Increased atherosclerosis and endothelial dysfunction in mice bearing constitutively deacetylated alleles of Foxo1 gene

Abstract

Complications of atherosclerosis are the leading cause of death of patients with type 2 (insulin-resistant) diabetes. Understanding the mechanisms by which insulin resistance and hyperglycemia contribute to atherogenesis in key target tissues (liver, vessel wall, hematopoietic cells) can assist in the design of therapeutic approaches. We have shown that hyperglycemia induces FoxO1 deacetylation and that targeted knock-in of alleles encoding constitutively deacetylated FoxO1 in mice (Foxo1(KR/KR)) improves hepatic lipid metabolism and decreases macrophage inflammation, setting the stage for a potential anti-atherogenic effect of this mutation. Surprisingly, we report here that when Foxo1(KR/KR) mice are intercrossed with low density lipoprotein receptor knock-out mice (Ldlr(-/-)), they develop larger aortic root atherosclerotic lesions than Ldlr(-/-) controls despite lower plasma cholesterol and triglyceride levels. The phenotype is unaffected by transplanting bone marrow from Ldlr(-/-) mice into Foxo1(KR/KR) mice, indicating that it is independent of hematopoietic cells and suggesting that the primary lesion in Foxo1(KR/KR) mice occurs in the vessel wall. Experiments in isolated endothelial cells from Foxo1(KR/KR) mice indicate that deacetylation favors FoxO1 nuclear accumulation and exerts target gene-specific effects, resulting in higher Icam1 and Tnfα expression and increased monocyte adhesion. The data indicate that FoxO1 deacetylation can promote vascular endothelial changes conducive to atherosclerotic plaque formation.

FIGURE 1.

Metabolic analysis in chow-fed mice. A–D, plasma glucose, FFA, cholesterol, and TG in 14–16-month-old male Foxo1^KR/KR (KR/KR) mice and control littermates (WT) fasted for 48 h and re-fed 24 h. E and F, hepatic cholesterol and TG content in re-fed Foxo1^KR/KR (KR/KR) mice and control littermates (WT). *, p < 0.05, **, p < 0.01, n = 5–6. Data are presented as data as means ± S.E.

FIGURE 2.

Metabolic analysis in WTD-fed mice. A–D, plasma glucose, FFA, cholesterol, and TG in 14–16-month-old male Foxo1^KR/KR (KR/KR) mice and control littermates (WT) fasted for 48 h and re-fed 24 h. E and F, hepatic cholesterol and TG content in re-fed Foxo1^KR/KR (KR/KR) mice and control littermates (WT). G and H, plasma TG and cholesterol levels in 14–16-month-old male Foxo1^KR/KR (KR/KR) mice and control littermates (WT) after injection of P407. *, p < 0.05, **, p < 0.01, n = 6. Data are presented as means ± S.E.

FIGURE 3.

Metabolic and aortic root lesion analyses in WTD-fed Foxo1^KR/KR:Ldlr^−/− mice. A and B, FPLC analysis of TG and cholesterol content in lipoprotein fractions of pooled serum from Foxo1^KR/KR:Ldlr^−/− and control Ldlr^−/− mice fasted for 6 h. C, aortic root lesion size in Foxo1^KR/KR:Ldlr^−/− and control Ldlr^−/− mice without or following bone marrow transplantation (BMT). Data are presented as means ± S.E. *, p < 0.05, n = 9–21. D, apoptosis measurements in thioglycolate-elicited peritoneal macrophages isolated from WTD-fed male Foxo1^KR/KR:Ldlr^−/− mice (KR/KR) and control Ldlr^−/− mice (WT). A representative experiment is shown. E, quantification of apoptosis cells in D, n = 4. Data are presented as means ± S.E. Tg, thapsigargin; 7-KC, 7-ketocholesterol. NC, no challenge; FC, free cholesterol.

FIGURE 4.

Deacetylation of FoxO1 induced dysfunctions in vascular endothelial cells. A, human aortic endothelial cells were transduced with adenoviruses encoding GFP-tagged FoxO1 wild type (WT), Foxo1^KR/KR (KR), or S253A mutant at multiplicity of infection = 20, and FoxO1 localization was determined by fluorescence microscopy in cells cultured in the presence of 10% FBS. B, isolated lung endothelial cells were treated with oxLDL for 3 or 16 h and then stained with FoxO1 (green) and Hoechst (nuclei). C and D, gene expression analysis in mRNA isolated from lung endothelial cells derived from wild type (WT) or Foxo1^KR/KR mice (KR/KR). Cells were incubated in low glucose (LG, 5.5 mm) or high glucose (HG, 25 mm) for 11 h (C) or with oxLDL (0.1 mg/ml) for 7 h (D) prior to isolating RNA. *, p < 0.05, **, p < 0.01 versus untreated cells, n = 4 for each treatment condition. ctl, control; au, arbitrary units. E, monocyte adhesion assay on primary lung endothelial cells. Cells were pretreated with oxLDL (0.1 mg/ml) or TNFα (20 ng/ml) for 6 h before co-culture with WEHI-274.1 cells. F, quantification of adhering monocytes in E. *, p < 0.05, **, p < 0.01 versus EC from WT mice, n = 4. Data are presented as means ± S.E.