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Peroxisome Proliferator-Activated Receptor α,γ Coagonist LY465608 Inhibits Macrophage Activation and Atherosclerosis in Apolipoprotein E Knockout Mice Steven H. Zuckerman*, Raymond F. Kauffman, and Glenn F. Evans Division of Cardiovascular Research, Lilly Research Laboratories, Indianapolis, Indiana 46285

ABSTRACT: The apolipoprotein E (apoE) knockout mouse has provided an approach to the investigation of the effect of both cellular and humoral processes on atherosclerotic lesion progression. In the present study, pharmacologic modulation of both interferon gamma (IFNγ)-inducible macrophage effector functions, and atherosclerotic lesions in the apoE knockout mouse were investigated using the peroxisome proliferator-activated receptor (PPAR) α,γ coagonist LY465608. LY465608 inhibited, in a concentration-dependent manner, IFNγ induction of both nitric oxide synthesis and the β 2 integrin CD11a in elicited peritoneal macrophages from apoE knockout mice. Similar effects were observed ex vivo following 10 d of treating mice with 10 mg/kg of LY465608. Treatment of apoE knockout mice for 18 wk with LY465608 resulted in a statistically significant 2.5-fold reduction in atherosclerotic lesion area in en face aorta preparations. These effects were apparent in the absence of any reduction in total serum cholesterol or in lipoprotein distribution. Finally, treatment of apoE knockout mice with established atherosclerotic disease resulted in a modest but not statistically significant decrease in aortic lesional surface area. These results demonstrate the utility of PPAR coagonists in reducing the progression of the atherosclerotic lesion. Paper no. L8986 in Lipids 37, 487–494 (May 2002).

Novel therapeutic approaches to the prevention and mitigation of atherosclerotic disease include strategies in which more specific interventions toward the inflammatory response are being considered. The macrophage is involved in all aspects of the pathobiology of the atherosclerotic lesion and mediates its effects through both cellular and humoral processes including the elaboration of cytokines and oxidants (1–3). Macrophage activation has been observed within the atheroma and is believed to be due to the autocrine and *To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: ABC-1, ATB-binding cassette transporter-1; AP-1, activator protein-1; apo, apolipoprotein; CD11a, integrin αL; CD11b, integrin αM; CD36, member of the scavenger receptor family; CMC, carboxymethylcellulose; FITC, fluorescein isothiocyanate; FPLC, fast protein liquid chromatography; IFNγ, interferon γ; IL, interleukin; iNOS, inducible nitric oxide synthetase; KO, knockout; LDLR, LDL receptor; LY465608, 2-{4-[2-(2biphenyl-4-yl-5-methyl-oxazol-4-yl)-ethoxy]-phenoxy}-2-methyl-propionic acid; MMP 9, matrix metalloprotease 9; NF-κB, nuclear factor κB; PPAR, peroxisome proliferator-activated receptor; SR-BI, scavenger receptor class B, type I; TNF, tumor necrosis factor. Copyright © 2002 by AOCS Press

paracrine effects of cytokines as well as to the binding and ingestion of oxidized LDL (4–6). Interferon γ (IFNγ) represents one such macrophage-activating cytokine whose effects on apolipoprotein (apoE) secretion, ABC-1 and ACAT expression, cholesterol efflux, and metalloprotease production could contribute to the pathology associated with atherosclerosis (7–12). Inhibiting IFNγ signaling, for example, by crossing apoE knockout (KO) mice with mice lacking the IFNγ receptor, results in reduced atherosclerosis (13). These in vivo results suggest that pharmacologic agents that inhibit macrophage activation through IFNγ signaling could be useful in slowing or reversing lesion progression. The peroxisome proliferator-activated receptors (PPAR) represent nuclear receptors that function as ligand-activated transcription factors. PPAR subtypes include PPARα, PPARδ, and PPARγ; upon ligand activation, PPAR form heterodimeric structures with the retinoid X receptor to bind to and activate specific gene transcription through PPAR response elements (14–16). Anti-inflammatory effects both in vitro and in vivo have been reported for both PPARα- and PPARγ-specific agonists (17–21). PPARγ agonists, for example, have been reported to inhibit IFNγ-induced inducible nitric oxide synthetase (iNOS) activity, gelatinase, and scavenger receptor activity in vitro in mouse macrophages, matrix metalloprotease 9 (MMP 9) activity in human monocyte-derived macrophages, and both phorbol ester and lipopolysaccharide induction of tumor necrosis factor (TNF) and interleukin 6 (IL-6) production (17,22–25). PPARγ agonists also have been reported to increase both scavenger receptor class B type I (SR-BI) and CD36 expression in macrophages, thereby contributing to the process of reverse cholesterol transport (26–30). Similarly, PPARα agonists also have been reported to have anti-inflammatory effects including inhibiting the production of MMP 9 activity, IL-6 and endothelin-1 secretion, and cycloxygenase 2 and iNOS expression (19–21). PPAR activation appears to inhibit inflammatory processes by antagonizing both nuclear factor κB (NF-κB) and activator protein-1 (AP-1) pathways (20,21,31). Therefore, it would be expected that PPARα or -γ agonists should be beneficial in preclinical models of atherosclerosis. To this extent, the recent reports demonstrating that activation of the retinoid X receptor

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through the administration of rexinoids or a PPARα,γ coagonist reduced lesion development in the apoE KO mouse (32) suggest the utility of this approach. Separate studies with the PPARγ agonist troglitazone, rosiglitazone, or GW7845 also reported reductions in lesional areas in both apoE and LDL receptor KO mice as well as increases in CD36 and lipoprotein lipase message (33–35). LY465608 (2-{4-[2-(2-biphenyl-4-yl-5-methyl-oxazol-4yl)-ethoxy]-phenoxy}-2-methyl-propionic acid) represents a new class of PPARα,γ coagonists in which the pharmacophores of known PPARγ and PPARα selective agents were combined into a single compound. IC50 values for human PPARα,γ of 174 and 548 nM, respectively, were determined (36). Cotransfection studies demonstrated that this compound is an effective agonist against both receptors as well as the mouse PPARα. The specificity of this compound was also determined in transfection studies where it was demonstrated that the Ki for retinoid and rexinoid receptors, glucocorticoid, and thyroid receptors was greater than 10 µM. Finally, this compound has been demonstrated to lower both plasma glucose and TG levels in a diabetic mouse model at 30 mg/kg (36). These results as well as the nuclear receptor specificity of LY465608 suggested a potential therapeutic role for this PPAR coagonist in reducing atherosclerosis in the apoE KO mouse. The present study was initiated to evaluate the impact of LY465608 on macrophage effector functions in vitro systematically, to evaluate its effects on macrophage responses to IFNγ ex vivo, and to determine whether there would be any beneficial effect on lesion development and/or progression. LY465608 was demonstrated both in vitro and ex vivo to inhibit the ability of IFNγ to induce both iNOS and CD11a expression. Furthermore, when administered at the initiation of exposure of apoE KO mice to an atherogenic diet, LY465608 significantly reduced the extent of atherosclerotic lesion development. Finally, similar studies were performed on older apoE KO mice with established lesions to determine whether a similar beneficial effect could be observed in lesional surface area. These latter results, however, failed to achieve statistical significance. Collectively, the results provide further evidence for the therapeutic value of a PPAR coagonist in the treatment of atherosclerosis in a model in which a vascular effect is observed in the absence of a significant beneficial impact on serum lipoprotein profiles. MATERIALS AND METHODS Macrophage cultures and reagents. Peritoneal macrophages were obtained from thioglycolate-primed apoE KO mice (C57BL/6Tac-Apoetm1UncN11, male; Taconic Laboratories, Germantown, NY) and maintained in culture in RPMI 1640 supplemented with 2% FCS (Hyclone Laboratories, Logan, UT). Macrophages were activated with the designated concentrations of recombinant murine IFNγ (BioSource International, Camarillo, CA) for 48 h, and supernatants were quantified for nitrites and cells processed for flow cytometry. LY465608 at final concentrations between 1 nM and 10 µM Lipids, Vol. 37, no. 5 (2002)

were added to the macrophage cultures at the time of IFNγ addition. Flow cytometry and nitric oxide measurements. Following 48 h of stimulation with IFNγ, macrophage cultures were stained with a phycoerythrin-conjugated antibody against CD11a (integrin αL) or fluorescein isothiocyanate (FITC)conjugated antibody against CD11b (integrin αM) (Pharmingen, San Diego, CA). All flow cytometry experiments were evaluated on 10,000 individual cells gated for macrophages based on their forward and side light-scatter profiles. The mean fluorescence intensity was measured using a Becton Dickinson FacSort with Cellquest software (Becton Dickinson, San Jose, CA). Supernatants were quantified for nitric oxide levels using the Griess reaction as previously described (30). The optic density change at 10 min was measured at 550 nm and converted to micromoles of nitrites based on a sodium nitrite standard curve. Atherosclerosis model. Male apoE KO mice were shifted to a high-fat diet (21% milkfat, 0.15% cholesterol; Harlan Teklad, Madison, WI) and were dosed orally by gavage with 10 mg/kg of LY465608 [prepared in 1% carboxymethylcellulose (CMC) plus 1% Tween 80] daily. Mice were treated with LY465608, 10 mg/kg/d, for 18 wk starting at 8 wk of age when they were shifted to a high-fat diet. At the end of the 18 wk of treatment both vehicle and LY465608 mice were evaluated for lipoprotein profiles and atherosclerotic lesions. In studies designed to investigate the effect of LY465608 on established lesions, mice were maintained on the high-fat diet for 16 wk and then treated for an additional 12 wk with 10 mg/kg LY465608 prior to sacrifice. The control animals in all experiments were dosed with the CMC vehicle. All animal studies were in compliance with institutional guidelines. Lesion analysis. At sacrifice, animals were prepared for en face aorta evaluation by cutting the aorta below the bifurcation in the lower abdomen as previously described (37). Briefly, the aortas were perfused with PBS, cleaned of external fat, opened, flattened on a microscope slide, and covered with a coverglass. All samples were fixed in 4% formalin and kept at 4°C in humidified chambers until imaging. Samples were visualized using a Microtek ScanMaker 9600 XL (Microtek Lab, Inc., Redondo Beach, CA) and Adobe Photoshop LE (Adobe Systems, San Jose, CA). The images were analyzed using a custom-written macro (Mike Esterman, Jeff Hanson, Lilly Research Labs) in Image Pro Plus (Media Cybernetics, L.P., Silver Spring, MD) that uses the threshold tool to select lesion sites based on image intensity contrasted to the normal translucent arterial wall. Lesion area and total aortic area were expressed in square mm. Approximately 45 mm square of total aortic surface was evaluated per aorta for lesion quantification. Serum cholesterol and lipoprotein analysis. Total serum cholesterols were measured using the Cholesterol CII kit from Wako Pure Chemicals (Richmond, VA). Sera from each group at the designated times were pooled, and 25–100 µL aliquots were resolved by fast protein liquid chromatography (FPLC; Pharmacia, Bromma, Sweden) using tandem-linked Superose

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6 columns as previously described (38). Total cholesterol was quantified enzymatically (Wako Chemicals USA, Richmond, VA) from 100-µL aliquots of the FPLC fractions. The relative amount of cholesterol within each peak was determined by area quantification under the curves using the appropriate baseline modifications from the FPLC cholesterol tracings. Serum TG levels were measured by enzymatic assay (Sigma Chemicals, St. Louis, MO). Statistics. Statistical analysis was performed by unpaired (two-tailed) t-test. Values are reported as means ± SD. The 95% confidence limit was taken as significant (P < 0.05). Additonal statistical analysis was by a Dunnett’s multiple comparisons test. Analysis of the lesion area data was performed using chi square and Tukey’s biweight function robust method as well as by a Mann–Whitney U test, a nonparametric test. RESULTS Activation of primary peritoneal macrophages with IFNγ results in an increase in macrophage effector functions including the generation of nitric oxide through the induction of iNOS and the upregulation of the β 2 integrin CD11a. Consistent with previous studies using the less-specific PPARγ agonist 15-deoxy-∆12,14-prostaglandin J2 (30), 100 nM LY465608 resulted in a significant reduction both in nitric oxide production (Fig. 1A) and in CD11a induction (Fig. 1B) at all concentrations of IFNγ used for macrophage activation. In contrast, CD11b, which is already expressed on the macrophage surface, was only significantly modulated by LY465608 at the highest IFNγ concentration (Fig. 1C). The effects of the coagonist were concentration dependent, with significant effects apparent at 100 nM for both iNOS activity (Fig. 2A) and CD11a induction without any effect on CD11b expression until the highest concentration of 10 µM (Fig. 2B). These results suggested that a PPARα,γ coagonist was able to mitigate the macrophage effector functions induced by IFNγ. To determine whether a similar impact on macrophage effector functions could be demonstrated in vivo, apoE KO mice on standard laboratory chow were dosed at 10 mg/kg orally with LY465608 for up to 10 d. At day 10, elicited macrophages were harvested and stimulated in vitro with varying concentrations of IFNγ for an additional 48 h. In comparison with the vehicle group, elicited macrophages from LY465608-treated mice showed a reduced induction of CD11a at both optimal (500 units/mL) and suboptimal (50 units/mL) concentrations of IFNγ (Fig. 3A), and at optimal IFNγ concentrations, they showed an approximate 50% reduction in nitric oxide formation (Fig. 3B). There was also, in the LY465608-treated animals, an overall increase in total serum cholesterol; in the control cholesterol was 412 ± 89 vs. coagonist 591 ± 186 mg/dL. This increase in the coagonist group was statistically significant by a two-tailed unpaired ttest (P < 0.03). The demonstration ex vivo that LY465608 was able to modulate IFNγ-mediated macrophage effector functions

FIG. 1. Effects of LY465608 (2-{4-[2-(2-biphenyl-4-yl-5-methyl-oxazol4-yl)-ethoxy]-phenoxy}-2-methyl-propionic acid) on interferon γ (IFNγ)mediated induction of nitric oxide and CD11a in murine peritoneal macrophages. Macrophages from apoE KO mice were treated in vitro in triplicate, with 0–500 units/mL IFNγ with or without 100 nM LY465608 for 40–60 h. Cell supernatants were assayed for nitrites (A), and cells for the membrane integrins CD11a (B) and CD11b (C) by flow cytometry measuring the mean fluorescence intensity (MFI). Brackets represent the standard deviation of the mean from triplicate cultures. The difference between the untreated and LY465608- treated macrophages was significant by a two-tailed unpaired Student’s t test at P < 0.05 (#), P < 0.01 (*), or P < 0.001 (+). A standard two-way ANOVA was also performed, followed by a Bonferroni adjusted multiple comparison of treated vs. untreated at each IFNγ concentration. Significance was at the P < 0.001 (+,*) or P < 0.05 (#) levels.

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FIG. 3. Ex vivo effects of LY465608 on murine peritoneal macrophages. ApoE KO mice were dosed orally for 10 d with LY465608 or vehicle alone; and elicited peritoneal macrophages were collected, plated in 24-well plates, and evaluated for IFNγ-induced CD11a expression (A) and nitric oxide production (B). CD11a expression was measured following induction of macrophages with varying concentrations of IFNγ (5, 50, and 500 units/mL) and for nitric oxide production at 500 units/mL of IFNγ. Macrophages were not exposed to exogenous LY465608 during the 48–72 h in vitro phase of the experiment. Cells from three animals for each treatment were pooled before plating. Representative experiment of five; brackets indicate the SD of the mean. *Difference from the control was significant by a two-tailed unpaired Student’s t-test (P < 0.01). For abbreviations see Figures 1 and 2.

FIG. 2. LY465608 in a concentration-dependent manner inhibited nitric oxide and CD11a induction by IFNγ. Triplicate cultures of macrophages were treated with 1 nM to 10 µM LY465608 with 200 units/mL IFNγ. After 48 h incubation, supernatants were assayed for nitrites (A), and cells were evaluated for expression of CD11a and CD11b (B). *Difference from the control was significant by a two-tailed unpaired Student’s t-test (P < 0.01). A one-way ANOVA analysis was also performed, followed by a Dunnett’s multiple comparison of each concentration of LY465608 compared to vehicle. Significance was also at the P < 0.01 level. For abbreviations see Figure 1.

suggested it could have a favorable impact on atherosclerotic disease progression in the apoE KO mouse. Accordingly, mice were treated with LY465608, 10 mg/kg/d, for 18 wk starting at 8 wk of age, when they were shifted to a high-fat diet. At the end of the 18 wk of treatment, both vehicle and LY465608 mice were evaluated for cholesterol and lipoprotein profiles, and quantitation of aortic lesions was performed on the en face preparations. As demonstrated (Fig. 4A), LY465608 treatment reduced the rate of weight gain compared to the vehicle group. This decrease in weight gain was not due to a reduction in food consumption, which remained relatively constant in both groups through the duration of the study (approximately 25 g/mouse/wk). Interestingly, the LY465608-treated animals had a statistically significant (P < 0.01) increase in serum cholesterol relative to the vehicle group at sacrifice (996 ± 217 vs. 808 ± 208 mg/dL) which Lipids, Vol. 37, no. 5 (2002)

was evident in the VLDL peak of the lipoprotein profile (Fig. 4B). Serum TG were reduced in the LY465608 group (521 ± 191 vs. 591 ± 217 mg/dL); however, this reduction was not statistically significant. Although there were no significant reductions in the VLDL or LDL lipoproteins, and in fact there was a consistent increase in the former, there were significant reductions in aortic lesions in the LY465608-treated animals. En face evaluation of the lesion distribution revealed that the atherosclerotic lesions were primarily localized to the aortic arch and the surfaces around the bifurcations. Representative aortas en face (Fig. 5A) clearly demonstrate a decrease in the lesion surface area in the LY465608-treated animals that was statistically significant and apparent when the lesion distribution for each animal was quantified (Fig. 5B). The mean lesion area in the vehicle group was 31.5 ± 9.2 vs. 13.5 ± 4.9%, and this difference was significant (P < 0.0001). Furthermore, the vehicle group had 8 of 9 animals with >25% lesion area, whereas the LY465608 group had 0 of 10 animals with greater than 25% of the surface area containing lesions. These results demonstrate a significant reduction in lesion progression when apoE KO mice were treated with LY465608 at the time that they were shifted to the high-fat atherogenic diet. To determine whether the PPAR coagonist LY465608 could have a similar effect in apoE KO mice with established lesions, a final series of experiments was performed in which mice were allowed to consume the high-fat diet for 16 wk. Following 16 wk on diet, mice were randomized to receive either LY465608 at 10 mg/kg or vehicle and were continued on diet and treatments for an additional 12 wk prior to


FIG. 4. Effect of LY465608 on weight gain and serum cholesterol profile. ApoE KO mice, 10 per group, were placed on “western” high-fat diets and dosed with LY465608 or the vehicle for 18 wk. Body weights and food consumption were measured weekly. Body weights (A) and total serum cholesterol fast protein liquid chromatography profile (B) were determined on pooled serum samples at the termination of the study. Brackets represent the SD of the mean. The difference in weight between the vehicle- and LY465608-treated mice was significant by a two-tailed unpaired Student’s t-test (P < 0.01) by 6 wk treatment and remained so through the remainder of the study. The changes in weight were also analyzed by a repeated measures ANOVA, and the LY465608 group was compared to vehicle at each week using a Bonferroni adjusted multiple comparison test. By this analysis significance after week 0 was P < 0.0001. OD, optical density; for other abbreviations see Figure 1.

sacrifice. As demonstrated (Fig. 6A), apoE KO mice treated with the PPAR coagonist demonstrated a significant reduction in weight, which was apparent by 2 wk. As in the previous study, this reduction in weight gain was not due to a decrease in food consumption. Furthermore, serum cholesterol levels between the two groups were indistinguishable (1517 ± 503 vehicle and 1518 ± 590 mg/dL for LY465608) as were the lipoprotein profiles, which consisted primarily of a VLDL fraction with an LDL shoulder (data not shown). There was, however, a significant increase in serum TG in the LY465608 animals 884 ± 442 vs. 418 ± 130 mg/dL (P < 0.003). Quantification of lesion surface area revealed that 11 of 15 animals treated with LY465608 had less than 25% lesion area compared to the vehicle group with 6 of 15. The chi-squared test for the significance of this change in ratio, however, did not achieve statistical significance (P < 0.065). Although there

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FIG. 5. Effect of LY465608 on aortic lesion formation in apoE KO mice on “western” diet. Mice at 8 wk of age were placed on a high-fat diet and randomized to vehicle or 10 mg/kg LY465608 groups. After 18 additional weeks on the diet the animals were sacrificed, and the aortas were dissected and placed on microscope slides. Representative en face images of the aorta from control and LY465608-treated aortas (A) and the percentage of the aortic suface area covered with lesions are presented for each animal (n = 10 per group) (B). Note reduction in the lesion surface area from the LY465608-treated animals. This difference was significant by a two-tailed unpaired Student’s t-test (P < 0.0001). Scale bar represents 5 millimeters.

appeared to be a nonsignificant reduction in lesion area in this study on established lesions, there was one animal in the LY465608 group that had greater than 80% of the aortic surface area covered with lesion. Whereas this animal did have other problems detected at autopsy, i.e., fluid in the lungs, there was no apparent reason to treat this animal as an outlier and hence it was included in the data set. These results, however, do suggest that intervention fairly late in the process of lesion development can in fact reduce further increases in lesion accumulation through effects independent of serum cholesterol levels or lipoprotein profiles. DISCUSSION PPARα,γ agonists as well as coagonists are clearly demonstrating additional biologic responses beyond HDL elevation and insulin sensitization that would suggest a broader therapeutic role in the management of chronic inflammatory diseases (39–42). To explore these potential roles further, the PPARα,γ coagonist LY465608 was evaluated in a model of atherosclerosis where HDL represents a minor component of the lipoprotein profile and where insulin sensitivity is not recognized as part of the disease process. The apoE KO model represents such a model and is characterized by extremely high serum cholesterols (>1000 mg/dL when placed on a high-fat diet), primarily carried on VLDL and remnant particles, and significant lesions throughout the aortas (43). Lipids, Vol. 37, no. 5 (2002)

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FIG. 6. Therapeutic intervention with LY465608 on mice after 16 wk on a high-fat diet. ApoE KO mice were placed on the “western” diet for 16 wk and then randomized (n = 15 per group) to receive LY465608 orally at 10 mg/kg or vehicle alone for an additional 12 wk. The highfat diet was continued during this treatment phase. Weight (A) and food consumption were monitored weekly. At the termination of the experiment, the aortic surface area covered with atherosclerotic lesions was quantified and the distribution was presented for each animal (B). The difference in weight between the two groups was significant by a twotailed unpaired Student’s t-test (P < 0.01) as early as 2 wk on LY465608. The changes in weight were also analyzed by a repeated measures ANOVA, and the LY465608 group was compared to vehicle at each week using a Bonferroni adjusted multiple comparison test. By this analysis, significance after week 0 was P < 0.01. (B) Robust mean estimates are presented (horizontal bars) and were 26.3% in the vehicle and 21.6% for the LY465608 group. The chi-squared test for the significance of the difference in lesion surface area did not achieve statistical significance (P < 0.065).

The present study was designed to determine whether LY465608 treatment in vitro would mitigate the macrophage response to IFNγ using both iNOS and CD11a induction as indicators of macrophage activation. These two parameters can be linked to the pathology associated within the atherosclerotic lesion by inducing oxidant stress with the former and by promoting macrophage adherence and migration through the β 2 integrin pathway with the latter (44,45). The results of this study demonstrated a direct inhibitory effect of this coagonist on these parameters of macrophage activation. Furthermore, similar effects were also apparent ex vivo when apoE KO mice were pretreated with LY465608 for 10 d prior to harvesting elicited macrophages and subjecting them to in vitro stimulation with IFNγ. In additional studies, 4–5 d of oral treatment with LY465608 exhibited similar ex vivo results (data not shown). The ability of the coagonist to influence these effector functions suggested that LY465608 should favorably reduce Lipids, Vol. 37, no. 5 (2002)

atherosclerotic lesion progression, and, in fact, lesional surface area was reduced 2.5-fold in the experimental group. The extent of lesion reduction in the apoE KO mouse compares favorably with similar studies recently reported for the PPARγ agonist troglitazone (35), with troglitazone, rosiglitazone, or GW7845 in the LDL receptor KO (33, 34), and with a retinoid X receptor agonist LG100364 (32). This latter study reported a lesser effect on lesion reduction with the PPAR coagonist GW2331, and still lesser efficacy with the PPARγ agonist rosiglitazone. Although the results in reducing total lesional area were similar across these studies, some differences were observed. In the present study we detected a reduction in weight gain with chronic administration without an impact on food consumption. These effects were consistent with PPARα activity of the coagonist, as PPARα agonists have been reported to reduce body weight increase in other rodent models (46,47). It is, however, noteworthy that in one study troglitazone did reduce total body weight in the LDL receptor (LDLR) KO mice fed a high-fat diet (34). Chen et al. (35) reported a doubling in HDL cholesterol but no effect on total cholesterol or TG, whereas Claudel et al. (32) reported increases in total serum cholesterol and TG, primarily in the VLDL and remnant peak. In the present study we did not detect any increase in HDL cholesterol but did observe a significant increase in total cholesterol, through the VLDL and remnant peak, but no increase in serum TG as seen in the second study which had been designed to evaluate the effect of LY465608 on lesion development. Finally, to reflect the clinical situation more closely, we designed studies to determine whether LY465608 would have an effect on established lesions rather than initiating treatment at the time of exposure to the atherogenic diet. Accordingly, apoE KO mice were maintained on the atherogenic diet for 16 wk prior to initiating treatment with LY465608 for an additional 12 wk. These results, although failing to achieve statistical significance, did suggest reduced lesional surface areas in the LY465608 group when compared to the controls. This represents, to the best of our knowledge, the first study in which intervention with a potential anti-atherosclerotic compound was evaluated in apoE KO mice with established lesions. Whereas the precise mechanism(s) by which PPAR coagonists may limit the development of new lesions and affect established lesional mass remains unclear, it is likely to involve the inhibition of both NF-κB and AP-1 dependent transcriptional pathways (20,21). PPAR expression within atherosclerotic lesions in monocytes, macrophages, foam cells, and smooth muscle and endothelial cells provides a pathway for limiting lesion progression (45). PPARα agonists, for example, reportedly induce apoptosis of TNF-activated macrophages and limit monocyte recruitment in early lesions by inhibiting TNF induction of vascular cell adhesion molecule expression, presumably by serving as negative regulators of NF-κB (48,49). One such mechanism of inhibiting NF-κB activation is through the induction of IκBα by PPARα agonists and thus keeping the NF-κB complex inactive within


the cytoplasm (21). However, whereas many of the NF-κB and AP-1 dependent regulated genes are thought to be proinflammatory, such as MMP-9, IL-6, and TNF, there is some evidence that PPAR agonists may have confounding effects on lesion development. Lee et al. (50), for example, reported that the PPARα agonist Wy14643 stimulated the synthesis of IL-8 and monocyte chemotactic protein-1 in human aortic endothelial cells, whereas the PPARγ agonist troglitazone suppressed production of these chemokines. Furthermore, although PPAR agonists have been reported to upregulate CD36 and consequently the uptake of oxidized LDL (28,29), further studies have implicated a PPARγ pathway for promoting cholesterol efflux through the increase in ABC-1 expression via induction (26). These authors further demonstrated that PPARγ-null bone marrow cells transplanted into LDLR KO mice resulted in significant increases in atherosclerotic lesion mass. Collectively, these studies suggest that PPAR coagonists should favorably affect atherosclerotic disease through anti-inflammatory processes as well as by stimulating reverse cholesterol transport. The results of the in vitro and in vivo studies clearly demonstrate that the PPAR coagonist LY465608 can favorably affect atherosclerotic disease progression through processes unrelated to changes in serum lipoprotein profiles. These data demonstrate a clear role and direction for the next generation of agents to treat atherosclerosis, namely, targeting the vascular component.

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ACKNOWLEDGMENTS The authors wish to acknowledge Dr. Mary K. Peters for providing LY465608, and Mark Farmen for statistical support. The technical assistance of Pat Forlor, Jack Cochran, Phyllis A. Cross, Brian K. McKenney, Martin S. Cramer, Richard L. Tielking, Courtney Burch, Kat Andrzejewski, and Renee L. Grubbs is also gratefully acknowledged.

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[Received January 22, 2002, and in revised form March 29, 2002; revision accepted April 9, 2002]