Mertk receptor mutation reduces efferocytosis efficiency and promotes apoptotic cell accumulation and plaque necrosis in atherosclerotic lesions of apoe-/- mice

Abstract

Objective: Atherosclerotic plaques that are prone to disruption and acute thrombotic vascular events are characterized by large necrotic cores. Necrotic cores result from the combination of macrophage apoptosis and defective phagocytic clearance (efferocytosis) of these apoptotic cells. We previously showed that macrophages with tyrosine kinase-defective Mertk receptor (Mertk(KD)) have a defect in phagocytic clearance of apoptotic macrophages in vitro. Herein we test the hypothesis that the Mertk(KD) mutation would result in increased accumulation of apoptotic cells and promote necrotic core expansion in a mouse model of advanced atherosclerosis.

Methods and results: Mertk(KD);Apoe(-/-) mice and control Apoe(-/-) mice were fed a Western-type diet for 10 or 16 weeks, and aortic root lesions were analyzed for apoptosis and plaque necrosis. We found that the plaques of the Mertk(KD);Apoe(-/-) mice had a significant increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive apoptotic cells. Most importantly, there were more non-macrophage-associated apoptotic cells in the Mertk(KD) lesions, consistent with defective efferocytosis. The more advanced (16-week) Mertk(KD);Apoe(-/-) plaques were more necrotic, consistent with a progression from apoptotic cell accumulation to plaque necrosis in the setting of a defective efferocytosis receptor.

Conclusions: In a mouse model of advanced atherosclerosis, mutation of the phagocytic Mertk receptor promotes the accumulation of apoptotic cells and the formation of necrotic plaques. These data are consistent with the notion that a defect in an efferocytosis receptor can accelerate the progression of atherosclerosis and suggest a novel therapeutic target to prevent advanced plaque progression and its clinical consequences.

Figure 1. Total body weight and plasma lipoproteins of Apoe^−/− and Mertk^KD;Apoe^−/− mice

Body weight, total plasma cholesterol, and fast performance liquid chromatography gel-filtration profiles (from pooled plasma samples) from male Apoe^−/− and Mertk^KD;Apoe^−/− mice fed a Western-type diet for either (A) 10 wks or (B) 16 wks. n.s., statistically non-significant difference between the two groups of mice.

Figure 2. Mertk expression and aortic root lesion area in Apoe^−/− and Mertk^KD; Apoe^−/− mice after 10 wks and 16 wks on a Western-type diet

(A) Immunohistochemistry for Mertk and macrophages was performed as described in Materials and Methods. The isotype control IgG was matched for the anti-Mertk antibody. Sections were counterstained with hematoxylin. nec, necrotic area. (B) Aortic RNA from each genotype was subjected to RT-PCR for Mertk, as described in Materials and Methods. (C–D) Examples of hematoxylin and eosin-stained aortic root sections from each group of mice fed a Western-type diet for either (C) 10 wks or (D) 16 wks. Bar, 0.3 mm. Below each set of sections are graphs showing the quantified lesion area. n.s., statistically non-significant difference between the two groups of mice.

Figure 3. TUNEL-positive nuclei are increased in the aortic root lesions of Mertk^KD;Apoe^−/− mice

TUNEL analysis of aortic root sections from Mertk^+/+;Apoe^−/− and Mertk^−/−;Apoe^−/− mice fed a Western-type diet for either (A) 10 wks or (B) 16 wks. Micrographs display nuclei (blue; Hoechst), TUNEL-positive signal (red), and merged images. Bar, 10 μm. Quantified data are shown below the images (* indicates P<0.05). (C) Similar to the images in (B), but the right image in each group shows merged nuclear, TUNEL, and macrophage (green) staining. Bar, 10 μm.

Figure 4. Plaque necrosis is increased in the aortic root lesions of Mertk^KD;Apoe^−/− mice after 16 wks on a Western-type diet

The images show examples of sections of hematoxylin and eosin-stained aortic roots from each group of mice fed a Western-type diet for 16 wks. nec, necrotic areas. Bar, 100 μm. The graph shows quantification of necrotic areas (n = 10 per group; * indicates P<0.05).

Figure 5. Phagocytosis efficiency of apoptotic cells is decreased in Mertk^KD;Apoe^−/− plaques

(A) Examples of nuclear (blue; Hoechst), TUNEL (red), and macrophage (green) staining to differentiate macrophage-associated (left column of images) from free (right column of images) apoptotic cells in aortic root lesions of 16-wk diet-fed Apoe^−/− and Mertk^KD;Apoe^−/− mice, respectively. (B) Examples of merged nuclear, TUNEL, and macrophage-stained images differentiating macrophage-associated (top image) from free (bottom image) apoptotic cells in aortic root lesions of Apoe^−/− and Mertk^KD;Apoe^−/− mice, respectively. Arrows indicate apoptotic bodies. (C) Quantitation of the free-to-macrophage-associated ratio of apoptotic cells in the lesions of the two groups of mice (* indicates P<0.05).