Macrophage LRP-1 controls plaque cellularity by regulating efferocytosis and Akt activation

Abstract

Objective: The balance between apoptosis susceptibility and efferocytosis of macrophages is central to plaque remodeling and inflammation. LRP-1 and its ligand, apolipoprotein E, have been implicated in efferocytosis and apoptosis in some cell types. We investigated the involvement of the macrophage LRP-1/apolipoprotein E axis in controlling plaque apoptosis and efferocytosis. Method and Results- LRP-1(-/-) macrophages displayed nearly 2-fold more TUNEL positivity compared to wild-type cells in the presence of DMEM alone or with either lipopolysaccharide or oxidized low-density lipoprotein. The survival kinase, phosphorylated Akt, was barely detectable in LRP-1(-/-) cells, causing decreased phosphorylated Bad and increased cleaved caspase-3. Regardless of the apoptotic stimulation and degree of cell death, LRP-1(-/-) macrophages displayed enhanced inflammation with increased IL-1 beta, IL-6, and tumor necrosis factor-alpha expression. Efferocytosis of apoptotic macrophages was reduced by 60% in LRP-1(-/-) vs wild-type macrophages despite increased apolipoprotein E expression by both LRP-1(-/-) phagocytes and wild-type apoptotic cells. Compared to wild-type macrophage lesions, LRP-1(-/-) lesions had 5.7-fold more necrotic core with more dead cells not associated with macrophages.

Conclusions: Macrophage LRP-1 deficiency increases cell death and inflammation by impairing phosphorylated Akt activation and efferocytosis. Increased apolipoprotein E expression in LRP-1(-/-) macrophages suggests that the LRP-1/apolipoprotein E axis regulates the balance between apoptosis and efferocytosis, thereby preventing necrotic core formation.

Figure 1

Detection of apoE protein in the medium (A), apoE mRNA levels in cells (B), and immunohistochemistry for apoE (green) and nuclei (DAPI, blue) in WT (C) or LRP1^-/- (D) macrophages after 24h in serum-free DMEM. *p<0.05, Student's t test.

Figure 2

TUNEL positive macrophages (A and B) after 24h in DMEM alone or with LPS (50ng/ml) or oxidized LDL (50μg/ml). Annexin V positive cells (C) and medium cytokine levels (D) after 24h in DMEM with staurosporine (50μg/ml). *p<0.05 between the two groups, Student's t test (A-D). Data represent 2 (C-D) or 3 (A-B) experiments.

Figure 3

Peritoneal macrophages positive for annexinV alone(A) or annexinV and 7AAD (B). TUNEL analyses of aortic sections from WT (n=5) and MΦLRP1^-/- (n=4) BM recipient LDLr^-/- mice fed western diet for 16 weeks (C). Micrographs show nuclei (DAPI, blue), TUNEL positive staining (red) and merged images. Quantitation of TUNEL results (D) and co-localization of macrophage stain (green) in TUNEL-positive regions of arteries from mice with LRP1^-/- BM (E). *p<0.05, Mann-Whitney test

Figure 4

(A) Western blot of cell pAkt^{(serine 473)}, total Akt, pBad^{(serine 136)}, and β-actin and (B) Akt1 mRNA levels in WT or LRP1^-/- macrophages after 24h in DMEM alone or with LPS (50ng/ml). (C) Immunohistochemical detection of activated caspase-3 (red) after 24h in DMEM. DAPI (pink) staining identifies nuclei. *p<0.05, Student's t test (B) and all data represent 2 experiments.

Figure 5

Efferocytosis of apoptotic (CFDA-SE/green label) WT cells by WT (A) or LRP1^-/- (B) macrophages. DAPI (blue) staining identifies nuclei. (C) CFDA-SE positive phagocytes as percentage of total macrophages. (D) ABCA7 mRNA after 24h in DMEM. *p<0.05, Student's t test. (E) Phagocytosis of CFDA-SE labeled WT, apoE^-/-, or LRP1^-/- apoptotic macrophages by WT, apoE^-/-, or LRP1^-/- efferocytes. Differences (p<0.05) were marked as follows: * versus the WT/WT condition; # versus the apoE^-/-/apoE^-/- condition; & versus the apoE^-/-/WT condition. ANOVA with Bonferroni's post test. Data represent two (D, E) or four (C) experiments.

Figure 6

Percent of macrophages positive for efferocytosis of apoptotic CFDA-SE/labeled WT cells (A) 1h post injection into the peritoneal cavity of WT (n=3) or MΦLRP1^-/- (n=4) mice as determined by flow cytometry. Quantitation of the ratio of free versus macrophage associated TUNEL positive cells (B) in aortic sections from WT (n=5) and MΦLRP1^-/- (n=4) BM recipient LDLr^-/- mice fed western diet for 16 weeks. (C, D) TUNEL positive cells in aortic sections from WT (C) or MΦLRP1^-/- (D) BM recipient LDLr^-/- mice. Cells are either free (triangles) or associated with viable macrophages (arrows). (E) Quantitation of necrotic area in aortic sections from WT (n=5) and MΦLRP1^-/- (n=4) BM recipient LDLr^-/- mice. Images show hematoxylin and eosin staining of aortic root sections. NEC denotes necrotic area (E). *p<0.05, Mann Whitney test.