Hyperlipidemia and atherosclerotic lesion development in Ldlr-deficient mice on a long-term high-fat diet

Abstract

Background: Mice deficient in the LDL receptor (Ldlr(-/-) mice) have been widely used as a model to mimic human atherosclerosis. However, the time-course of atherosclerotic lesion development and distribution of lesions at specific time-points are yet to be established. The current study sought to determine the progression and distribution of lesions in Ldlr(-/-) mice.

Methodology/principal findings: Ldlr-deficient mice fed regular chow or a high-fat (HF) diet for 0.5 to 12 months were analyzed for atherosclerotic lesions with en face and cross-sectional imaging. Mice displayed significant individual differences in lesion development when fed a chow diet, whereas those on a HF diet developed lesions in a time-dependent and site-selective manner. Specifically, mice subjected to the HF diet showed slight atherosclerotic lesions distributed exclusively in the aortic roots or innominate artery before 3 months. Lesions extended to the thoracic aorta at 6 months and abdominal aorta at 9 months. Cross-sectional analysis revealed the presence of advanced lesions in the aortic sinus after 3 months in the group on the HF diet and in the innominate artery at 6 to 9 months. The HF diet additionally resulted in increased total cholesterol, LDL, glucose, and HBA1c levels, along with the complication of obesity.

Conclusions/significance: Ldlr-deficient mice on the HF diet tend to develop site-selective and size-specific atherosclerotic lesions over time. The current study should provide information on diet induction or drug intervention times and facilitate estimation of the appropriate locations of atherosclerotic lesions in Ldlr(-/-) mice.

Figure 1. Plasma lipids levels and body weights of Ldlr ^−/− mice fed HF or regular chow diets over time.

Plasma total cholesterol, LDL and HDL levels in HF diet-fed (A), and regular chow diet-fed (C) male Ldlr ^−/− mice. Body weights of HF diet-fed (B) and regular chow diet-fed (D) mice. Data are presented as mean values ± SEM (n = 10).

Figure 2. Atherosclerotic lesion progression and location in HF diet-fed Ldlr ^−/− mice.

Analysis of atherosclerotic lesions was performed using the en face method. Representative images of Sudan IV-stained aorta in Ldlr ^−/− mice fed the HF diet for 0 to 12 months (A). Lesion-occupied areas in full-length aorta (A), aortic arch (C), innominate artery (D), thoracic aorta (E), and abdominal aorta (F) were quantified and expressed as a percentage of the lumen area in Ldlr ^−/− mice on the HF diet at 0 month (•), 3 months (▴), 6 months (▾), 9 months (△), and 12 months (▽).

Figure 3. Aortic sinus lesion morphology in Ldlr ^−/− mice.

(A) Representative sections in Ldlr ^−/− mice fed a regular chow diet (0 M) or HF diet for 1 to 12 months (1 M, 3 M, 6 M, 9 M, and 12 M) stained with Oil Red-O and hematoxylin, original magnification: ×100 or ×200, as indicated; (B) Lesion area was quantified using computer software IPP6.0.

Figure 4. Atherosclerotic lesion development in the aortic sinus of Ldlr ^−/− mice.

A and B, Representative examples of H&E, Movat's pentachrome, VSMC-specific Actin and macrophage antibody (Mac-2)-stained aortic sinus sections are provided. In Movat's pentachrome stained sections, black represents nuclei and elastin fibers, blue represents ground substance and mucin, yellow represents collagen and reticular fibers, red represents muscle, and intense red represents fibrinoid and fibrin. The asterisk indicates the classical cholesterol cleft, while NC stands for necrotic core. The arrow signifies representative regions staining positively for VSMC and macrophage. The bar indicates 200 µm in (A) and 50 µm in (B), respectively.