Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice (original) (raw)
Intervention studies were performed in LDLR–/– mice fed a Western-style diet for 10 weeks, starting at age 8–12 weeks. To reduce the possibility that effects of a single PPARγ ligand on atherosclerosis resulted from PPARγ-independent mechanisms, two distinct PPARγ agonists were used: rosiglitazone and GW7845. Rosiglitazone is a member of the TDZ class of insulin sensitizers that was developed using rodent models of type 2 diabetes. It has an effective concentration of 50% (EC50) for murine PPARγ of 76 nM (33). GW7845 is a member of the tyrosine-based class of insulin sensitizers that was developed using human PPARγ as a molecular target. It has an EC50 for murine PPARγ of 1.2 nM (33). Both drugs are highly specific for PPARγ, with EC50 for PPARα and PPARδ in excess of 10 μM (33). We initially performed a pilot study using a calculated dose of 20 mg rosiglitazone/kg/day to establish appropriate dietary cholesterol content and extent of atherosclerosis. Rosiglitazone exerted a significant antiatherogenic effect in male mice in this study, but not in female mice (data not shown). However, because the 1.25% added dietary cholesterol resulted in serum cholesterol levels in excess of 2,000 mg/dL, a potential protective effect in females could have been overwhelmed. Two subsequent intervention studies were therefore carried out in which the added cholesterol was reduced to 0.01%. Each experiment resulted in the same pattern of responses to dietary and drug treatments, and the data from the two studies were pooled to increase statistical power.
At a dose of 20 mg/kg/day, rosiglitazone plasma levels averaged 6.4 plus or minus 0.06 μg/mL in male mice and 5.1 plus or minus 0.69 μg/mL in female mice at 10 weeks. GW7845 levels averaged 3.2 plus or minus 0.39 μg/mL in male mice and 3.2 plus or minus 0.46 μg/mL in female mice after 10 weeks of treatment. These serum levels are sufficient to exert inhibitory effects on proinflammatory gene expression in vitro (29). All animals appeared healthy throughout the study. Serum aspartate aminotransferase and alkaline phosphatase levels were used to assess potential liver toxicity and were not altered at the end of the study (data not shown). Histologic analysis of the bone marrow indicated a significant increase in percentage of marrow fat, and marked extramedullary hematopoiesis was observed in both male and female mice (data not shown). There were no significant changes in complete blood counts or hemoglobin. Data for body weight, total cholesterol, triglycerides, and HDLc at specific time points are presented in Table 1. The body weights in all groups increased during the intervention period, but the relative weight gain in males was greater than that in females. The Western diet resulted in a marked increase in total cholesterol within 1 month; the total cholesterol then remained constant at approximately 1,500 mg/dL in males. There was a slight increase in cholesterol levels of treated females, but this effect only reached statistical significance (P = 0.05) in the rosiglitazone treatment group after 10 weeks. Triglycerides were significantly increased and HDLc levels were decreased in female mice treated with rosiglitazone or GW7845. A decrease in HDLc levels was seen in male mice treated with rosiglitazone only.
Average weights, cholesterol, triglyceride, and HDLc levels
PPARγ ligands inhibit the development of atherosclerosis in male mice. Atherosclerosis at the aortic origin was determined by computer-assisted image analysis as described previously (32). Male and female control animals exhibited similar levels of atherosclerosis. Lesions were observed underneath most of the valve leaflets, with some lesions exhibiting areas of central necrosis (Figure 1a). Macroscopically detectable lesions were generally absent from the thoracic or abdominal aorta (data not shown). Markedly fewer and smaller lesions were found in male mice that were treated with either rosiglitazone or GW7845, with quantitative analysis indicating a 60 to 80% reduction in lesion area (Figure 1b). In contrast, the extent of atherosclerosis in female mice treated with either rosiglitazone or GW7845 was not statistically different from that in control mice, confirming the findings of the initial pilot study.
Atherosclerosis in LDLR–/– mice fed a high-fat, cholesterol-enriched Western diet for 10 weeks. (a) Sections through the aortic root at the levels of the aortic valves were stained for elastin to highlight the medial boundaries of atherosclerotic lesions. (b) Quantitative analysis of lesion areas in control mice (C), mice treated with rosiglitazone (Ro), and mice treated with GW7845 (GW). For male mice, means ± SEM were: C, 0.161 ± 0.067 mm2/section (n = 10); Ro, 0.037 ± 0.014 mm2/section (n = 12); GW, 0.063 ± 0.027 mm2/section (n = 10). For female mice, means ± SEM were: C, 0.131 ± 0.035 mm2/section (n = 10); Ro, 0.183 ± 0.0.088 mm2/section (n = 10); GW, 0.181±0.0091 mm2/section (n = 10). NS, not statistically significant.
Metabolic effects of PPARγ ligands. To investigate possible mechanisms accounting for antiatherogenic effects of PPARγ ligands in male mice and lack of these effects in female mice, lipoprotein levels were evaluated in control and treatment groups. Fast-performance liquid chromatography (FPLC) analysis of pooled terminal serum samples indicated that GW7845 and rosiglitazone had no effect on the lipoprotein profile in male mice (Figure 2a). In contrast, in female mice the VLDL, IDL, and LDL fractions were increased and the HDL fraction decreased in both the rosiglitazone and GW7845 treatment groups (Figure 2b).
Size distribution of lipoprotein particles in LDLR–/– mice fed a high-fat, cholesterol-enriched diet and treated with solvent (control; diamonds), rosiglitazone (squares), or GW7845 (triangles) for 10 weeks. Plasma was pooled from four mice from each treatment group and fractionated by FPLC. Mean cholesterol content in each fraction was determined in duplicate.
Effects of PPARγ ligands on serum glucose, insulin, HbA1c, and NEFA levels are presented in Table 2. The Western diet itself did not significantly alter glucose, HbA1c, or NEFA levels, but insulin levels rose in both male and female mice. Rosiglitazone and GW7845 treatment resulted in a significant decrease in insulin levels in male mice but had no significant effect on insulin levels in female mice (Table 2). HbA1c decreased in males treated with rosiglitazone and GW7845.
Average glucose, insulin, HbA1c, NEFA levels
To further investigate the effects of rosiglitazone and GW7845 on glucose homeostasis, the response to an oral glucose challenge was assessed in LDLR–/– mice fed the Western diet for 8 weeks. LDLR–/– mice fed a normal chow diet were used as additional control groups. Mice were fasted for 4 hours before being given an oral glucose load of 0.75 mg/g. Blood samples were taken at 0, 15, 30, 60, and 90 minutes for measurement of glucose and insulin levels. In male mice, the Western diet had relatively little effect on glucose levels in response to the oral glucose challenge (Figure 3a). In female mice, after glucose administration, the Western diet resulted in modest elevations in glucose that were normalized by treatment with either rosiglitazone or GW7845 (Figure 3b). Striking differences in the insulin responses to oral glucose challenge were noted between male and female mice treated with rosiglitazone and GW7845. The Western diet resulted in increased fasting insulin levels in both male and female mice (Figure 3, c and d), compared with the chow-fed controls. Treatment with rosiglitazone or GW7845 resulted in normalization of the fasting insulin levels and the insulin response to glucose challenge in male mice, but not in female mice (Figure 3, c and d), consistent with changes in insulin levels observed in the intervention studies (Table 2).
Glucose and insulin responses to an oral glucose challenge in LDLR–/– mice fed the normal chow (circles); high-fat, cholesterol-enriched diet and solvent (control; diamonds); rosiglitazone (squares); or GW7845 (triangles). Blood glucose and plasma insulin levels were determined at base line (after a 4-hour fast) and 15, 30, 60, and 90 minutes after oral administration of 0.75 mg glucose/g body weight. Samples were taken from eight animals per group. Data are expressed as the mean ± SEM. A_P_ < 0.0001, B_P_ < 0.002, C_P_ < 0.015, and D_P_ < 0.04, drug treatment group vs. control group.
Effects of PPARγ ligands on gene expression. To investigate potential effects of PPARγ ligands on patterns of gene expression within the arterial wall, RNA analysis was performed in LDLR–/– mice fed a Western diet for 10 weeks in the absence or presence of rosiglitazone or GW7845 as described for the intervention studies. RNA was isolated from the base of the heart containing the aortic origin affected by atherosclerosis and analyzed for TNF-α, MCP-1, VCAM-1, and gelatinase B mRNA levels, using quantitative real-time PCR. TNF-α and gelatinase B mRNA levels were significantly lower in male mice treated with rosiglitazone or GW7845 (Figure 4). Decreases in TNF-α and gelatinase B were smaller in female mice and did not reach statistical significance in the case of gelatinase B. Levels of VCAM-1 and MCP-1, which are thought to be involved in monocyte adhesion to the vessel wall and migration into the lesion, respectively (34), did not change significantly among the groups (Figure 4). Reductions in TNF-α and gelatinase B mRNA levels were also observed in RNA prepared from the apex of the heart, suggesting general effects of the PPARγ ligands (data not shown). Differences in the responses of TNF-α and gelatinase B genes to PPARγ ligand between male and female mice were not likely due to differences in PPARγ expression, because PPARγ mRNA levels were approximately two times higher in female tissues (data not shown).
Expression of TNF-α, MCP-1, VCAM-1, and gelatinase B mRNA in the aortic root. The mRNA levels were quantitated using real-time RT-PCR. Six to seven samples per group were analyzed. C, control; Ro, rosiglitazone; GW, GW7845. Data are expressed as mean ± SEM. A_P_ < 0.05, B_P_ < 0.01, and C_P_ < 0.001, drug treatment groups vs. cholesterol group.
Because there were significant differences in lesion size in male controls and animals treated with solvent, rosiglitazone, or GW7845, we also investigated whether PPARγ ligands altered levels of gene expression in the artery wall under conditions of equivalent degrees of atherosclerosis. LDLR–/– male mice were fed a 1.25% cholesterol and 21% milk-fat diet for 16 weeks to induce significant atherosclerosis in the aortic arch. Mice were then treated with rosiglitazone, GW7845, or control solvent for 2 weeks while maintaining the high-fat, high-cholesterol diet. Aortas were dissected and weighed to confirm comparable levels of atherosclerosis (35). As an additional control group, mRNA was isolated from aortas of normocholesterolemic animals. The aortas from each group were pooled, and mRNA was isolated for analysis of macrosialin, CD36, SR-A, MCP-1, TNF-α, and VCAM-1 gene expression (Figure 5). Macrosialin is a macrophage-specific membrane glycoprotein that serves as a marker of tissue macrophages (36). Macrosialin expression was low in normal aortas and markedly increased in atherosclerotic aortas, as expected. Macrosialin levels were not significantly altered by 2 weeks of treatment with rosiglitazone or GW7845, consistent with our observation that PPARγ ligands do not alter macrosialin expression in peritoneal macrophages (data not shown) and reflecting comparable levels of atherosclerosis in these three groups. SR-A and MCP-1 mRNA levels were also elevated in atherosclerotic aortas, as expected. Surprisingly, the mRNA levels for VCAM-1 remained unchanged. In contrast to previous findings in cell-culture models (29, 37), mRNA levels for these genes were not decreased by treatment with PPARγ ligands. However, treatment with rosiglitazone or GW7845 significantly increased CD36 expression and inhibited TNF-α expression, indicating actions of PPARγ on gene expression in the artery wall. The effects on CD36 expression were tissue specific, because no increase in CD36 expression was observed in cardiac tissue of mice treated with rosiglitazone or GW7845 (data not shown).
Expression of macrosialin, CD36, SR-A, MCP-1, TNF-α, and VCAM-1 mRNA in the aorta. Male LDLR–/– mice were fed either a normal chow diet (N) or a high-cholesterol diet for 4 months to induce the development of atherosclerosis (Athero). Animals fed the high-cholesterol diet were then treated with either solvent control, rosiglitazone, or GW8745 for 2 weeks. The mRNA levels were quantitated using real-time RT-PCR. Data represent pooled aortas with an average weight of 3.86 ± 0.16 mg/aorta for normal chow (N) (n = 11); 5.75 ± 0.67 mg/aorta for high cholesterol (C)(n = 6); 5.67 ± 0.56 mg/aorta for high cholesterol/rosiglitazone (Ro) (n = 6); and 5.80 ± 0.70 mg/aorta for high cholesterol/GW7845 (GW) (n = 6). Data are in triplicates and expressed as mean ± SEM.