Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis

Abstract

Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE-deficient mice, demonstrating that CysC deficiency (CysC(-/-) ) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC(-/-) mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7-oxysterol-mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol-mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE-deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.

Keywords: autophagy; cystatin C; lysosomal membrane permeabilization; macrophage cell death.

Figure 1

Decreased autophagy is associated with reduced CysC levels in progression of human atheroma. Representative photographs of autofluorescence of ceroid, Atg5 (brown), double immunohistochemistry of p62/SQSTM1 (brown) and CD68 (red), ubiquitin (brown) and CysC (brown) in situ in early lesions (left panels) and advanced human carotid lesions (middle panels); bars = 50 μm. Note: In early lesion areas, there are very low levels of ceroid, significantly higher levels of Atg5 and significantly lower levels of p62/SQSTM1 in CD68‐positive macrophages and low levels of ubiquitin, while advanced lesions show pronounced ceroid accumulation, low Atg5 and massive accumulation of CD68‐positive macrophages with significant increased levels of p62/SQSTM1 and ubiquitin and lower levels of CysC. Histograph show quantification of immunopositive areas of Atg5, p62/SQSTM1, ubiquitin and CysC. *P < 0.05, **P < 0.01.

Figure 2

CysC deficiency in apoE^−/− mice results in reduced autophagy and increased apoptotic cell death. Lesions from apoE^−/−CysC^+/+ (n = 6) and apoE^−/−CysC^−/− mice (n = 4) were immunostained with Atg5 or LC3β. Apoptosis was determined by the TUNEL technique, and images were analysed as described in the Methods section. The stained sections were counterstained with haematoxylin. (A) Representative photographs of Atg5, LC3β and TUNEL (stained in red) from apoE^−/−CysC^+/+ mice and apoE^−/−CysC^−/−, bars = 25 μm. (B–D) Quantification of Atg5, LC3β and TUNEL in the two groups of mice. *P < 0.05 versus apoE^−/− CysC^+/+ mice.

Figure 3

7‐oxysterols induce CysC and macrophages from CysC‐deficient mice (CysC^−/−) show less autophagy activity and are more sensitive to 2mix‐mediated apoptosis by LMP. (A) THP‐1 cells were treated with 7‐oxysterol for 24 or 48 hrs, immunostained with CysC and analysed with flow cytometry (MFI: mean fluorescence intensity). *P < 0.05 versus control cells from the same time point (n = 2–6, mean ± S.D.). (B) Peritoneal macrophages from wild‐type (WT) or CysC^−/− mice were treated with 2mix for 12 hrs or pre‐treated with rapamycin for 1 hr and then exposed to the 2mix for 12 hrs, immunostained with anti‐p62/SQSTM1 antibody, photographed with confocal microscopy and quantified by image analysis. Note: in WT cells (white bars), p62/SQSTM1 was increased following 2mix exposure, which was reversed by rapamycin pre‐treatment (**P < 0.01). In CysC^−/− cells (hatched bars), basal levels of p62/SQSTM1 were already higher in untreated control cells as compared with the cells from WT mice. Furthermore, rapamycin pre‐treatment had less significant effect on 2mix‐induced p62/SQSTM1 in CysC^−/− cells (*P < 0.05). (C and D) Cells were first stained with lysosomal AO and then treated as indicated for 6 hrs and analysed by confocal microscopy (C, representative photographs of AO‐stains, bars = 10 μm), and cytosolic/nuclear AO green fluorescence intensity was analysed by Photoshop image analysis and was identified as cells with LMP (D, *P < 0.05). Note in C: Red fluorescence indicates AO located in lysosomes, whereas green florescence indicates relocation of AO from lysosomes to cytosol or nuclei.

Figure 4

2mix‐induced lipid accumulation is minimized by 2 μg human recombinant cystatin C. THP‐1 differentiated macrophages were treated with or without 2mix for 24 hrs in the presence or absence of CysC. Cells were stained with Oil red O and counterstained with haematoxylin and analysed by light microscopy; bars = 20 μm. Percentage of oil Red O positive areas: control 0.55 ± 0.16, 2mix 3.47 ± 0.55, CysC 1 μg + 2mix 3.20 ± 0.61 and CysC 2 μg + 2mix 0.80 ± 0.07.