EFFECTS OF PPARγ LIGANDS ON ATHEROSCLEROSIS AND CARDIOVASCULAR DISEASE

EFFECTS OF PPARγ LIGANDS ON ATHEROSCLEROSIS AND CARDIOVASCULAR DISEASE C. Fiévet and B. Staels, Institut Pasteur de Lille, Département d Athérosclérose, Lille, F- 59019 France, Inserm, U545, Lille, F-59019 France, Université de Lille 2, Lille, F-59006 France Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors regulating the expression of genes that control lipid and glucose homeostasis, modulating thus the major metabolic disorders predisposing to atherosclerosis and subsequent cardiovascular diseases [1]. Moreover, PPARs exert additional anti-inflammatory and lipid-modulating effects in the arterial wall, being therefore interesting molecular targets for the treatment of atherosclerosis [2]. Finally, PPARs regulate genes that control thrombogenicity which occurs after the rupture of atherosclerotic plaques [3,4]. Three different PPARs have been identified (PPARα, PPARβ/δ, PPARγ) each displaying distinct tissue distribution patterns [5]. PPARs are activated by natural ligands (such as fatty acids and eiconasoids) and by pharmacological agonists. Whereas ligands for PPARβ/δ are still in the experimental stage, PPARα and PPARγ are the targets of two classes of drugs currently used in clinical practice: fibrates and thiazolidinediones (TZDs) respectively. Pioglitazone and rosiglitazone are TZDs currently used for the treatment of type 2 diabetes as insulin sensitizing agents. These drugs have not only significant hypoglycemic effects, but also potential positive effects on lipid metabolism, the endothelium, oxidative stress, and vascular inflammation [6]. These additive actions of TZDs might reduce the development of atherosclerosis. Preclinical development of PPAR ligands that would act on metabolic factors which enhance atherosclerosis includes the analysis of their effects in suitable murine models. However, the development of a reliable experimental animal model is not easy because ideally the regulatory pathways in these models need to be similar to those in humans [7]. PPARγ and Atherosclerosis in Humans Whereas the effects of PPARα activation by fibrate treatment on atherogenesis and coronary events in humans are fairly well documented through clinical intervention studies [8], until recently no study data were available answering the questions whether treatment with TZDs translates into a therapeutic benefit in atherosclerotic cardiovascular disease. Most clinical studies performed to date, assessing the effects of TZDs in diabetic patients, suggested vascular protective effects of PPARγ ligands related to improved insulin-sensitivity, decreased vascular and systemic markers of inflammation, reduced carotid wall thickness and neointima formation, and depending on the TZD, corrected dyslipoproteinemia [6]. Moreover, interestingly, even in non-diabetic patients with coronary artery disease, rosiglitazone treatment induced beneficial effects on the vascular endothelium, exerted anti-platelet activities, and reduced carotid intimamedia thickness progression [9-13]. All these reports argued for potent anti-atherogenic effects of TZDs, raising moreover the possibility of their additional use in clinical conditions other than diabetes. The PROactive study [14] is a prospective trial addressing the role of the PPARγ activator pioglitazone in the prevention of macrovascular events in patients with type 2 diabetes and pre-existing cardiovascular disease. The results of this study were just reported [15]. Although the primary endpoint, which was composed of a combination of disease-related

(mortality, non-fatal myocardial infarction, stroke, and acute coronary syndrome) and procedural (coronary and leg revascularisation and leg amputation) endpoints, did not reach significance, pioglitazone treatment significantly reduced the principal secondary endpoint (all cause mortality, non-fatal myocardial infarction, and stroke) by 16%. Moreover, the results from a first post-hoc analysis, presented at the recent AHA scientific sessions in Dallas, in the subgroup of patients with prior myocardial infarction showed that pioglitazone decreased recurrent cardiovascular events by 19%, fatal and non-fatal myocardial infarction by 28%, and acute coronary syndrome by 37% (www.proactive-results.com). Altogether, treatment with pioglitazone did not result in major adverse events, with the exception of an increase of reported, non-adjudicated heart failure. Since death due to heart failure was similar in the placebo and pioglitazone groups, it is likely that the high reported number in the pioglitazone group is due to the well-known increase in edema and thus does not represent bona fide heart failure. These results thus further indicate that pioglitazone treatment, on top of optimal cardiovascular treatment, reduces cardiovascular risk in type 2 diabetics with prior cardiovascular disease. The next trial in type 2 diabetic patients to report is the RECORD study with rosiglitazone which is still running [16]. PPARγ and Mouse Models of Atherosclerosis Previous studies performed in different mouse models (LDL receptor- or apo E-deficient mice) all demonstrated an atheroprotective effect of PPARγ activation by TZDs [17]. From these studies, it could be plausibly concluded that the overall beneficial action of the TZDs occurred independently from systemic lipid changes, and, possibly, from the insulin sensitizer activity of the compounds. Recently, we investigated the effect of two different PPARγ ligands (rosiglitazone and pioglitazone) on atherogenesis in a non-diabetic murine model which displays mixed dyslipidemia and develops atherosclerotic lesions consisting mainly of foam cells, i.e. homozygous human apolipoprotein (apo) E2 knock-in mice (E2-KI mice) that express human apoe2 instead of mouse apoe [18]. Surprisingly, when using this model, we demonstrated that PPARγ ligands did not protect E2-KI mice from atherosclerosis and foam cell accumulation. Also no significant changes in lipoprotein metabolism were observed in rosiglitazone- or pioglitazone-treated mice. Although glucose homeostasis was improved in the mice, this improvement was not sufficient to induce a delay in atherosclerosis development in this mouse model of normal glucose homeostasis. PPARγ is expressed in all cell types of the arterial wall, including endothelial cells, monocytes/macrophages and vascular smooth muscle cells (SMCs) [2] and besides its activity on macrophage lipid homeostasis with direct consequences for atherosclerosis development [19], PPARγ modulates the earliest step of the atherosclerotic lesion by inhibiting the expression of certain cytokines involved in the recruitment of monocytes/macrophages by endothelial cells [20]. Through these properties, PPARγ may exert cardiopreventive activities by decreasing foam cell formation. In our study in E2-KI mice, the lack of effect of PPARγ activation on macrophage accumulation contrasts with previous in vivo animal studies showing protective effects [17]. Although the exact reasons are unclear, several explanations can be evoked. Atherosclerosis in the E2-KI mice, in contrast to other mouse models, is characterized by the almost exclusive presence of macrophages. It is possible that, in the context of severe, uncorrected dyslipidemia, PPARγ activation in macrophages is insufficient to reverse the proatherogenic program. Moreover, a substantial controversy exists on the role of PPARγ in

macrophage cholesterol metabolism in mice. In vitro, PPARγ ligands may induce macrophage foam cell formation through CD36 induction and increased uptake of oxidized LDL [21]. In vivo rosiglitazone treatment in obese insulin-resistant ob/ob mice resulted in a decrease of peritoneal macrophage CD36 protein content likely due to the insulin sensitizing activity of the compound [22]. However, LDL receptor-deficient mice that display only mild insulin-resistance showed an increase in CD36 protein after rosiglitazone treatment in vivo [22]. One can note the proatherogenic role of the scavenger receptor CD36 has been recently challenged [23], nevertheless we can suggest that the effects of rosiglitazone on foam cell formation and atherogenesis could differ depending on the degree of insulin resistance. E2-KI mice are non-diabetic and are not insulin resistant, and therefore the impact of PPARγ agonists on macrophages may not be sufficient to improve atherosclerosis in this mouse model. Whether these observations can be extended to the human situation is unclear. Previous results on human and murine macrophages have demonstrated significant species-differences in the control of cholesterol homeostasis by PPARγ ligands [19,20]. For instance, whereas PPARγ activation results in the induction of the cholesterol efflux transporter ABCA1 in human macrophages [19], this effect is much less pronounced in murine macrophages [20]. Finally, it is possible that the atheroprotective effects of PPARγ are mediated via other cell-types that do not participate in atherogenesis in E2-KI mice such as SMCs. In this respect, it is interesting to note that TZD treatment has been shown to improve endothelial function and decrease intima-media thickness also in non-diabetic patients [9-13]. Such activities may be mediated, in part, via their effects on the proliferation and migration of vascular SMCs and the anti-inflammatory activities in these and other cell types involved in atherogenesis [24,25]. Moreover, PPARα and PPARγ ligands may influence thrombogenesis and inhibit proteins implicated in plaque rupture resulting in plaque stabilization [3,4]. Therefore, we cannot exclude that determining the effects of TZDs on advanced atherosclerosis may prove to be a stronger predictor of their potential clinical benefit and further testing their effects in other atherosclerosis models and in humans is therefore warranted. References 1. Marx N, Duez H, Fruchart J-C, Staels B. Peroxisome proliferators-activated receptors and atherogenesis: regulators of gene expression in vascular cells. Circ Res 2004;94:1168-78. 2. Marx N, Libby P, Plutzky J. Peroxisome proliferator-activated receptors (PPARs) and their role in the vessel wall: possible mediators of cardiovascular risk? J Cardiovasc Risk 2001;8:203-10. 3. Li D, Chen K, Sinha N, et al. The effects of PPAR-gamma ligand pioglitazone on platelet aggregation and arterial thrombus formation. Cardiovasc Res 2005;65:907-12. 4. Kockx M, Gervois P, Poulain P, et al. Fibrates suppress fibrinogen gene expression in rodents via activation of the peroxisome proliferator-activated receptor-alpha. Blood 1999;93:2991-98. 5. Desvergne B, Wahli W. Peroxisome proliferators-activated receptors: nuclear control of metabolism. Endocr Rev1999;20:649-88. 6. Verges B. Clinical interest of PPARs ligands. Diabetes Metab 2004;30:7-12. 7. Tailleux A, Torpier G, Mezdour H, Fruchart JC, Staels B, Fievet C. Murine models to investigate pharmacological compounds acting as ligands of PPARs in dyslipidemia and atherosclerosis. Trends Pharmacol Sci 2003;24:530-34. 8. Robins SJ, Collins D, Wittes JT, for the VA-HIT Study Group: Relation of gemfibrozil treatment and lipid levels with major coronary events VA-HIT: a randomised controlled trial. JAMA 2001;285:1585-91.

9. Sidhu JS, Cowen D, Kaski JC. The effects of rosiglitazone, a PPARgamma agonist, on markers of endothelial cell activation, C-reactive protein, and fibrinogen levels in non-diabetic coronary artery disease patients. J Am Coll Cardiol 2003;42:1757-63. 10. Sidhu JS, Cowan D, Tooze JA, Kaski JC. Peroxisome proliferator-activated receptor-γ agonist rosiglitazone reduces circulating platelet activity in patients without diabetes mellitus who have coronary artery disease. Am Heart J 2004;147:e-25. 11. Sidhu JS, Cowan D, Kaski JC. Effects of rosiglitazone on endothelial function in men with coronary artery disease without diabetes mellitus. Am J Cardiol 2004;94:151-56. 12. Sidhu JS, Kaposzta Z, Markus HS, Kaski JC. Effect of rosiglitazone on common carotid intimamedia thickness progression in coronary artery disease patients without diabetes mellitus. Arterioscler Thromb Vasc Biol 2004;24:930-34. 13. Marx N, Wöhrle J, Nusser T, et al. Pioglitazone reduces neointima volume after coronary stent implantation. Circulation 2005;112:2792-98. 14. Charbonnel B, Dormandy J, Erdmann E, Massi-Benedetti M, Skene A. The prospective pioglitazone clinical trial in macrovascular events (PROactive): can pioglitazone reduce cardiovascular events in diabetes? Study design and baseline characteristics of 5238 patients. Diabetes Care 2004;27:1647-53. 15. Dormandy J, Charbonnel B, on behalf of the PROactive investigators. Secondary prevention of macrovascular events in patients with type 2 diabetes in the PROactive Study (PROspective pioglitazone Clinical Trial In macrovascular Events): a randomised controlled trial. Lancet 2005;366:1279-89. 16. Home PD, Pocock SJ, Beck-Nielsen H, et al. Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD): study design and protocol. Diabetologia 2005;48:1726-35. 17. Zhang L, Chawla A. Role of PPARγ in macrophage biology and atherosclerosis. Trends Endocrinol Metab 2004;15:500-505. 18. Hennuyer N, Tailleux A, Torpier G, et al. PPARα, but not PPARγ, activators decrease macrophage-laden atherosclerotic lesions in a nondiabetic mouse model of mixed dyslipidemia. Arterioscler Thromb Vasc Biol 2005;25:1897-1902. 19. Chinetti G, Lestavel S, Bocher V, et al. PPARα and PPARγ activators induce cholesterol removal from human macrophages foam cells through stimulation of the ABCA1 pathway. Nat Med 2001;7:53-58. 20. Li AC, Binder CJ, Gutierrez A, et al. Differential inhibition of macrophage foam-cell formation and atherosclerosis in mice by PPARα, β/δ, and γ. J Clin Invest 2004;114:1564-76. 21. Tontonoz P, Nagy L, Alvarez JGA, Thomazy VA, Evans RM. PPARγ promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 1998;93:241-52. 22. Liang CP, Han S, Okamoto H, et al. Increased CD36 protein as a response to defective insulin signalling in macrophages. J Clin Invest 2004;113:764-73. 23. Moore KJ, Kunjathoor VV, Koehn SL, et al. Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice. J Clin Invest 2005;115:2192-201. 24. Marx N, Schonbeck U, Lazar MA, Libby P, Plutzky G. Peroxisome proliferator-activated receptor γ activators inhibit gene expression and migration in human vascular smooth muscle cells. Cir Res 1998;83:1097-103. 25. Zahradka P, Yurkova N, Litchie B, Moon MC, Del Rizzo DF, Taylor CG. Activation of peroxisome proliferator-activated receptors alpha and gamma1 inhibits human smooth muscle cell proliferation. Mol Cell Biochem 2003;246:105-10. Address correspondence to: Catherine Fiévet

UR 545 INSERM Institut Pasteur de Lille 1, rue Calmette BP245 59019 Lille, France Tel: 33-3-20-87 77 54 Fax: 33-3-20-87 73 60 E-mail: catherine.fievet@pasteur-lille.fr