Low ambient oxygen prevents atherosclerosis

Abstract

Large population studies have shown that living at higher altitudes, which lowers ambient oxygen exposure, is associated with reduced cardiovascular disease mortality. However, hypoxia has also been reported to promote atherosclerosis by worsening lipid metabolism and inflammation. We sought to address these disparate reports by reducing the ambient oxygen exposure of ApoE-/- mice. We observed that long-term adaptation to 10% O2 (equivalent to oxygen content at ∼5000 m), compared to 21% O2 (room air at sea level), resulted in a marked decrease in aortic atherosclerosis in ApoE-/- mice. This effect was associated with increased expression of the anti-inflammatory cytokine interleukin-10 (IL-10), known to be anti-atherogenic and regulated by hypoxia-inducible transcription factor-1α (HIF-1α). Supporting these observations, ApoE-/- mice that were deficient in IL-10 (IL10-/- ApoE-/- double knockout) failed to show reduced atherosclerosis in 10% oxygen. Our study reveals a specific mechanism that can help explain the decreased prevalence of ischemic heart disease in populations living at high altitudes and identifies ambient oxygen exposure as a potential factor that could be modulated to alter pathogenesis. Key messages: Chronic low ambient oxygen exposure decreases atherosclerosis in mice. Anti-inflammatory cytokine IL-10 levels are increased by low ambient O2. This is consistent with the established role of HIF-1α in IL10 transactivation. Absence of IL-10 results in the loss of the anti-atherosclerosis effect of low O2. This mechanism may contribute to decreased atherosclerosis at high altitudes.

Keywords: Anti-inflammatory; Atherosclerosis; Hypoxia; IL-10; Oxygen.

Fig. 1

Low ambient oxygen reduces atherosclerosis and inflammation in ApoE−/− mice. ApoE−/− mice (5–6 wk old) were maintained in room air (21% O₂) or placed in a hypoxia chamber (10% O₂) for 22 wk prior to analyses (except for 1e). a The aortas were longitudinally dissected and atherosclerotic plaque areas (red) were quantified after staining with Sudan IV. Shown are representative images of the aortic arch where most of the plaques develop (n=11). Scale bars: 1 mm. b Aortic root cross sections were stained with Oil Red O and the stained plaque areas were measured (n=10–11). Representative aortic root sections are shown. Scale bars: 200 μm. c ApoE−/− mice were exposed to the indicated ambient oxygen for 22 wk and the proinflammatory cytokine level in plasma were measured by multiplex immunoassay kit (Meso Scale Discovery) (n=5). Cytokines that can serve as Th1 markers are indicated in brackets. d Levels of plasma cytokines that can serve as markers of other Th subtypes (indicated in brackets) were measured by individual immunoassay kits (Thermo and eBioscience) (n=5). e Effect of hypoxia on both basal and LPS-stimulated plasma cytokine levels in ApoE−/− mice. These mice were exposed to the indicated ambient oxygen for 5 wk prior to i.p. injection of LPS (n=5–11). f Hypoxia responsive VEGF and anti-inflammatory cytokine IL10 gene expression in aortic tissue were measured by real-time RT-PCR (n=6). Values shown as mean ± SEM, *p < 0.05.

Fig. 2

Hypoxia increases anti-inflammatory cytokine IL-10 expression in vivo and in vitro. a Mouse CD4+ T cells were isolated from spleens of ApoE−/− mice chronically adapted to the indicated ambient oxygen condition and the mRNA levels of the following genes were measured as markers of the respective T cell subtypes: INF-γ (Th1); IL-4 (Th2); IL-10 (Th2 and Treg); IL-17 (Th17); and FOXP3 (Treg) (n=6). b Effect of hypoxia (5% O₂, 1 to 3 d exposure) on IL-10 mRNA levels in cultured ApoE−/− mouse CD4+ T cells. Relative hypoxia was confirmed by the increase in HIF-1α protein and the expression of its target gene VEGF (n=3). c Effect of hypoxia on the time course of IL-10 expression in cultured mouse bone marrow-derived macrophages (BMDM) after lipopolysaccharide (LPS) stimulation (n=3). d IL-10 expression in human peripheral blood T cells cultured for 3 d under the indicated oxygen conditions (n=3). Values shown as mean ± SEM, *p < 0.05.

Fig. 3

IL10 gene is regulated by HIF-1α. a HIF-1α protein was stabilized by deferoxamine (DFO) in mouse CD4+ T cells in a dose-dependent manner and IL-10 mRNA levels quantified by real-time RT-PCR (n=3). b Hypoxia (5% O₂) induced HIF-1α protein was knocked down in mouse CD4+ T cells using shRNA and IL-10 mRNA levels were measured. Nonspecific shRNA (NS) was used as control (n=3). c Hypoxia (5% O₂) induced HIF-1α protein was knocked down in the Raw 264.7 macrophage cells using shRNA and IL-10 mRNA levels were measured. Nonspecific shRNA (NS) was used as control (n=3). d The sites of candidate hypoxia response elements (HRE, consensus sequence Pu(A/G)CGTG) in the promoter and intron 4 sequences of the mouse IL10 gene are numbered in shaded boxes. HIF-1α interaction with the putative HRE sites was examined by chromatin immunoprecipitation and quantified using real-time PCR in mouse CD4+ cells cultured in a 5% O₂ incubator. Values shown as mean ± SEM, *p < 0.05.

Fig. 4

IL-10 is necessary for the decrease in atherosclerosis in low ambient oxygen. a Effect of IL-10 on aortic atherosclerotic plaque development in ApoE−/− mice chronically adapted to hypoxia. Mice of the indicated genotypes were maintained in either 21% O₂ or 10% O₂ for 22 wk. Atherosclerosis was quantified by staining whole aorta with Sudan IV (red, representative images). Scale bars: 1 mm. b Representative cross sections of the aortic root at the valve leaflet level were stained with H&E to show differences in the arterial wall (neointima and smooth muscle layers) depending on ambient oxygen exposure and IL-10 genotype. Scale bars: 200 μm. c Plasma IFN-γ levels were measured as marker of inflammatory activity in mice exposed to 10% or 21% O₂ for 22 wk (n=5–7). **p< 0.01, one-way ANOVA with a Tukey posttest.