 inhibits expression of scavenger receptors in mouse macrophages in vitro and substantially reduces atherosclerotic lesion formation in apoE -deficient mice in vivo; moreover, we previously showed that it inhibits neointima formation in a vascular injury model. 12 Am80, which specifically binds to RAR- /ß, has been used safely to treat acute promyelocytic leukemia, 25 which would seem to make it an attractive candidate drug with which to treat and prevent atherosclerosis.
Our findings demonstrate that Am80 inhibits IL-6 expression and the signaling elicited by IL-6 in macrophages. The central role played by IL-6 in cardiovascular disease is suggested by clinical studies showing that serum levels of the cytokine are increased in patients with unstable angina, 5 that it is expressed in atherosclerotic lesions, and that it colocalizes with Ang II in the macrophage-rich shoulder region of plaques. 4 IL-6 is thought to be a pivotal regulator of extracellular matrix deposition and reorganization. 5 In addition, IL-6 has been shown to be involved in foam cell formation, and Keider et al reported that Ang II does not stimulate CD36 expression in peritoneal macrophages taken from IL-6- deficient mice, indicating that IL-6 is an important component of the signaling pathways that control CD36 expression. 6 The results of the present study suggest that inhibition of IL-6 expression and signaling by Am80 reduces SR-A and CD36 expression in macrophages in vitro ( Figure 3 ). Recent studies have shown that the JNK signaling is important for IL-6 expression in response to free cholesterol loading. 26 However, Am80 did not alter JNK1/2 phosphorylation 26 induced by the ACAT inhibitor (TMP-153) treatment 27 in Raw264 cell (unpublished observations, Takeda and Manabe, 2005), suggesting that the JNK pathway is not involved in the inhibition of IL-6 expression by Am80.
Considering its various functions, Am80?s inhibition of IL-6 would be expected to affect both matrix degradation and foam cell formation in vivo. Systemically treating apoE -deficient mice with Am80 reduced accumulation of not only foam cells within atherosclerotic lesions but also extracellular matrix components ( Figure 5 ). These findings suggest that inhibition of IL-6 production is a key mechanism by which Am80 suppresses plaque formation in apoE -deficient mice. Still, we and others have shown that Am80 also affects the functions of other cell types that play important roles in atherogenesis, including SMCs and T cells. For instance, Am80 suppresses expression of PDGF-A in SMCs by inhibiting KLF5, 12 and it induces IL-10 secretion in T-cells. 28 In addition, atRA has been shown to promote fibrinolysis and to inhibit thrombosis and platelet aggregation. 29 Given that atherogenesis is an integral of a variety of cellular activities involving multiple cell types and various growth factors and cytokines, it is very likely that it is Am80?s cumulative effects on all affected cell types that leads to reduced plaque formation in apoE -deficient mice.
Somewhat surprisingly atherosclerotic plaque formation was recently found to be enhanced in apoE -/- IL-6 -/- double knockout mice. 30 Those investigators noted several findings that may be related to the reported enhancement of plaque formation, however. First, serum total cholesterol, LDL, and VLDL levels were all significantly higher in the double knockout mice than in single apoE knockout mice. Second, expression of the anti-inflammatory cytokine IL-10 was much reduced in the double knockout mice. Third, matrix metalloproteinase (MMP) activity was enhanced leading to disintegration of extracellular matrix and alteration of its assembly within the vessel wall, which could affect plaque development. 31 In the context of those changes, the effects of Am80 treatment appear to differ from those of total ablation of IL-6 gene. For instance, IL-6 production was reduced but not completely blocked in Am80-treated animals ( Table ), and total cholesterol and LDL levels were unaffected. It is also noteworthy that levels of IL-10 were not reduced in Am80-treated animals. As mentioned above, Am80 affects the functions of a variety of cell-types, presumably via both IL-6-independent and -dependent mechanisms. These differences in effects of Am80 and null mutation of IL-6 are likely to have led to differential effects on plaque formation in apoE -/- mice.
The present findings are also at variance with earlier studies showing that RA induces CD36 in human monocytic THP-1 cells, 32-34 and IL-6 inhibits SR-A expression in THP-1 cells and human peripheral monocytes. 35 This discrepancy may reflect differences between the models, the species, and the differentiation state of the cells. THP-1 cells are a monocytic cell line in which expression of SR-A and CD36 accompanies differentiation into macrophages. Raw264 cells and peritoneal macrophages, by contrast, are mouse macrophages and express CD36 even under basal culture conditions. Previous studies have shown that atRA promotes macrophage differentiation of THP-1 cells. 36 We found that Am80 also promotes macrophage differentiation of THP-1 cells (unpublished observations, Takeda and Manabe, 2005). It is therefore plausible that the upregulation of CD36 seen in Figure 3 A reflects differentiation, though the exact mechanisms underlying the differential effects of Am80 on CD36 remain unknown.
The results of our reporter assays suggest that Am80 suppresses IL-6 production at least in part at the level of transcription by inhibiting C/EBPß-dependent transactivation of the IL-6 promoter. In Raw264 cells, PMA induced both IL-6 and C/EBPß ( Figure 1 and Figure I), whereas Am80 inhibited the PMA-induced IL-6 expression but not the C/EBPß expression. This suggests that Am80 in some way interferes with the function of C/EBPß. Consistent with that idea, C/EBPß-dependent gene transcription is similarly inhibited by RA in adipocytes, although C/EBPß expression is not. 37
Tontonoz et al recently demonstrated that CD36 gene is controlled by PPAR via the PPAR /RXR-responsive element (PPRE) within -274/-263-bp region of the CD36 promoter. 14 However, we found that the PPRE was dispensable for inhibition of the promoter activity by Am80. Moreover, Am80 did not affect the activity of PPRE-dependent minimal promoter 38 (Takeda and Manabe, unpublished observations, 2005). These results suggest that Am80 inhibits the CD36 transcription via mechanisms independent of PPAR. It is noteworthy to mention that the CD36 promoter has neither RARE nor the C/EBP binding motif. It would be important to determine the molecular mechanisms by which Am80 inhibits CD36 transcription in future studies for better understanding the role played by RAR in the control of macrophage function. Of particular importance will be RAR?s interactions with other transcription factors.
In conclusion, our findings support the notion that modulation of the function of inflammatory and vascular cells using synthetic retinoids is a promising strategy for the treatment and prevention of vascular diseases, including atherosclerosis.
Acknowledgments
This work was supported in part by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science and from Ministry of Education, Culture, Sports, Science, and Technology (to R.N. and I.M.), Grant-in-aid from National Institute of Biomedical Innovation, Japan (to R.N.), and research grants from the Tokyo Biochemical Research Foundation and Kato Memorial Bioscience Foundation (to I.M.).
【参考文献】
Glass CK, Witztum JL. Atherosclerosis. The road ahead. Cell. 2001; 104: 503-516.
Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med. 2002; 8: 1235-1242.
Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S, Rhee JS, Silverstein R, Hoff HF, Freeman MW. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002; 277: 49982-49988.
Schieffer B, Schieffer E, Hilfiker-Kleiner D, Hilfiker A, Kovanen PT, Kaartinen M, Nussberger J, Harringer W, Drexler H. Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation. 2000; 101: 1372-1378.
Rattazzi M, Puato M, Faggin E, Bertipaglia B, Zambon A, Pauletto P. C-reactive protein and interleukin-6 in vascular disease: culprits or passive bystanders? J Hypertens. 2003; 21: 1787-1803.
Keidar S, Heinrich R, Kaplan M, Hayek T, Aviram M. Angiotensin II administration to atherosclerotic mice increases macrophage uptake of oxidized ldl: a possible role for interleukin-6. Arterioscler Thromb Vasc Biol. 2001; 21: 1464-1469.
Huber SA, Sakkinen P, Conze D, Hardin N, Tracy R. Interleukin-6 exacerbates early atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 1999; 19: 2364-2367.
Nagy L, Thomazy VA, Shipley GL, Fesus L, Lamph W, Heyman RA, Chandraratna RA, Davies PJ. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines. Mol Cell Biol. 1995; 15: 3540-3551.
Wang T, Niwa S, Bouda K, Matsuura S, Homma T, Shudo K, Nagai H. The effect of Am-80, one of retinoids derivatives on experimental allergic encephalomyelitis in rats. Life Sci. 2000; 67: 1869-1879.
Niwa S, Ochi T, Hirano Y, Wang T, Inagaki N, Shudo K, Nagai H. Effect of Am-80, a retinoid derivative, on 2,4-dinitrofluorobenzene-induced contact dermatitis in mice. Pharmacology. 2000; 60: 208-214.
Nagai H, Matsuura S, Bouda K, Takaoka Y, Wang T, Niwa S, Shudo K. Effect of Am-80, a synthetic derivative of retinoid, on experimental arthritis in mice. Pharmacology. 1999; 58: 101-112.
Shindo T, Manabe I, Fukushima Y, Tobe K, Aizawa K, Miyamoto S, Kawai-Kowase K, Moriyama N, Imai Y, Kawakami H, Nishimatsu H, Ishikawa T, Suzuki T, Morita H, Maemura K, Sata M, Hirata Y, Komukai M, Kagechika H, Kadowaki T, Kurabayashi M, Nagai R. Kruppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling. Nat Med. 2002; 8: 856-863.
Eickelberg O, Pansky A, Mussmann R, Bihl M, Tamm M, Hildebrand P, Perruchoud AP, Roth M. Transforming growth factor-beta1 induces interleukin-6 expression via activating protein-1 consisting of JunD homodimers in primary human lung fibroblasts. J Biol Chem. 1999; 274: 12933-12938.
Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM. PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell. 1998; 93: 241-252.
Pfitzner E, Kirfel J, Becker P, Rolke A, Schule R. Physical interaction between retinoic acid receptor and the oncoprotein myb inhibits retinoic acid-dependent transactivation. Proc Natl Acad Sci U S A. 1998; 95: 5539-5544.
Oishi Y, Manabe I, Tobe K, Tsushima K, Shindo T, Fujiu K, Nishimura G, Maemura K, Yamauchi T, Kubota N, Suzuki R, Kitamura T, Akira S, Kadowaki T, Nagai R. Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation. Cell Metab. 2005; 1: 27-39.
Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, Kishimoto T, Akira S. Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A. 1993; 90: 10193-10197.
Nagpal S, Cai J, Zheng T, Patel S, Masood R, Lin GY, Friant S, Johnson A, Smith DL, Chandraratna RA, Gill PS. Retinoid antagonism of NF-IL6: insight into the mechanism of antiproliferative effects of retinoids in Kaposi?s sarcoma. Mol Cell Biol. 1997; 17: 4159-4168.
Natsuka S, Akira S, Nishio Y, Hashimoto S, Sugita T, Isshiki H, Kishimoto T. Macrophage differentiation-specific expression of NF-IL6, a transcription factor for interleukin-6. Blood. 1992; 79: 460-466.
Shiffman D, Mikita T, Tai JT, Wade DP, Porter JG, Seilhamer JJ, Somogyi R, Liang S, Lawn RM. Large scale gene expression analysis of cholesterol-loaded macrophages. J Biol Chem. 2000; 275: 37324-37332.
Nicholson AC. Expression of CD36 in macrophages and atherosclerosis: the role of lipid regulation of PPARgamma signaling. Trends Cardiovasc Med. 2004; 14: 8-12.
Feng J, Han J, Pearce SF, Silverstein RL, Gotto AM Jr, Hajjar DP, Nicholson AC. Induction of CD36 expression by oxidized LDL and IL-4 by a common signaling pathway dependent on protein kinase C and PPAR-gamma. J Lipid Res. 2000; 41: 688-696.
Ralph P, Nakoinz I. Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J Immunol. 1977; 119: 950-954.
Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. 1980; 26: 171-176.
Tobita T, Takeshita A, Kitamura K, Ohnishi K, Yanagi M, Hiraoka A, Karasuno T, Takeuchi M, Miyawaki S, Ueda R, Naoe T, Ohno R. Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid. Blood. 1997; 90: 967-973.
Li Y, Schwabe RF, DeVries-Seimon T, Yao PM, Gerbod-Giannone MC, Tall AR, Davis RJ, Flavell R, Brenner DA, Tabas I. Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor- alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent inflammation in advanced atherosclerosis. J Biol Chem. 2005; 280: 21763-21772.
Sugiyama Y, Ishikawa E, Odaka H, Miki N, Tawada H, Ikeda H. TMP-153, a novel ACAT inhibitor, inhibits cholesterol absorption and lowers plasma cholesterol in rats and hamsters. Atherosclerosis. 1995; 113: 71-78.
Iwata M, Eshima Y, Kagechika H. Retinoic acids exert direct effects on T cells to suppress Th1 development and enhance Th2 development via retinoic acid receptors. Int Immunol. 2003; 15: 1017-1025.
van Giezen JJ, Boon GI, Jansen JW, Bouma BN. Retinoic acid enhances fibrinolytic activity in-vivo by enhancing tissue type plasminogen activator (t-PA) activity and inhibits venous thrombosis. Thromb Haemost. 1993; 69: 381-386.
Schieffer B, Selle T, Hilfiker A, Hilfiker-Kleiner D, Grote K, Tietge UJ, Trautwein C, Luchtefeld M, Schmittkamp C, Heeneman S, Daemen MJ, Drexler H. Impact of interleukin-6 on plaque development and morphology in experimental atherosclerosis. Circulation. 2004; 110: 3493-3500.
Lijnen HR. Metalloproteinases in development and progression of vascular disease. Pathophysiol Haemost Thromb. 2003; 33: 275-281.
Wuttge DM, Romert A, Eriksson U, Torma H, Hansson GK, Sirsjo A. Induction of CD36 by all-trans retinoic acid: retinoic acid receptor signaling in the pathogenesis of atherosclerosis. FASEB J. 2001; 15: 1221-1223.
Han S, Sidell N. Peroxisome-proliferator-activated-receptor gamma (PPARgamma) independent induction of CD36 in THP-1 monocytes by retinoic acid. Immunology. 2002; 106: 53-59.
Szanto A, Nagy L. Retinoids potentiate peroxisome proliferator-activated receptor gamma action in differentiation, gene expression, and lipid metabolic processes in developing myeloid cells. Mol Pharmacol. 2005; 67: 1935-1943.
Liao HS, Matsumoto A, Itakura H, Doi T, Honda M, Kodama T, Geng YJ. Transcriptional inhibition by interleukin-6 of the class A macrophage scavenger receptor in macrophages derived from human peripheral monocytes and the THP-1 monocytic cell line. Arterioscler Thromb Vasc Biol. 1999; 19: 1872-1880.
Matikainen S, Hurme M. Comparison of retinoic acid and phorbol myristate acetate as inducers of monocytic differentiation. Int J Cancer. 1994; 57: 98-103.
Schwarz EJ, Reginato MJ, Shao D, Krakow SL, Lazar MA. Retinoic acid blocks adipogenesis by inhibiting C/EBPbeta-mediated transcription. Mol Cell Biol. 1997; 17: 1552-1561.
Vu-Dac N, Schoonjans K, Kosykh V, Dallongeville J, Fruchart JC, Staels B, Auwerx J. Fibrates increase human apolipoprotein A-II expression through activation of the peroxisome proliferator-activated receptor. J Clin Invest. 1995; 96: 741-750.
作者单位：Department of Cardiovascular Medicine (N.T., H.I., S.I., R.N.) and Nano Bioengineering Education Program (I.M.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Matsumoto, Japan (T.S.), School of Bi