PPARγ Agonists Enhance Human Vascular Endothelial Adhesiveness by Increasing ICAM-1 Expression

Biochemical and Biophysical Research Communications 263, 718 –722 (1999) Article ID bbrc.1999.1437, available online at http://www.idealibrary.com on

PPARg Agonists Enhance Human Vascular Endothelial Adhesiveness by Increasing ICAM-1 Expression Neng-Guin Chen, 1 Stephen F. Sarabia, Peter J. Malloy, Xiao-Yan Zhao, David Feldman, and Gerald M. Reaven Department of Medicine, Stanford University School of Medicine, Stanford, California 94305

Received August 17, 1999

Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium. Oxidized LDL (oxLDL) stimulates the adhesion of human monocytes to endothelial cells, in part, by increasing expression of ICAM-1. However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. The peroxisome proliferatoractivated receptor g (PPARg), a member of the nuclear receptor superfamily, is expressed in vascular endothelial cells. Whether it can be activated by a synthetic ligand, troglitazone, as well as by natural ligands, oxLDL and its lipid components (i.e., 9- and 13-HODE), has not yet been explored. This study was undertaken to determine whether PPARg is expressed in ECV304 human vascular endothelial cells and if so to define the biological effects of its activation by these agonists. Our results demonstrate that PPARg mRNA is expressed in ECV304 cells, and transfected cells with a PPARE luciferase construct respond to these agonists. In addition, ligand-dependent PPARg activation increased ICAM-1 protein expression and enhanced adherence of monocytes to ECV304 cells by two- to threefold. These findings suggest that the PPARg signaling pathway might contribute to the atherogenicity of oxLDL in vascular endothelial cells. © 1999 Academic Press Key Words: PPARg; oxidized LDL; endothelium; intercellular adhesion molecule-1 (ICAM-1).

Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium (1). A growing body of evidence suggests that oxidized LDL (oxLDL), a potent atherogenic particle, stimulates the adhesion of human monocytes to endothelial cells in vitro (2– 4). However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. 1 To whom correspondence should be addressed at Department of Medicine, Stanford University, S-005, 300 Pasteur Dr., Stanford, CA 94305-5103. Fax: 650-725-7085. E-mail: [email protected].

0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

The adhesion of monocytes is mediated by adhesion molecules expressed on the surface of the endothelial cells. Adhesion molecule intercellular adhesion molecule-1 (ICAM-1) interacts with monocytes by binding to the surface membrane b 2 integrin molecule, i.e., CD11/CD18 and mainly participates in firm adhesion and transmigration of monocytes into subendothelial space. ICAM-1 expression is induced in vitro by cytokines and other external stimuli such as shear stress and oxLDL to endothelial cells (4, 5– 8). Immunohistochemistry of human atherosclerotic arteries has demonstrated expression of ICAM-1 in all subtypes of atherosclerotic lesions, except fibrous plaques (9). ICAM-1 promoter activity and expression has been shown to be enhanced by oxLDL and one of its components, lysophosphatidylcholine (4, 10). Whether other lipid components also play a role in oxLDL stimulated ICAM-1 expression is not well defined. The presence of oxidized fatty acids, mainly linoleic acid, and cholesterol metabolites from oxLDL particles in atherosclerotic lesions (11, 12) supports a central role for oxLDL and its lipid components in the pathogenesis of atherosclerosis. Peroxisome proliferator-activated receptorg (PPARg), a ligand-dependent nuclear transcription factor regulates the expression of several genes important for lipid metabolism (13–15). In human monocytes and macrophages, PPARg is activated by a synthetic ligand, troglitazone, a new class of antidiabetic drug, as well as by the natural ligands oxLDL and its lipid components (i.e., 9-hydroxy-(S)-10,12-octadecadienoic acid [9(S)-HODE] and [13(S)-HODE], and 15-deoxy-D 12,14-prostaglandin J 2 (15-d PGJ 2) (16, 17). These ligands have been shown to promote the differentiation of human monocytes to macrophages and to facilitate oxLDL uptake through transcriptional induction of the scavenger receptor CD36. These events facilitate foam cell formation, an early step in the process of atherogenesis (1). PPARg has also been shown to be expressed in foam cells of atherosclerotic lesions (17). These observations strongly suggest that PPARg plays an important role in the pathogenesis of atherosclerosis.

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The role of PPARg in vascular endothelial cells has not yet been established. It has recently been shown to express and regulate gene expression of plasminogen activator inhibitor type-1 (PAI-1) expression in human endothelial cells, thus potentially promoting thrombosis (18). We postulate that oxLDL and its lipid components may exert their atherogenic effects by acting as natural ligands for PPARg activation. The ECV304 has previously been shown to express ICAM-1 but not VCAM-1 and E-selectin in response to cytokines (19 – 21). Because of the special characteristic of this cell line, we examined the expression and activity of PPARg in ECV304 cells, and the effect of PPARg agonists on ICAM-1 expression and endothelial cell adhesiveness. Our results demonstrate that PPARg mRNA is expressed in ECV304 human vascular endothelial cells, and both oxLDL and 13-HODE activated the endogenous PPARg. Furthermore, our data suggest that on monocyte adhesion and ICAM-1 expression induced by oxLDL, at least, in part, is mediated by PPARg signaling pathway in these cells. METHODS Materials. Chemical compounds were purchased from Sigma (St. Louis, MO). Troglitazone was obtained from Sankyo (Paysippany, NJ). 15d-PGJ 2, 9-hydroxy-(S)-10,12-octadecadienoic [9(S)-HODE] and [13(S)-HODE] were purchased from Cayman Chemical (Ann Arbor, MI). Cell culture. A spontaneously transformed human umbilical vein endothelial cell line (ECV 304) was obtained from ATCC (Rockville, MD). Cells were maintained in M199 media (Irvine Scientific) containing 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technologies, Grand Island, NY). Murine monocytoid cells (WEHI78/24) were obtained from ATCC and grown in DMEM containing 10% FBS. Reverse-transcriptase polymerase chain reaction (RT-PCR). The RT-PCR procedure of Inoue et al. (22) was modified for the detection and semi-quantitative analysis of PPARg mRNA expression in human endothelial cells. Total RNA was extracted using Trizol. RNA (1.5 mg) was mixed with 0.5 mg of oligo (dT) primer and heated at 65°C for 5 min. For reverse transcription, the reaction contained 100 mM Tris–HCl (pH 8.3), 50 mM MgCl 2, 10 mM DTT, 0.5 mM dNTPs. The reaction was initiated by addition of 200 units Superscript RT (Life Technologies, Grand Island, NY) and incubated at 42°C for 1 h. The reaction was terminated by heating at 95°C for 5 min. Human PPARg gene specific oligonucleotide primers (59-GCA TTA TGA GAC ATC CCC AC-39 and 59-TCT CTC CGT AAT GGA AGA CC-39), (Operon Technologies, Inc., Alameda, CA) were used to amplify both PPARg1 and PPARg2 spliced variants to produce a single 474-bp PCR product (23). Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA served as an internal control during RT-PCR. Two mL of the RT reaction was amplified in a 25 mL PCR containing 10 mM Tris–HCl (pH 8.3), 100 mM KCl, 2 mM MgCl 2, 0.2 mM dNTPs, 1.5 units of Taq DNA polymerase (Qiagen Inc., Valencia, CA), 0.4 mM PPARg primers, and 1 mM GAPDH primers. Amplification was carried out with a Perkin–Elmer Cetus DNA Thermal Cycler 480 (Emeryville, CA) using an initial denaturation at 94°C for 30 s, followed by 30 cycles at 94°C for 1 min, 58°C for 45 s, and 72°C for 1.5 min. PCR products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining.

Preparation of oxLDL. Plasma from normolipidemic fasting volunteers was obtained by venipuncture. The antioxidant, butylated hydroxytoluene (BHT), and EDTA were immediately added to the plasma to achieve a final concentration of 20 mM and 0.1%, respectively. Plasma samples were then pooled and placed at 4°C for 15 min and then the LDL prepared by sequential gradient ultracentrifugation as described by Pritchard et al. (24). The LDL solution was dialyzed against 0.9% saline with 0.01% EDTA overnight to eliminate the BHT. LDL oxidation was initiated by addition 5 mM CuSO 4 and incubating for 24 h at 4°C. The oxidation was terminated by adding BHT (20 mM) before using the oxLDL for the experiments. Transient transfection assay. ECV304 cells were plated in 6-well plates at 100,000 cells/well in M199 media (Irvine Scientific) containing 10% fetal calf serum. After growth at 37°C for 20 h, TK-luc or pPPAREx1-TK-luc plasmids (Promega, Madison, WI) were transfected using Superfect (Qiagen, Valencia, CA) according to the manufacturer’s directions. The control plasmid pSV-Renilla was cotransfected to monitor transfection efficiency. All values are corrected for Renilla. pPPAREx1-TK-luc reporter vector was a gift from Dr. Mitchell Lazar. After 20 h of transfection, medium was changed and troglitazone or oxLDL were added and the incubation continued for an additional 24 h. Cells were then harvested and luciferase activities were determined using the Dual Luciferase assay (Promega). Each transfection was carried out in triplicate and experiments were repeated at least four times. Monocyte– endothelial cell binding assay. ECV304 cells were seeded in six well plates three days prior to assays, and confluence confirmed prior to binding assay. Cells were treated with PPARg agonists at the time of culture initiation. Monocytes (WEHI 78/24 cells) 1,000,000 cells/ml were added to six-well plates containing confluent and treated endothelial cell monolayers. The plates were transferred to a rocking platform for 30 min at ambient temperature. Nonadherent cells were removed by washing three times with binding buffer containing Hank’s balanced salt solution (HBSS), and plates rocked for an additional 5 min with fresh binding buffer. Binding buffer was then replaced with HBSS containing 2% glutaraldehyde to fix the remaining cells. Adherent cells were enumerated by videomicroscopy using a computer-aided image analysis system (Image Analyst, Automatrix Corp., Boston, MA) (25). Determination of ICAM-1 by enzyme-linked immunosorbent assay (ELISA). Surface expression of ICAM-1 and total cell proteins were obtained using protein extraction reagent (Pierce Chemical, Rockford, IL). ICAM-1 was measured by ELISA (R & D Systems, MN). Protein concentration was determined by Bradford assay (Pierce Chemical, Rockford, IL). Data were expressed as ng ICAM-1 per total cell protein. Statistical analysis. All data are shown as mean 6 SEM. Statistical significance was tested in all experiments using analysis of variance (ANOVA) followed by Tukey’s post-hoc comparisons. Statistical significance was assumed for P , 0.05.

RESULTS PPARg is expressed in ECV304 cells. To determine whether PPARg is expressed in ECV304 cells, RT-PCR was used to detect PPARg mRNA. As shown in Fig. 1, ECV304 cells express PPARg mRNA (lane 3) and the amount of PPARg mRNA expressed in these cells is comparable to MCF-7 human breast cancer cells (lane 2), a cell line known to express PPARg mRNA (26). No amplification was obtained in the samples in which the reverse transcriptase was omitted (lane 4). Primer specificity for each PCR product (i.e., the 474 bp for PPARg or 600 bp for GAPDH gene product) was

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FIG. 1. Detection of PPARg mRNA by RT-PCR. RT- PCR products of PPARg (474 bp) and GAPDH (600 bp) are observed in ECV304 cells (lane 3). As a positive control (lane 2), PPARg mRNA expression is shown in MCF-7 human breast cancer cells. Lane 1 contains standard marker.

checked by observing only one product when a primer set for PPARg or GAPDH, was used individually in the PCR (data not shown). Ligand activation of endogenous PPARg in ECV304 cells. To test whether PPARg agonists activate the endogenous PPARg, ECV vascular endothelial cells were transiently transfected with a PPARE-reporter plasmid and treated with various ligands. As show in Fig. 2, both troglitazone and oxLDL enhanced reporter activity in a dose-dependent manner ranging from a 1.5- to 3-fold increment. More interestingly, 9- and 13-(S)-HODE also stimulated luciferase activity up to 3- to 4-fold (treated cells vs control, P , 0.01). These results indicate the presence of functional PPARg activity in this cell type and demonstrate activation by oxLDL as well as known PPARg ligands. PPARg ligands enhance monocyte binding to ECV304 cells. To study the potential role of PPARg on endothelial adhesiveness, monocytes and endothelial interaction was examined by a functional adhesion assay. ECV304 cells were treated with troglitazone (10 mM), oxLDL (50 mg/ml), 13(S)-HODE (20 mg/ml), TNF-a (100 U/ml) and LPS (10 ng/mL) for 20 h prior to the study. Results are shown in Fig. 3. Monocyte adherence to endothelial cells was significantly increased up to two-fold by troglitazone, oxLDL and 13(S)-HODE (treated cells vs control, P , 0.01, n 5 8). Cells treated with TNF-a (100 U/ml) and LPS (10 ng/ml) as positive controls were also shown to increase monocyte adhesion to ECV304 cells by 3-fold (P , 0.01, n 5 8). PPARg ligands increase the expression of ICAM-1 in ECV endothelial cells. Since PPARg ligands enhance monocyte binding to ECV340 cells, the effect of PPARg

FIG. 2. Ligand activation of endogenous PPARg. ECV304 cells were transiently transfected with a PPARE-TK-luc reporter plasmid. Various PPARg ligands including 9(S)-HODE (20 mg/ml) and 13(S)HODE (20 mg/ml) were added to the transfected cells and normalized reporter activity was determined. Error bars indicate mean 6 SEM of four determinations. *Significant difference (P , 0.01) from the control group as determined by one-way ANOVA plus Tukey’s posthoc comparison.

ligands on ICAM-1 expression was examined. ICAM-1 levels were measured in total protein extracts of ECV cells. As shown in Fig. 4, ICAM-1 expression was significantly increased as a result of PPARg ligand treatment. Nearly 2- to 3-fold induction of ICAM-1 by troglitazone (10 mM), oxLDL (50 mg/ml) and 13(S)-HODE (20 mg/ml) were observed when compared to control cells (P , 0.01, n 5 8). Our present data demonstrate a threefold increment in ICAM-1 expression at low doses of TNF-a (100 U/ml) and LPS (10 ng/ml) as positive controls (P , 0.01, n 5 8). In addition, we also

FIG. 3. Effects of PPARg ligands on monocyte– endothelial cell interactions. ECV304 cells were treated with DMSO-treated control, or troglitazone (10 mM), oxLDL (50 mg/ml), 13(S)-HODE (20 mg/ml) as well as TNF-a (100 U/ml) and LPS (10 ng/ml) as positive controls for 20 h. Monocyte binding assays were then performed. The data were the mean number of monocyte bound per 70 high power field 6 SEM of different experiments. (Significant difference (P , 0.01) from the control group as determined by one-way ANOVA in conjunction with Tukey’s post-hoc comparison.

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FIG. 4. Effect of PPARg ligands on the expression of ICAM-1. ECV304 cells were treated with DMSO-treated control, or troglitazone (10 mM), oxLDL (50 mg/ml), 13(S)-HODE (20 mg/ml) as well as TNF-a (100 U/ml) and LPS (10 ng/ml) as positive controls for 20 h. *Significant difference (P , 0.01) from the control group as determined by one-way ANOVA in conjunction with Tukey’s post-hoc comparison.

examined induction of both VCAM-1 and E-selectin. Both VCAM-1 and E-selectin levels did not change in response to either TNF-a or PPARg agonists (data not shown), which is consistent with the observations of others (21). DISCUSSION Although PPARg mRNA is expressed predominantly in white adipose tissue (13–15), it also expresses in kidney, intestine, retina and several cancer cells (26, 27). More recently, PPARg was shown to be expressed in both human monocyte/macrophages (16, 17), and vascular endothelial cells (18) where it appears to participate in the pathogenesis of atherosclerosis. Our results are consistent with the findings of other (18) indicating that vascular endothelial cells express PPARg. The role of PPARg in vascular endothelial cells is beginning to be elucidated. PPARg ligands have been shown to be novel regulators of PAI-1 gene expression in vascular endothelial cells (18). However, the natural ligands for PPARg in human vascular endothelial cells have not been well defined. Marx et al. (18) have shown that 15d-PGJ 2 activates PPARg in a dose-dependent manner in bovine aortic endothelial cells. Since 15dPGJ 2 is not very selective for PPARg (28 –30) we choose PPARg-specific ligands in this study such as troglitazone, oxLDL and 13(S)-HODE. Our findings suggest that both oxLDL and 13(S)-HODE serve as natural ligands for PPARg in ECV304 human vascular endothelial cells. The cellular mechanism whereby oxLDL exerts its effect as a natural ligand on PPARg activation is not well understood. It is likely that oxLDL may be taken up by scavenger receptors or by a novel receptor for

oxLDL, known as the C-type lectin-like oxLDL receptor-1 (LOX-1) (31–34) on the surface of endothelial cells. The binding to either scavenger receptors or LOX-1 may lead to the internalization of oxLDL, proteolytic degradation and the release of 13-HODE inside these cells. Elevation of cellular 13-HODE serves a natural ligand for PPARg activation and lead to upregulate ICAM-1 expression and increased monocyteendothelial cell interaction. The possibility that these effects may be mediated via PPARg has not been addressed previously and needs further investigation. In addition to oxLDL and 13-HODE, we also demonstrated that troglitazone, the synthetic PPARg ligand, enhances ICAM-1 expression and increases monocyte binding to vascular endothelial cells. Taken together, it is likely that induction of ICAM-1 expression in vascular endothelial cells is, in part, dependent upon PPARg activation. As mentioned in the Introduction, induction of ICAM-1 in vascular endothelial cells is mainly dependent upon the stimulation of TNF-a, oxLDL as well as shear stress. In this study, a twofold increase in ICAM-1 expression and monocyte binding stimulated by PPARg ligands is significant and comparable to the effects of other recognized stimuli such as shear stress (8) or cyclic strain (35). Although it has been reported that the degree of induction of ICAM-1 in response to TNF-a is less in ECV cells than human umbilical vein endothelial cells (29), this is not surprising, since it is often observed that primary cells yield stronger signals than established cell lines. To understand the physiological role of PPARg in vascular endothelial cells in vivo, animals studies will be required. In conclusion, we have shown that PPARg is expressed in ECV304 human vascular endothelial cells and that it can be activated by various PPARg ligands including oxLDL. Activation of endogenous PPARg in these cells is associated with increased expression of ICAM-1 and enhancement of monocyte binding to endothelial cells. These findings suggest that the PPARg signaling pathway in vascular endothelial cells may contribute to oxLDL-induced atherogenesis. ACKNOWLEDGMENTS This work was supported by research grants (HL-08506 and DK42482). Dr. N-G. Chen is a recipient of a National Research Service Award (HL-07708) from the National Institutes of Health.

REFERENCES

721

1. Ross, R. (1993) Nature 362, 801– 809. 2. Quinn, M. T., Parthasarathy, S., Fong, G. L., and Steinberg, D. (1987) Proc. Natl. Acad. Sci. USA 84, 2995–2998. 3. Berliner, J. A., Territo, M. C., Sevanian, A., Ramin, S., Kim, J. A., Bamshad, B., Esterson, M., and Fogelman, A. M. (1990) J. Clin. Invest. 85, 1260 –1266.

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4. Erl, W., Weber, P. C., and Weber, C. (1998) Atherosclerosis 136, 297–303. 5. Ledebur, H. C., and Parks, T. P. (1995) J. Biol. Chem. 270, 933–943. 6. Walpola, P. L., Gotlieb, A. I., Cybulsky, M. I., and Langille, B. L.(1995) Arterioscler. Thromb. Vasc. Biol. 15, 2–10. 7. Nagel, T., Resnick, N., Atkinson, W. J., and Dewey, C. F., Jr., and Gimbrone, M. A., Jr. (1994) J. Clin. Invest. 94, 885– 891. 8. Chiu, J. J., Wung, B. S., Shyy, J. Y. J., Hsieh, H. J., and Wang, D. L. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 3570 –3577. 9. Poston, R. N., Haskard, D. O., Coucher, J. R., Gall, N. P., and Johnson-Tidey, R. R. (1992) Am. J. Pathol. 140, 665– 673. 10. Zhu, Y., Lin, J. H.-C., Liao, H.-L., Verna, L., and Stemerman, M. B. (1997) Biochem. Biophys. Acta 1345, 93–98. 11. Belkner, J., Wiesner, R., and Kuhn, H. (1992) Agents Actions Suppl. 37, 78 – 84. 12. Kuhn, H., Belkner, J., Wiesner, R., Schewe, T., Lankin, V. Z., and Tikhaze, A. K. (1992) Eicosanoids 5, 17–22. 13. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. (1995) Cell 83, 835– 839. 14. Lemberger, T., Desvergne, B., and Wahli, W. (1996) Annu. Rev. Cell Dev. Biol. 12, 335–363. 15. Schoonjans, K., Staels, B., and Auwerx, J. (1996) Biochim. Biophys. Acta 1302, 93–109. 16. Nagy, L., Tontonoz, P., Alvarez, J. G. A., Chen, H., and Evans, R. M. (1998) Cell 93, 229 –240. 17. Tontonoz, P., Nagy, L., Alvarez, J. G. A., Thomazy, V. A., and Evans, R. M. (1998) Cell 93, 241–252. 18. Marx, N., Bourcier, T., Sukhova, G. K., Libby, P., and Plutzky, J. (1999) Arterioscler. Thromb. Vasc. Biol. 19, 546 –551. 19. Takahashi, K., and Sawasaki, Y. (1992) In Vitro Cell Dev Biol. 28A, 380 –382. 20. Stannard, A., Bradley, N. J., and Owen, J. S. (1997) Biochem. Soc. Trans.. 486S, 25. [abstract] 21. Kalogeris, T. J., Laroux, F. S., Cockrell, A., Ichikawa, H.,

22. 23.

24.

25. 26.

27.

28. 29. 30. 31. 32. 33.

34.

35.

722

Okayama, N., Phifer, T. J., Alexander, J. S., and Grisham, M. B. (1999) Am. J. Physiol. 276(Cell Physiol. 45), C856 –C864. Inoue, I., Shino, K., Noji, S., Awata, T., and Katayama, S. (1998) Biochem. Biophys. Res. Commun. 246, 370 –374. Auboeuf, D., Rieusset, J., Fajas, L., Vallier, P., Frering, V., Riou, J. P., Staels, B., Auwerx, J., Laville, M., and Vidal, H. (1997) Diabetes 46, 1319 –1327. Pritchard, K. A., Groszek, L., Smalley, D. M., Sessa, W. C., Wu, M., Villalon, P., Wolin, M. S., and Stemerman, M. B. (1995) Circ. Res. 77, 510 –518. Tsao, P. S., Buitrago, R., Chan, J. R., and Cooke, J. P. (1996) Circulation 94, 1682–1689. Lenberger, T., Braissant, O., Juge-Aubry, C., Keller, H., Saladin, R., Stael, B., Auwerx, J., Burger, A., Meier, C. A., and Wahli, W. (1996) Ann. N.Y. Acad. Sci. 486, 231–251. Mueller, E., Sarraf, P., Tontonoz, P., Evans, R. M., Martin, K. J., Zhang, M., Fletcher, C., Singer, S., and Spiegelman, B. M. (1998) Mol Cell 1, 465– 470. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M., and Evans, R. M. (1995) Cell 83, 803– 812. Kliewer, S. A., Lenhard, J. M., Willson, T. M., Patel, I., Morris, D. C., and Lehmann, J. M. (1995) Cell 83, 813– 819. Brun, R. P., Tontonoz, P., Forman, B. M., Ellis, R., Chen, J., Evans, R. M., and Spiegelman, B. M. (1996) Genes Devel. 10, 974 –984. Krieger, M., Acton, S., Ashkenas, J., Pearson, A., Penman, M., and Resnick, D. (1993) J. Biol. Chem. 268, 4569 – 4572. Bickel, P. E., and Freeman, M. W. (1992) J. Clin. Invest. 90, 1450 –1457. Sawamura, T., Kume, N., Aoyama, T., Moriwaki, H., Hoshikawa, H., Aiba, Y., Tanaka, T., Miwa, S., Katsira, Y., Kita, T., and Masaki, T. (1997) Nature 386, 73–77. Moriwaki, H., Kume, N., Sawamura, T., Aoyama, T., Hoshikawa, H., Ochi, H., Nishi, E., Masaki, T., and Kita, T. (1998) Arterioscler. Thromb. Vasc. Biol. 18, 1541–1547. Cheng, J.-J., Wung, B.-S., Chao, Y.-J., and Wang, D. L. (1996) Hypertension 28, 386 –391.