Chronic intermittent hypoxia induces atherosclerosis

Abstract

Rationale: Obstructive sleep apnea, a condition leading to chronic intermittent hypoxia (CIH), is associated with hyperlipidemia, atherosclerosis, and a high cardiovascular risk. A causal link between obstructive sleep apnea and atherosclerosis has not been established.

Objectives: The objective of the present study was to examine whether CIH may induce atherosclerosis in C57BL/6J mice.

Methods: Forty male C57BL/6J mice, 8 weeks of age, were fed either a high-cholesterol diet or a regular chow diet and subjected either to CIH or intermittent air (control conditions) for 12 weeks.

Measurements and main results: Nine of 10 mice simultaneously exposed to CIH and high-cholesterol diet developed atherosclerotic lesions in the aortic origin and descending aorta. In contrast, atherosclerosis was not observed in mice exposed to intermittent air and a high-cholesterol diet or in mice exposed to CIH and a regular diet. A high-cholesterol diet resulted in significant increases in serum total and low-density lipoprotein cholesterol levels and a decrease in high-density lipoprotein cholesterol. Compared with mice exposed to intermittent air and a high-cholesterol diet, combined exposure to CIH and a high-cholesterol diet resulted in marked progression of dyslipidemia with further increases in serum total cholesterol and low-density lipoprotein cholesterol (124 +/- 4 vs. 106 +/- 6 mg/dl; p < 0.05), a twofold increase in serum lipid peroxidation, and up-regulation of an important hepatic enzyme of lipoprotein secretion, stearoyl-coenzyme A desaturase-1.

Conclusions: CIH causes atherosclerosis in the presence of diet-induced dyslipidemia.

Figure 1.

Effect of chronic intermittent hypoxia (CIH) or intermittent air (IA) on fasting serum levels of (A) total cholesterol (TC), (B) low-density lipoprotein cholesterol (LDL-C), (C) high-density lipoprotein cholesterol (HDL-C), (D) phospholipids (PL), and (E) triglycerides (TG) in C57BL/6J mice on regular chow and a high-cholesterol diet. Solid bars, CIH; open bars, IA control. *p < 0.05, ^†p < 0.01, and ^‡p < 0.001, for the difference between IH and IA.

Figure 2.

Characterization of serum lipoproteins in C57BL/6J mice on a high-cholesterol diet after exposure to chronic intermitten hypoxia or intermittent air. Lipoproteins were examined by fast protein liquid chromatography, using ÄKTAprime system (GE Healthcare Life Sciences, Piscataway, NJ) followed by cholesterol measurement by gas chromatography in each fraction. Each profile represents pooled serum from eight mice. HDL = high-density lipoprotein; LDL = low-density lipoprotein; VLDL = very-low-density lipoprotein.

Figure 3.

Tumor necrosis factor (TNF)-α in the livers of C57BL/6J mice on a regular chow diet or on a high-cholesterol diet and exposed to chronic intermittent hypoxia (CIH) or intermittent air (IA) for 12 weeks. (A) Hepatic TNF-α mRNA levels by real-time reverse transcription–polymerase chain reaction; (B) TNF-α protein levels by ELISA with total liver lysate. Solid bars = CIH; open bars = IA control. *p < 0.05 for the difference between CIH and IA.

Figure 4.

Effect of chronic intermittent hypoxia (CIH) on serum lipid peroxidation, using the malondialdehyde (MDA) assay. Solid bars = CIH; open bars = intermittent air control. *p < 0.05 for the difference between CIH and intermittent air control.

Figure 5.

Representative cross-sections of the ascending aorta (sinus of Valsalva) in C57BL/6J mice exposed to (A) intermittent air (IA) control conditions and regular diet, (B) chronic intermittent hypoxia (CIH) and regular diet, (C) IA and a high-cholesterol diet, or (D) CIH and a high-cholesterol diet. Transverse frozen sections of the aorta were stained with oil red O and hematoxylin. Original magnification: ×100. The thick arrow points at the atherosclerotic plaque with a necrotic core. The thin arrow points at the fatty streak.

Figure 6.

Representative images of the thoracic (aortic arch and descending aorta) and abdominal aorta in C57BL/6J mice by the en face method. Sudan IV staining; original magnification: (A–D) ×10, water immersion; (E and F) ×20, dry preparation. (A) Intermittent air (IA) control conditions and regular diet; (B and E) chronic intermittent hypoxia (CIH) and regular diet; (C) IA and high-cholesterol diet; (D and F) CIH and high-cholesterol diet. Arrows point at atherosclerotic lesions.

Figure 7.

Analysis of stearoyl-coenzyme A desaturase-1 (SCD-1) in the livers of C57BL/6J mice on a regular chow diet and on a high-cholesterol diet and exposed to chronic intermittent hypoxia (CIH) or intermittent air (IA) for 12 weeks. n = 10 per group. (A) Hepatic SCD-1 mRNA levels by real-time reverse transcription–polymerase chain reaction; (B and C) SCD-1 protein levels by immunoblot with total liver lysate. (B) shows SCD-1 and β-tubulin bands in representative samples. (C) shows mean optical density of SCD-1 bands normalized to β-tubulin. (A and C) Solid bars = CIH; open bars = IA control. *p < 0.05 and ^†p < 0.01 for the difference between CIH and IA.