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. Author manuscript; available in PMC: 2013 Mar 7.
Published in final edited form as: Cell Metab. 2012 Mar 7;15(3):372–381. doi: 10.1016/j.cmet.2012.01.018

FoxOs Integrate Pleiotropic Actions Of Insulin In Vascular Endothelium To Protect Mice From Atherosclerosis

Kyoichiro Tsuchiya 1, Jun Tanaka 2, Yu Shuiqing 1, Carrie L Welch 1, Ronald A DePinho 3, Ira Tabas 1,4,5, Alan R Tall 1,4,5, Ira J Goldberg 1, Domenico Accili 1
PMCID: PMC3315846  NIHMSID: NIHMS355382  PMID: 22405072

Abstract

Atherosclerotic cardiovascular disease is the leading cause of death in insulin-resistant (type 2) diabetes. Vascular endothelial dysfunction paves the way for atherosclerosis through impaired nitric oxide availability, inflammation, and generation of superoxide. Surprisingly, we show that ablation of the three genes encoding isoforms of transcription factor FoxO in endothelial cells prevents atherosclerosis in Low-density lipoprotein receptor knockout mice by reversing these sub-phenotypes. Paradoxically, the atheroprotective effect of FoxO deletion is associated with a marked decrease of insulin-dependent Akt phosphorylation in endothelial cells, owing to reduced FoxO-dependent expression of the insulin receptor adaptor proteins, Irs1 and Irs2. These findings support a model in which FoxO is the shared effector of multiple atherogenic pathways in endothelial cells. FoxO ablation lowers the threshold of Akt activity required for protection from atherosclerosis. The data demonstrate that FoxO inhibition in endothelial cells has the potential to mediate wide-ranging therapeutic benefits for diabetes-associated cardiovascular disease.

Treatment advances in type 2 diabetes have significantly curtailed the prevalence, severity, and costs associated with its microvascular complications (National Institute of Diabetes and Digestive and Kidney Diseases, 2005). Disappointingly, progress in treating atherosclerotic macrovascular complications lags behind, partly because–unlike microvascular disease–their development appears to be impervious to glycemia control (U.K. Prospective Diabetes Study Group, 1998). Indeed, considerable epidemiological evidence indicates that insulin resistance can promote atherosclerosis independent of hyperglycemia (Despres et al., 1996). Endothelial dysfunction is the bellwether of atherosclerosis. And a wealth of cellular studies has shown that insulin acts in endothelial cells (EC) to generate nitric oxide (NO), prevent inflammation, inhibit reactive oxygen species, and decrease monocyte recruitment (Du et al., 2006; Kim et al., 2006; Kuboki et al., 2000). These findings are borne out by the demonstration that insulin resistance caused by genetic ablation of insulin receptor (InsR) predisposes to endothelial dysfunction and aortic lipid accumulation in Apoe−/− mice (Rask-Madsen et al., 2010), as does Akt1 ablation (Fernandez-Hernando et al., 2007). Given the pleiotropic actions of insulin, and the panoply of Akt substrates, it seems that the anti-atherogenic effects of Akt could be mediated by multiple substrates.

FoxO transcription factors (encoded by Foxo1, Foxo3a, and Foxo4) are Akt substrates that regulate metabolic pathways in different tissues (Accili and Arden, 2004). In EC, FoxO1 and FoxO3a repress transcription of endothelial nitric oxide synthase (eNos) (Potente et al., 2005), and promote inducible Nos (iNos) expression in response to oxidative stress, leading to generation of peroxynitrite and endothelial dysfunction (Tanaka et al., 2009). These observations suggest that FoxO mediates aspects of endothelial dysfunction that are conducive to atherogenesis. But a direct test of this hypothesis, and genetic analyses of physiologically relevant FoxO substrates in vivo have not been conducted thus far. Three factors have prevented rigorous insight into this issue: the presence of three FoxO isoforms (1, 3a, and 4) with overlapping functions in EC; the developmental role of FoxOs in angiogenesis (Kitamura et al., 2007), which might be expected to thwart the successful generation of EC-specific gene deletions; and the pleiotropism of InsR and Akt functions, which are unlikely to be relayed exclusively through FoxOs.

Seeking to fill this gap in knowledge, we generated triple mutant mice lacking FoxO1, 3a, and 4 in endothelial cells, and demonstrate that the triple knockout largely prevents aortic lesion development in mice lacking Ldl receptor (Ldlr), indicating that FoxO integrate Akt signals to mediate multiple actions of insulin required for endothelial function.

Results

Western diet promotes early-onset insulin resistance and FoxO activation

We first interrogated changes in aortic FoxO activity during the progression of atherosclerosis in Ldlr−/− mice fed Western-type diet (WTD) for various lengths of time. Six weeks of WTD resulted in hyperglycemia, hyperinsulinemia, dyslipidemia (Figure 1A, Table S1), and aortic lipid accumulation, as detected by Oil Red-O staining in Ldlr−/− mice (Figures 1B and 1C). In aortae excised from WTD-fed Ldlr−/− mice following insulin stimulation, InsR, Akt, FoxO1, and FoxO3a phosphorylation were significantly lower than in chow-fed mice, while Erk phosphorylation was enhanced. The ratio of phosphorylated eNOS to total eNOS was also decreased (Figures 1D and 1E). Irs1 and Irs2 protein levels tended to decrease. After 20 weeks on WTD, metabolic abnormalities (Figure 1F, Table S1) and lipid accumulation worsened considerably (Figures 1G and 1H), and were associated with further reductions of Akt, eNOS, FoxO1, and FoxO3a phosphorylation, as well as Irs1 and Irs2 levels (Figures 1I and 1J). These data suggest that FoxO activation in response to vascular insulin resistance occurs early in the pathogenesis of atherosclerosis. To pinpoint mechanisms leading to decreased FoxO1 phosphorylation, we performed experiments in primary cultures of murine aortic EC. Incubation with LPS or FFA inhibited insulin-induced Akt, eNOS (Kim et al., 2005), and FoxO1 phosphorylation, increasing the latter's nuclear localization (Figures S1A–D), and indicating that FoxO1 activation can occur in response to endothelial cell stress.

Figure 1. Insulin signaling in aortae of WTD-fed Ldlr−/− mice.

Figure 1

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(A) Glucose tolerance tests.

(B-C) Representative Oil Red-O staining of en face aorta preparations (B) and quantification of lesion area (C) (n=3-4 for each genotype).

(D-E) Representative immunoblots (D) and quantification (E) of insulin signaling, eNOS, and Erk1/2 in aortae isolated 5 min after intravenous insulin injection from mice fed standard (SD) or Western diet (WTD) for 6 weeks. We determined band intensity from three independent experiments.

(F-J) We carried out similar experiments in mice fed SD or WTD for 20 weeks. Data indicate means ± SEM. * P< 0.05, ** P< 0.01, *** P< 0.001 vs. chow.

Mice lacking FoxO in EC are protected from vascular dysfunction and atherosclerosis

To probe the role of FoxO in atherosclerosis, we used Tie2-cre–mediated recombination to generate mice lacking all FoxO isoforms in vascular EC (henceforth, Vascular Endothelial Cell KnockOut, VECKO) (Paik et al., 2007). Genotyping and mRNA analyses indicated successful recombination at the three loci and a >96% reduction of the 3 mRNAs (data not shown), and of FoxO1 and FoxO3a protein levels in aortic (Figure 2B), cardiac, and lung EC (data not shown). FoxO4 protein levels were undetectable by western blot in control Ldlr−/− mice, even though mRNA studies indicated that the gene is transcribed.

Figure 2. Glucose metabolism, vascular function, and atherosclerosis in VECKO; Ldlr−/− mice.

Figure 2

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(A-B) Foxo1, 3a, and 4 mRNA (A) or FoxO1 and 3a protein levels (B) in cultured aortic EC from VECKO and control mice.

(C-D) Glucose (C) and insulin tolerance tests (D) in VECKO; Ldlr−/− and Ldlr−/− mice after 18-19 weeks on WTD (n=7-10).

(E-F) Acetylcholine– (E) and Na nitroprusside–induced (F) vasorelaxation in femoral arteries from VECKO; Ldlr−/− and Ldlr−/− mice after 18 weeks on WTD (n=5). Data indicate means ± SEM.

* P< 0.05, ** P< 0.01, *** P< 0.001 vs. WT or Ldlr−/−.

We investigated the effects of FoxO ablation in EC on vascular function and atherosclerosis by generating double mutant VECKO; Ldlr−/− mice and assessing their response to WTD. Following 18-20 weeks on WTD, VECKO; Ldlr−/− and Ldlr−/− controls had similar glucose tolerance, insulin sensitivity (Figures 2C and 2D), body weight, plasma insulin, total and HDL cholesterol, triglyceride, and FFA levels (Table S2). These data indicate that, unlike endothelial Irs2 ablation (Kubota et al., 2011), Foxo ablation doesn't affect systemic insulin sensitivity.

We didn't detect differences in systolic blood pressure before or after WTD (not shown). But WTD-fed VECKO; Ldlr−/− mice showed enhanced eNOS-dependent arterial relaxation, with a 4.5-fold decrease in the EC50 of endothelial NO-dependent vasodilatation in response to acetylcholine (Ach) compared to Ldlr−/− littermates (7.9 ± 1.6 vs. 36 ± 6.3 nM, P= 0.003) (Figure 2E). In contrast, we detected no differences in endothelium-independent vasodilatation in response to Na nitroprusside (9.6 ± 2.3 vs. 12 ± 3.9 nM in VECKO; Ldlr−/− vs. Ldlr−/−) (Figure 2F). These data indicate that FoxO ablation increases NO-dependent vasodilatation following WTD.

Multiple analyses consistently indicated a striking decrease of atherosclerosis in VECKO; Ldlr−/− mice compared to Ldlr−/− controls. Oil Red-O staining of en face dissected aortae revealed a 77% reduction in lesion area, with comparable reductions in the aortic arch and descending aorta (76% and 74%, respectively) (Figures 3A and 3B). In addition, we detected lower collagen content in thoracic and brachiocephalic aorta (Figure 3C). Plaques in the thoracic aorta of Ldlr−/− mice demonstrated evidence of smooth muscle cell migration and macrophage infiltration, as detected by immunostaining with α-smooth muscle actin and Mac-3, respectively (Figure 3D). In contrast, VECKO; Ldlr−/− mice showed little evidence of either increased smooth muscle cells or lesional macrophages (Figure 3D), In addition, we detected lower Vcam1 immunoreactivity in lesional endothelium of aortae from VECKO; Ldlr−/− mice (Figure 3E), consistent with reduced expression of cell adhesion molecules. The number of coronary lesions decreased by 80% in VECKO; Ldlr−/− mice (Figure 3F).

Figure 3. Aortic and coronary immunohistochemistry.

Figure 3

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(A-B) Representative en face Oil Red-O staining (A) and quantification (B) of lesion area in total, aortic arch, and descending thoracic aortae in VECKO; Ldlr−/− and Ldlr−/− mice after 20 weeks WTD (n=11-13).

(C) Histological analysis of the brachiocephalic and thoracic aorta with hematoxylin and eosin (HE) and Masson-Trichrome staining. Values indicate quantification of atherosclerotic lesions and positive area as % of total area, respectively (n=4).

(D) Immunostaining of thoracic aorta with α-smooth muscle actin and Mac-3 antibodies.

(E) Immunostaining of thoracic aorta with Vcam-1 antibody. Values indicate quantification of immunoreactive areas as % of total area (n=4).

(F) Histological analysis of coronaries by HE staining. Values indicate plaque-positive coronaries as % of total coronaries scored in 4 independent sections of each mouse (n=8). Data indicate means ± SEM.* P< 0.05, ** P< 0.01, and *** P< 0.001 vs. Ldlr−/−. Scale bars: 200μm

In view of the reported off-target recombination induced by Tie2-Cre in bone marrow (BM)-derived cells (Murdoch et al., 2007), we determined the contribution of bone marrow-derived cells to the observed phenotype. Proteins levels of FoxO1 and FoxO3a were within normal range in BM and peripheral blood mononuclear cells (Figures S2A and S2B), as were Foxo1, 3a and 4 mRNA levels in peritoneal macrophages (Figure S2C). To rule out a contribution from BM-derived cells to the observed atheroprotective phenotype, we carried out BM transplantation experiments. We transferred BM obtained from Ldlr−/− mice into VECKO; Ldlr−/− or control Ldlr−/− mice. After 14 weeks on WTD, VECKO; Ldlr−/− mice that received BM from Ldlr−/− mice showed substantial reduction in total lesion area compared to Ldlr−/− mice that received heterologous BM from Ldlr−/− mice (Figures S2D and S2E). The extent of reduction was the same as in the experiments in Figure 3. These results exclude potential involvement of hematopoietic cells and macrophages in protecting VECKO; Ldlr−/− mice from atherosclerosis.

These observations indicate that endothelial Foxo ablation exerts atheroprotective effects in Ldlr−/− mice, independent of plasma lipid or glucose levels, and in a BM-independent fashion. The histopathology data suggest that pleiotropic mechanisms account for this difference–including increased NO production and decreased macrophage content in lesions. To clarify the underlying mechanisms, we performed further experiments in primary EC cultures.

Increased NO production in EC from VECKO mice

Consistent with the increased Ach-induced (NO-dependent) vascular relaxation, we saw a marked increase of eNos mRNA and protein in VECKO aortic EC (Figures 4A-C), aortic extracts (Figures 4F and 4G), heart (Figures S3A-C), and cardiac EC (Figures S3D and S3E). Relative amounts of eNos dimer, a readout of functional eNos (Zou et al., 2002), were comparable between groups (Figures 4D and 4E). Basal eNos phosphorylation on Ser1176 was increased (Figures 4B, 4C, 4F and 4G), while levels of insulin-induced phosphorylation were similar between the two genotypes. As a result, the ratio of eNos phosphorylated on Ser1176 to total eNos decreased in aortic EC and whole aortae from VECKO mice (Figures 4B, 4C, 4F and 4G). Similar results were observed in whole heart and cardiac EC (Figures S3B, S3C, S3E and S3F). Basal NO production was markedly higher in aortic (Figures 4H and 4I) and cardiac EC (Figures S3G and S3H) from VECKO mice, but was not stimulated further by insulin. Pretreatment with the eNOS inhibitor L-NAME inhibited insulin-stimulated NO production in aortic EC (Figures 4H and 4I), suggesting that increased NO production in VECKO ECs is eNOS-dependent.

Figure 4. eNOS expression and NO production in aortic EC and aorta.

Figure 4

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(A) eNos levels in cultured aortic EC from VECKO and control mice (n=4).

(B-C) Representative immunoblots (B) and quantification (C) of p-eNOS-S1176 and total eNOS in cultured aortic EC from VECKO and control mice following insulin treatment (n = 3). The loading control is shown in Figure 6A.

(D-E) Representative immunoblots (D) and quantification (E) of eNOS dimer and monomer in cultured aortic EC from VECKO and control mice analyzed by low-temperature SDS-PAGE immunoblotting (n = 4).

(F-G) Representative immunoblots (F) and quantification (G) of p-eNOS-S1176 and total eNOS in cultured aortic EC from VECKO and control mice (n = 3). The loading control of Akt is shown in Figure 6A. Scale bar: 200μm.

(H-I) Insulin-stimulated NO production in cultured aortic EC from VECKO and control mice pretreated with L-NAME (0.5mM) or vehicle. NO production visualized by DAF2-DA fluorescence (H) and quantified in (I). Data indicate means ± SEM. AU= arbitrary mRNA units. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. controls.

Blunted expression of cell adhesion molecules in VECKO EC

Monocyte recruitment by activated EC promotes atherosclerosis and is prevented by insulin (Nigro et al., 2006). We measured basal and LPS-induced levels of mRNA encoding adhesion molecules. LPS-induced Icam1 and Vcam1 expression was blunted in cells from VECKO mice (Figure 5A). To establish a causal relationship between FoxO loss-of-function and Vcam or Icam levels, we probed the ability of a FoxO1 gain-of-function mutant to regulate these genes in primary cultures of Human Aortic Endothelial Cells (HAEC). The constitutively active FoxO1 mutant lacking key phosphorylation sites, FoxO1-ADA (Nakae et al., 2003), increased Icam1 and Vcam1 expression in a dose-dependent manner (Figures S4A and S4B). To assess the functional consequences of these increases, we measured monocyte adhesion to cultured HAEC. FoxO1-ADA expression enhanced adhesion of THP-1 cells to HAEC, and neutralizing antibodies to Icam1 and Vcam1 reversed this effect (Figures S4C and S4D). To determine whether Icam1 and Vcam1 are direct targets of FoxO1, we performed reporter gene and chromatin immunoprecipitation (ChIP) assays. FoxO1-ADA increased expression of a reporter gene under the control of ICAM1 and VCAM1 promoters in human umbilical vein EC, whereas a DNA-binding deficient FoxO1-ADA (FoxO1-ADA-DBD) (Kitamura et al., 2005) did not (Figures S4E and S4F). Furthermore, ChIP assays showed that FoxO1 binds to cis-acting DNA elements in the ICAM1 and VCAM1 promoters in intact chromatin isolated from HAEC (Figure S4G).

Figure 5. Pro-inflammatory cytokine expression, NF-κB signaling, and oxidative stress in aortic or lung EC.

Figure 5

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(A) Time courses of Icam1, Vcam1, Mcp1, iNos, IL-1β, and IL-6 induction in aortic EC from VECKO and control mice. Basal (without LPS stimulation) gene expression in control cells is set to 1.

(B) Immunoblot analysis of lung EC from VECKO and control mice following stimulation with LPS for the indicated times.

(C) NF-κB luciferase activity in lung EC from VECKO and control mice transduced with adenovirus encoding Nf-κB–responsive luciferase reporter construct followed by LPS stimulation (n=4).

(D-E) Free cholesterol (FC)- and H2O2-stimulated reactive oxygen species (ROS) production in aortic EC from VECKO and control mice visualized by DAF2-DA fluorescence (D) and quantified from 4 independent wells in (E).

(F-G) H2O2-stimulated superoxide production in aortic EC from VECKO and control mice visualized by DHE fluorescence (F) and quantified in (G).

(H) Expression of NADPH oxidase subunits in aortic EC from VECKO and control mice (n=4). Data indicate means ± SEM. AU= arbitrary mRNA units. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. controls. Scale bar: 250μm.

Reduced inflammation and reactive oxygen species in VECKO aortic EC

iNos is a FoxO1 target that promotes generation of reactive oxygen species in response to oxidative stress in vascular endothelium (Tanaka et al., 2009). Its expression was markedly blunted in aortic EC from VECKO mice, as was expression of inflammatory cytokines Mcp-1, IL-1β, and IL-6 (Figure 5A). LPS-induced Akt phosphorylation promotes Nf-κB activation through Ikk phosphorylation (Romashkova and Makarov, 1999; Salmeron et al., 1996) and was likewise reduced in EC from VECKO mice (Figure 5B). Consistently, LPS-induced p105 degradation, IκBα phosphorylation, and Nf-κB-luciferase activity were attenuated (Figures 5B and 5C). These data indicate that Foxo deletion in EC suppresses inflammation, possibly via direct transcriptional regulation and/or its ability to decrease NF-κB activity (Senokuchi et al., 2008).

Production of superoxide and ROS by EC is proatherogenic, and can be brought about by oxidative stress (Du et al., 2006). Although in later stages of atherosclerosis macrophages are the main source of ROS, production of ROS can be demonstrated in early atherosclerotic lesions prior to macrophage recruitment (Napoli et al., 1997). Moreover, eNos overexpression in EC can cause eNos dysfunction, increase superoxide production and promote atherogenesis in Apoe−/− mice (Ozaki et al., 2002). We therefore asked whether the elevated basal levels of eNos in aortic EC from VECKO mice affected ROS production. ROS/peroxinitrite production by VECKO aortic EC in response to free cholesterol (FC) and H2O2 was significantly lower than controls (Figures 5D and 5E), as was H2O2–induced superoxide generation (Figures 5F and 5G). In addition, mRNAs encoding NADPH oxidase subunits p22phox, p67phox, and gp91phox–whose activity is required for ROS generation–were significantly decreased (Figure 5H). These data support the view that increased eNos levels in VECKO mice are atheroprotective, and that the failure to induce iNos prevents ROS generation. Thus, it's likely that the increased sensitivity of VECKO arteries to Ach-induced vasodilatation is secondary to greater NO bioavailability.

Decreased inflammatory and oxidative responses in VECKO; Ldlr−/− mice

We measured expression of a panel of inflammatory cytokines, chemokines, adhesion proteins, and NADPH oxidase components on RNA isolated from aortae of VECKO; Ldlr−/− and Ldlr−/− mice. iNos and Vcam1 were significantly reduced in VECKO; Ldlr−/− aortae compared to Ldlr−/− controls (Figure S4H), as were IL-6 and NADPH oxidase subunits p22phox and gp91phox. Icam1 expression tended to be lower in VECKO; Ldlr−/− aortae, but not significantly so. Vcam1 and iNos protein levels were similarly reduced (Figures S4I and S4J). Aortic TBARS content was significantly lower in VECKO; Ldlr−/− compared to controls (Figure S4K), whereas plasma TBARS concentrations were comparable (data not shown). Thus, FoxO deletion in EC reduced iNos and Vcam1, decreased inflammation, and curtailed focal oxidative stress in aortae. These observations indicate that, in addition to increasing NO production, FoxO ablation exerts atheroprotective effects by dampening oxidative stress, inflammation, and monocyte recruitment.

Delayed cellular senescence and decreased apoptosis in EC from VECKO mice

Foxo can inhibit cellular proliferation by increasing expression of cell cycle inhibitors (Medema et al., 2000; Paik et al., 2007). Moreover, eNos-derived NO can delay cellular senescence (Hayashi et al., 2006) and prevent apoptosis (Dimmeler et al., 1997). Consistently, aortic EC from VECKO mice proliferated more rapidly (Figures S5A and S5Ba), and showed an 80% decrease in cellular senescence (Figures S5A and S5C). Apoptosis induced by Tnf–α, FFA, H2O2, 7-keto-cholesterol (7–KC), or FC decreased on average by 50% in VECKO cells (Figure S5D), and was associated with lower levels of pro-apoptotic Bim, p21, p53, and proangiogenic Spry2 than controls (Figure S5E).

Paradoxical decrease of Akt phosphorylation in FoxO–deficient aortic EC

The effects described so far are remarkably broad, and suggest that insulin sensitivity in FoxO-deficient EC should be increased. But when we characterized insulin signaling in aortic EC from VECKO mice, we found the opposite. Total levels and insulin-induced tyrosine phosphorylation of the insulin receptor were preserved. But expression of the main InsR substrates, Irs1 and Irs2, was nearly undetectable (Figures 6A and 6B). Consequently, insulin-stimulated Akt phosphorylation on Ser473 and Thr308 and Erk phosphorylation were greatly decreased (Figures 6A and 6B). Likewise, levels of Igf1R were decreased, possibly contributing to the decrease in Akt and Erk phosphorylation, since insulin receptor and Igf1R engage in hybrid receptor formation in EC (Chisalita and Arnqvist, 2004). To rule out artifacts occurring during EC isolation, we also measured these parameters in vivo in whole aorta extracts, and detected decreases of Irs1 and Igf1R expression, and attenuated insulin-induced Akt phosphorylation (Figures S6A and S6B). The changes in whole aortae are not as marked as in cultured EC, reflecting the heterogeneous composition of this tissue.

Figure 6. Insulin signaling in aortic EC of VECKO mice.

Figure 6

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(A-B) Representative immunoblots (A) and quantification (B) of insulin signaling in cultured aortic EC from VECKO following insulin treatment.

(C) Irs1, Irs2 and Igf1r expression in cultured aortic EC from VECKO and control mice. AU= arbitrary mRNA units.

(D) Illustration of conserved putative FoxO-binding sequences (red) in the Irs1 promoter and primers used for amplification of region A and B during ChIP. TSS: transcription start site.

(E) ChIP assay of cultured aortic EC using immunoprecipitation (IP) with rabbit IgG or antibody against FoxO1 and RNA polymerase (pol) II. Size of predicted PCR products sizes is 88 and 208 bp, respectively. The asterisk on the gel indicates a non-specific band. Data indicate means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. controls.

Irs2 is a known FoxO target (Ide et al., 2004), and single FoxO1 knockout in liver decreases its expression (Matsumoto et al., 2007). The transcription factors that regulate Irs1 expression are unknown (Kubota et al., 2011). Irs1 mRNA levels were significantly decreased in aortic EC from VECKO mice, as were Irs2 and Igf1R levels (Figure 6C). We investigated whether FoxO binds to the Irs1 promoter. The mouse Irs1 promoter contains three conserved forkhead DNA binding sites in two regions, A and B (Figure 6D). ChIP analysis revealed that FoxO1 binds to both regions in aortic EC (Figure 6E). In cardiac microvascular EC from VECKO mice the decrease of Irs1 was less marked than in aortic EC (Figures S6C and S6D), and ChIP analysis showed that FoxO1 binds exclusively to region A (Figure S6E). These data reveal that Irs1 is a FoxO target gene in aortic endothelial cells.

Discussion

Atherosclerotic macrovascular complications account for nearly half of diabetes-related deaths and expenditures, and show an indifferent response to available anti-hyperglycemic drugs (Caro et al., 2002). In addition, regulatory requirements for registration of new anti-diabetic drugs in the U.S. and Europe have made it unlikely that further progress in diabetes treatment can be divorced from advances in treating its cardiovascular comorbidities (Joffe et al., 2010). This study advances our pathophysiological understanding and potential therapeutic thinking on the atherosclerotic complications of diabetes in two ways: it discloses an unexpectedly broad role of FoxO as mediators of insulin action on NO biogenesis, inflammation, lesional macrophage content, and ROS or superoxide production. And it provides a critical test of the hypothesis that treating endothelial insulin resistance may be sufficient to reduce atherosclerosis.

The preventive effect of FoxO ablation on atherosclerosis likely ensues from its ability to increase cell survival and NO availability, and dampen inflammation, chemotaxis, and superoxide generation. The effect of FoxO on eNos has been reported (Potente et al., 2005), but its pathophysiological consequences on atherosclerosis were hardly predictable. In fact, eNos overexpression has been shown to cause eNos dysfunction and superoxide production in Apoe−/− mice, resulting in accelerated atherosclerosis (Ozaki et al., 2002), while another study showed reduced lesion formation (van Haperen et al., 2002). Our findings indicate that eNos induction is beneficial in the context of FoxO ablation, possibly because of FoxO's dual role as repressor of eNos transcription and activator of iNos transcription. In VECKO mice, combined inhibition of iNos and NAPDH oxidase subunits that are required for ROS production during oxidative stress (Giacco and Brownlee, 2010; Tanaka et al., 2009), likely contributes to reduced superoxide production. Thus, any NO generated in FoxO-deficient cells is unlikely to be used for ROS production. Interestingly, expression of dominant-negative InsR in EC of transgenic mice increased NADPH oxidase expression and superoxide production (Duncan et al., 2008), consistent with a FoxO-dependent effect.

An additional component of the atheroprotective mechanism of FoxO ablation is its antiinflammatory effect, leading to decreased NF-κB activity and Icam1 and Vcam1 expression. The latter probably result from a combination of direct transcriptional effects of FoxO, and decreased NF-κB, which has been shown to modulate their expression (Neish et al., 1992). Increased NO can also contribute to the anti-inflammatory responses in VECKO EC by stabilizing the NF-κB/IκBα complex (Peng et al., 1995). Furthermore, previous reports demonstrated that Erk mediates MCP-1 and Vcam-1 expression in HUVEC (Cho et al., 2002; Modur et al., 1996). Therefore decreased Erk phosphorylation in VECKO EC can also contribute to attenuate inflammatory responses.

The association of these sub-phenotypes with the cellular effect of FoxO ablation on insulin signaling is most remarkable, as it provides prima facie evidence of a healthy EC that is virtually devoid of insulin signaling through Akt and FoxO. While previous genetic rescue experiments certainly pointed to this conclusion–for example, in the rescue of Irs1/lrs2 double mutants by FoxO1 ablation (Dong et al., 2008)–our findings add a pathophysiological dimension to these observations by demonstrating that Irs1 and Irs2 are FoxO targets. These findings can be construed to suggest that Akt's primary role is to keep FoxO in check, a conclusion supported by the compensatory increase in Akt activity observed in FoxO gain-of-function experiments (Chen et al., 2010; Matsumoto et al., 2006). But what is FoxO's role, then? We can extrapolate from work on pancreatic β-cells to propose that FoxO provides acute, but not chronic reprieve from metabolic stress (Kitamura et al., 2005). Thus, insulin signaling through FoxO could be viewed evolutionally as an acute stress response that goes awry when inciting factors (hyperglycemia, dyslipidemia, or inflammation) become entrenched. Physiologically, the likeliest role of this mechanism is to fine-tune insulin signaling, buffering EC against fluctuating plasma insulin levels. This interpretation is consistent with the fact that, while triple Foxo ablation is atheroprotective, FoxO1 activation by glucose, cytokines or FFA increases iNos production and ROS/superoxide generation, indicating that the three FoxO are likely responsible for accelerated atherosclerosis in mice lacking InsR (Rask-Madsen et al., 2010) or Akt1 (Fernandez-Hernando et al., 2007), as well as for endothelial dysfunction elicited by oxidative stress (Duncan et al., 2008; Vicent et al., 2003).

Our findings are also consistent with and expand the conclusions reached in studies of Irs2 EC-specific knockouts (Kubota et al., 2011). These workers proposed that reduced insulin-induced eNOS phosphorylation in EC-specific Irs2−/− mice causes insulin resistance via impaired capillary recruitment in skeletal muscle. In VECKO mice, Irs1 and Irs2 expression decreases, but eNOS expression is increased. The increase in total eNos levels offsets the decrease in Akt-induced eNos phosphorylation and likely allows FoxO-deficient EC to maintain elevated levels of NO production. The phosphor-eNos to total eNOS ratio is 60% lower in VECKO EC than controls, possibly due to reduced Akt activity. But actual NO production is increased, reflecting absolute levels of phosphorylated eNos, rather than phosphor-eNos/eNOS ratios. These data indicate that the biological outcome–NO production–depends on absolute phosphor-eNOS levels, not on the ratio of phosphorylated to total eNos (Fulton et al., 1999). Thus, we propose that the increase of eNOS in EC from VECKO mice protects them from systemic insulin resistance by increasing NO production, despite decreased Irs1 and 2 expression. Whether the increase in NO generation has additional local effects on insulin signaling in other tissues (e.g., liver, pancreatic islets, CNS) is a question of paramount interest that will be addressed systematically in future studies.

In conclusion, we demonstrate a remarkable atheroprotective effect of FoxO inhibition in mouse EC, and identify endothelial FoxO as a potential target for atherosclerosis prevention and treatment.

Experimental Procedures

Additional details of the Experimental Procedures are included in the Supplemental Information.

Animals

Foxo1F/F3F/F4F/F mice have been described (Haeusler et al., 2010; Paik et al., 2007). We generated vascular EC-specific triple FoxOs knockout mice (VECKO) by mating Foxo1F/F3F/F4F/F mice with Tie2-Cre male mice (Kisanuki et al., 2001) to avoid non-specific deletion of floxed alleles by germ-line activation (de Lange et al., 2008). We then crossed VECKO and Ldlr−/−mice to generate VECKO:Ldlr−/− mice. Atherosclerosis was induced by Western diet (WTD, 0.2% cholesterol, 42% from fat adjusted calorie diet, TD 88137, Harlan Tekland) for indicated periods. All experiments were conducted using only male Cre(+) mice and littermate Cre(−) (control) mice. The Columbia University Animal Care and Utilization Committee approved all procedures.

Cell culture

Aortic EC isolation and culture were performed according to protocols as described previously with some modifications (Harja et al., 2008; Kevil and Bullard, 2001; Kobayashi et al., 2005; Rask-Madsen et al., 2010). Cardiac and lung microvascular EC isolation and culture were performed as described (Rask-Madsen et al., 2010).

Metabolic Analysis

We measured blood glucose with a glucometer (One Touch Ultra, Lifescan), plasma insulin by ELISA (Millipore), triglyceride (Cayman chemical), cholesterol, and nonesterified fatty acids by colorimetric assays (Cholesterol E and NEFA C, Wako Pure Chemicals). Intraperitoneal glucose (1g kg−1) and insulin (1U kg−1) tolerance tests were performed as described (Nakae et al., 2002).

Atherosclerotic Lesion Analysis

Aortas were pinned on silicon dishes and stained for lipids using Oil Red-O. Areas were quantified using Image J software and expressed as the percentage of the total aorta area. Hearts were isolated and fixed in phosphate-buffered formalin then dehydrated and embedded in paraffin.

Statistical Analysis

Comparisons were made using paired or unpaired t test, as appropriate. Bonferroni post-hoc tests were utilized. Results are presented as means ± SEM or 95% CI as appropriate. P < 0.05 was considered statistically significant.

Supplementary Material

01

Highlights.

  • Knockout of the three Foxo genes prevents atherosclerosis in mice

  • FoxO inactivation increases NO production, reduces inflammation and oxidative stress

  • Akt activation by insulin is impaired in FoxO-deficient endothelial cells

  • FoxOs control Irs1 and Irs2 expression in endothelial cells

Acknowledgments

K.T. is the recipient of a postdoctoral fellowship for research abroad from the Japan Society for the Promotion of Science. This work was supported by National Institutes of Health grants HL-87123 to DA, IAT, and ART, and DK63608 (Columbia University Diabetes Research Center). We thank C. Rask-Madsen and G. King (Joslin Diabetes Center) for help with aortic endothelial cell isolation, M. Westerterp for help with en face aortic preparations, and members of the Accili, Tabas and Tall laboratories for discussion of the data and critical reading of the manuscript.

Footnotes

Authors' Contributions: K.T. designed and executed experiments, analyzed data, performed statistical analyses and wrote the manuscript. J.T and S.Y performed experiments, analyzed the data. C.L.W, R.A.D, I.A.T., A.R.T., I.J.G, and D.A. designed experiments, analyzed the data, oversaw research, wrote the manuscript.

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