Impact of peroxisome proliferator-activated receptor γ on angiotensin II type 1 receptor-mediated insulin sensitivity, vascular inflammation and atherogenesis in hypercholesterolemic mice

Abstract

Introduction: The angiotensin II type 1 receptor (AT1R) and the peroxisome proliferator-activated receptor γ (PPARγ) have been implicated in the pathogenesis of atherosclerosis. A number of studies have reported that AT1R inhibition or genetic AT1R disruption and PPARγ activation inhibit vascular inflammation and improve glucose and lipid metabolism, underscoring a molecular interaction of AT1R and PPARγ. We here analyzed the hypothesis that vasculoprotective anti-inflammatory and metabolic effects of AT1R inhibition are mediated by PPARγ.

Material and methods: Female ApoE(-/-)/AT1R(-/-) mice were fedwith a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE(-/-) mice served as a control group. After treatment for 4 weeks mice were sacrificed and read-outs (plaque development, vascular inflammation and insulinsensitivity) were performed.

Results: Using AT1R deficient ApoE(-/-) mice (ApoE(-/-)/AT1R(-/-) mice) we found decreased cholesterol-induced endothelial dysfunction and atherogenesis compared to ApoE(-/-) mice. Inhibition of PPARγ by application of the specific PPARγ antagonist GW9662 significantly abolished the anti-atherogenic effects of AT1R deficiency in ApoE(-/-)/AT1R(-/-) mice (plaque area as % of control: ApoE(-/-): 39 ±5%; ApoE(-/-)/AT1R(-/-): 17 ±7%, p = 0.044 vs. ApoE(-/-); ApoE(-/-)/AT1R(-/-) + GW9662: 31 ±8%, p = 0.047 vs. ApoE(-/-)/AT1R(-/-)). Focusing on IL6 as a pro-inflammatory humoral marker we detected significantly increased IL-6 levels in GW9662-treated animals (IL-6 in pg/ml: ApoE(-/-): 230 ±16; ApoE(-/-)/AT1R(-/-): 117 ±20, p = 0.01 vs. ApoE(-/-); ApoE(-/-)/AT1R(-/-) + GW9662: 199 ±20, p = 0.01 vs. ApoE(-/-)/AT1R(-/-)), while the anti-inflammatory marker IL-10 was significantly reduced after PPARγ inhibition in GW9662 animals (IL-10 in pg/ml: ApoE(-/-): 18 ±4; ApoE(-/-)/AT1R(-/-): 55 ±12, p = 0.03 vs. ApoE(-/-); ApoE(-/-)/AT1R(-/-) + GW9662: 19 ±4, p = 0.03 vs. ApoE(-/-)/AT1R(-/-)). Metabolic parameters of glucose homeostasis (glucose and insulin tolerance test) were significantly deteriorated in ApoE(-/-)/AT1R(-/-) mice treated with GW9662 as compared to vehicle-treated ApoE(-/-)/AT1R(-/-) mice. Systolic blood pressure and plasma cholesterol levels were similar in all groups.

Conclusions: Genetic disruption of the AT1R attenuates atherosclerosis and improves endothelial function in an ApoE(-/-) mouse model of hypercholesterolemia-induced atherosclerosis via PPARγ, indicating a significant role of PPARγ in reduced vascular inflammation, improvement of insulin sensitivity and atheroprotection of AT1R deficiency.

Keywords: angiotensin II type 1 receptor; atherosclerosis; inflammation; insulin sensitivity; peroxisome proliferator-activated receptor γ.

Figure 1

Experimental setting. Female ApoE^–/–/AT1R^–/– mice were fed with a high-fat and cholesterol-rich diet and received continuous treatment with the selective PPARγ antagonist GW9662 or vehicle at a rate of 700 ng/kg/min for 4 weeks using subcutaneously implanted osmotic mini-pumps. Additionally, one group of female ApoE^–/– mice served as a control group and was treated with vehicle through implanted osmotic minipumps. After treatment for 4 weeks mice were sacrificed and read-outs were performed

Figure 2

Insulin sensitivity. Intraperitoneal glucose (A) and insulin tolerance tests (B) (ipGTT and ipITT) were conducted after treatment with the selective PPARγ antagonist GW9662 or vehicle in ApoE^–/–/AT1R^–/– mice that were concomitantly fed a high-fat, cholesterol-rich diet for 4 weeks. The positive effect of AT1R deficiency in ApoE^–/– mice on glucose homeostasis was abolished in GW9662-treated ApoE^–/–/AT1R^–/– mice (ipGTT (A): ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.01, ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/–/AT1R^–/– + GW9662, *p = 0.01, ipITT (A): ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.04, ApoE^–/–/AT1R^–/–+ vehicle vs. ApoE^–/–/AT1R^–/– + GW9662, *p = 0.04). Mean ± SEM, n = 4–5 per group

Figure 3

Endothelial function and atherosclerotic lesion formation. After 4 weeks aortic segments of vehicle-treated ApoE^–/–- and vehicle- and GW9662-treated ApoE^–/–/AT1R^–/– mice were isolated and their functional performance was assessed in organ chamber experiments. Endothelium-dependent vasodilation induced by carbachol is shown in Figure 3 A. Vehicle-treated ApoE^–/– mice displayed severe impairment of endothelial function compared to vehicle-treated ApoE^–/–/AT1R^–/– mice. Treatment of ApoE^–/–/AT1R^–/– mice with GW9662 antagonized the protective vascular effects of AT1R deficiency (ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.01, ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/–/AT1R^–/– + GW9662, *p = 0.01). Vehicle-treated ApoE^–/– mice displayed increased atherosclerotic lesion formation. AT1R deficiency in ApoE^–/– mice resulted in a significantly reduced area of atherosclerotic lesions, whereas GW9662 antagonized the atheroprotective effects of AT1R deficiency. Representative histological cross-sections of the aortic root were stained with oil red O to analyze atherosclerotic plaque development. B – Quantitative analysis of atherosclerotic lesion formation indicated as plaque area in % of total area is depicted in Figure 3 C (ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.044, ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/–/AT1R–/– + GW9662, *p = 0.047). Mean ± SEM, n = 4–5 per group

Figure 4

Monocyte recruitment and vascular inflammation. For immunohistochemical analysis, cryosections were assessed for the monocyte/macrophage marker MOMA-2 with an indirect immunoenzymatic method. Figure 4 A shows representative aortic root preparations of MOMA-2 staining from all animal groups and Figure 4 B shows quantification of monocyte recruitment in percent. In contrast to vehicle-treated ApoE^–/–/AT1R^–/– mice, monocyte content in atherosclerotic plaques had a tendency to more monocyte recruitment in GW9662-treated animals. Plasma IL-6 (C) and IL-10 levels (D) were determined using an ELISA kit specific for mouse. The pro-inflammatory marker IL-6 was significantly increased in GW9662-treated animals (ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.01, ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/–/AT1R^–/– + GW9662, *p = 0.01), while the anti-inflammatory marker IL-10 was significantly reduced after PPARγ inhibition (ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/– + vehicle, *p = 0.03, ApoE^–/–/AT1R^–/– + vehicle vs. ApoE^–/–/ AT1R^–/– + GW9662, *p = 0.03). Mean ± SEM, n = 4–5 per group