Sonodynamic therapy-induced foam cells apoptosis activates the phagocytic PPARγ-LXRα-ABCA1/ABCG1 pathway and promotes cholesterol efflux in advanced plaque

Abstract

In advanced atherosclerotic plaques, defective efferocytosis of apoptotic foam cells and decreased cholesterol efflux contribute to lesion progression. In our previous study, we demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (SDT) could induce foam cells apoptosis via the mitochondrial-caspase pathway. In the current research, we sought to explore ALA-SDT-induced apoptosis of phagocytes and the effects of cholesterol efflux and efferocytosis in advanced apoE^-/- mice plaque. Methods: apoE^-/- mice fed western diet were treated with ALA-SDT and sacrificed at day 1, day 3, day 7 and day 28 post treatment. THP-1 macrophage-derived foam cells were treated with ALA-SDT. 5 hours later, the supernatant was collected and added to fresh foam cells (phagocytes). Then, the lipid area, efferocytosis, cholesterol efflux, anti-inflammatory reactions and PPARγ-LXRα-ABCA1/ABCG1 pathway were detected in plaque in vivo and in phagocytes in vitro. Results: We found that ALA-SDT induced foam cells apoptosis coupled with efferocytosis and upregulation of Mer tyrosine kinase (MerTK) both in vivo and in vitro. The lipid content in plaque decreased as early as 1 day after ALA-SDT and this tendency persisted until 28 days. The enhancement of phagocytes cholesterol efflux was accompanied by an approximately 40% decrease in free cholesterol and a 24% decrease in total cholesterol in vitro. More importantly, anti-inflammatory factors such as TGFβ and IL-10 were upregulated by ALA-SDT treatment. Finally, we found that PPARγ-LXRα-ABCA1/ABCG1 pathway was activated both in vivo and in vitro by ALA-SDT, which could be blocked by PPARγ siRNA. Conclusions: Activation of PPARγ-LXRα-ABCA1/ABCG1 pathway induced by ALA-SDT treatment engages a virtuous cycle that enhances efferocytosis, cholesterol efflux and anti-inflammatory reactions in advanced plaque in vivo and in phagocytes in vitro.

Keywords: atherosclerosis; cholesterol efflux; foam cells; phagocyte; sonodynamic therapy.

Figure 1

Schematic diagram of the experimental procedure applying ALA-SDT treatment in vivo (A) and in vitro (B).

Figure 2

ALA-SDT treatment enhances cholesterol efflux both in vivo and in vitro. (A) Representative microscopic photographs of lipid in mice plaques stained by Oil Red O. (B) Quantitative analysis of lipid area per plaque. (n=6). (C) Quantitative analysis of plaque area. (n=6). (D-E) After incubation with supernatants from foam cells treated by control, ultrasound or SDT for 4 h, the phagocytes were incubated for an additional 12 h in serum-free media containing apoA1 and HDL. (D) Representative microscopic photographs of phagocytes stained with Oil Red O. (E) Quantification of the relative OD value in phagocytes in (D). (F) Quantitative analysis of intracellular total cholesterol (TC) and free cholesterol (FC) levels in phagocytes in (D). The assessment was performed in triplicate. Data are expressed as mean ± SD; *P<0.05 vs. control group, **P<0.01 vs. control group.

Figure 3

ALA-SDT treatment induces foam cell mitochondria pathway apoptosis and decreases ER stress in mouse advanced atherosclerotic plaque. (A) Representative fluorescence micrographs of aortic root stained with TUNEL (red) for apoptotic cells and counterstained with DAPI (blue) for the nuclei. (B) Quantification of TUNEL-positive apoptotic cells per mm² plaque area (n=9). (C, E, G) Representative fluorescence micrographs of aortic root double stained with CD68 and cytochrome c (C), cleaved caspase 3 (E) or CHOP (G) (arrow) and counterstained with DAPI for nuclei. (D, F, H) Quantification of corresponding double-positive expression area in the aortic root of each group in (C, E, G) (n=9). (I) Aorta were collected 1 day after ALA-SDT treatment and cytochrome c, cleaved caspase 3 and CHOP expressions were detected by western blot. (J-L) Quantification of the protein expression of cytochrome c (J), cleaved caspase 3 (J) and CHOP (L) in aorta 1 day after treatment (n=6). Data are expressed as mean ± SD; **P<0.01 compared with control group.

Figure 4

ALA-SDT treatment increases efferocytosis both in vivo and in vitro. (A) Representative fluorescence micrographs of aortic root double-stained with TUNEL (red) and CD68 (green). Efferocytosis was defined as the merging of TUNEL-positive/CD68-positive (yellow, arrow) images. (B) Quantitative analysis of phagocytotic rate in plaques after ALA-SDT treatment (n=9). (C) Representative fluorescence micrographs of aortic root double-stained with CD68 and MerTK (yellow, arrow). (D) Quantitative analysis of MerTK-positive expression area rate in plaques (n=9). (E-G) Five hours after SDT treatment, the supernatants of foam cells were collected and added to fresh foam cells (phagocytes) for 4 h. Then, the phagocytes were washed twice and fresh apoptotic cells (AC) labeled with pHrodo TM Red SE were added to phagocytes. 1 h later, the non-ingested AC were removed from the phagocytes. (E-F) Phagocytes were incubated with Percp-anti-CD11b and detected by flow cytometry. Representative flow cytograms (E) and quantification (F) revealing phagocytosis of phagocytes in different groups. (G) MerTK expression in phagocytes after co-incubation with supernatants of foam cells from different treatment groups was detected by western blot. The assessment was performed in triplicate. Data are expressed as mean ± SD; **P<0.01 vs. control group, ^^P<0.01 vs. ultrasound group.

Figure 5

ALA-SDT treatment increases ABCA1 and ABCG1 expression both in vivo and in vitro. (A, C) Representative fluorescence micrographs of aortic root double-stained with CD68 and ABCA1 (A) or ABCG1 (C) (arrows). (B, D) Quantification of positive area of ABCA1 (B) and ABCG1 (D) in atherosclerotic lesions (n=9). (E) ABCA1 and ABCG1 expressions in atherosclerotic lesions were detected by western blot. (F-G) Quantification of ABCA1 (F) and ABCG1 (G) expressions in atherosclerotic lesions (n=6). (H) ABCA1 and ABCG1 expressions in phagocytes after co-incubation with supernatants of foam cells from different treatment groups were detected by western blot in vitro. (I-J) Quantification of ABCA1 (I) and ABCG1 (J) expressions in (H). The assessment was performed in triplicate. Data are expressed as mean ± SD; *P<0.05 vs. control group, **P<0.01 vs. control group, ^^P<0.01 vs. ultrasound group.

Figure 6

ALA-SDT treatment activates anti-inflammation reactions both in vivo and in vitro. (A, C) Representative fluorescence micrographs of aortic root double-stained with CD68 and IL10 (A) or TGFβ (C) (arrow). (B, D) Quantitative analysis of IL10-positive (B) or TGFβ-positive (D) expression area rate in mice plaques in vivo (n=9). (E-F) IL10 (E) and TGFβ (F) levels in culture medium of phagocytes after co-incubation with supernatants of foam cells treated with ALA-SDT were detected by ELISA in vitro. Data are expressed as mean ± SD; *P<0.05 and **P<0.01 vs. 0 h. (G-H) IL10 (G) and TGFβ (H) levels in culture medium of phagocytes after co-incubation with supernatants of foam cells from different treatment groups for 12 h were detected by ELISA in vitro. The assessments were performed in triplicate. Data are expressed as mean ± SD; *P<0.05 vs. control group, **P<0.01 vs. control group.

Figure 7

ALA-SDT treatment induces PPARγ and LXRα expression. (A, C) Representative fluorescence micrographs of aortic root double-stained with CD68 and PPARγ (A) or LXRα (C) (arrows). (B, D) Quantification of positive area of PPARγ (B) and LXRα (D) expression in mice plaques (n=9). (E) PPARγ and LXRα expressions in atherosclerotic lesions were detected by western blot. (F-G) Quantification of PPARγ (F) and LXRα (G) expressions in atherosclerotic lesions (n=6). (H) PPARγ and LXRα expressions in phagocytes after co-incubation with supernatants of foam cells from different treatment groups were detected by western blot in vitro. (I-J) Quantification of PPARγ (I) and LXRα (J) expressions in (H). The assessment was performed in triplicate. Data are expressed as mean ± SD; *P<0.05 vs. control group, **P<0.01 vs. control group, ^^P<0.01 vs. ultrasound group.

Figure 8

Promotion of cholesterol efflux induced by ALA-SDT is PPARγ dependent. (A-D) PPARγ was knocked down in phagocytes before exposing them to the supernatant of foam cells treated with ALA-SDT in vitro. (A-B) Expressions of PPARγ, LXRα, ABCA1 and ABCG1 in phagocytes were detected by western blot. (C-D) Quantification of total cholesterol (C) and free cholesterol (D) contents in phagocytes. (E-I) Phagocytes were pretreated with 10 μM of the PPARγ antagonist GW9662 for 1 h before exposing them to the supernatant of foam cells treated with ALA-SDT in vitro. (E-F) After incubation with supernatants from foam cells treated by ALA-SDT for 4 h, the phagocytes were incubated for an additional 12 h in serum-free medium containing apoA1 and HDL. (E) Representative microscopic photographs of phagocytes stained with Oil Red O. (F) Quantification of the relative OD value in phagocytes in (E). (G) Comparison of phagocytotic rate in phagocytes preincubated with GW9662. (H-I) Comparison of TGFβ (H) and IL10 (I) levels in culture medium of phagocytes preincubated with GW9662 after co-incubation with supernatants of foam cells stimulated by ALA-SDT. The assessment was performed in triplicate. Data are expressed as mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

Figure 9

A proposed theoretical model of the anti-atherosclerosis mechanism of ALA-SDT. ALA-PpIX accumulates in mitochondria of foam cells in the advanced plaque. Once exposed to ultrasound, the foam cells undergo apoptosis via mitochondria-caspase pathway. This process activates the phagocytic PPARγ-LXRα-ABCA1/ABCG1 pathway and results in the enhancement of efferocytosis, cholesterol efflux as well as anti-inflammatory responses, thus eventually ameliorating atherosclerosis. The red arrow indicates cholesterol influx into the plaque; the green arrow indicates cholesterol efflux out of the plaque by apoA1 and HDL.