SIRT1 inhibition promotes atherosclerosis through impaired autophagy

Abstract

SIRT1, a highly conserved NAD⁺-dependent protein deacetylase, plays a pivotal role in the pathogenesis and therapy of atherosclerosis (AS). The aim of this study is to investigate the potential effects of SIRT1 on AS in ApoE^-/- mice and the underlying mechanisms of autophagy in an ox-LDL-stimulated human monocyte cell line, THP-1. In vivo, the accelerated atherosclerotic progression of mice was established by carotid collar placement; then, mice were treated for 4 weeks with a SIRT1-specific inhibitor, EX-527. The atherosclerotic lesion size of EX-527-treated mice was greatly increased compared to that of the mice in the control group. Immunostaining protocols confirmed that the inhibition of SIRT1 during plaque initiation and progression enhanced the extent of intraplaque macrophage infiltration and impaired the autophagy process. In vitro cultured THP-1 macrophages exposed to ox-LDL were utilized to study the link between the SIRT1 function, autophagy flux, pro-inflammatory cytokine secretion, and foam cell formation using different methods. Our data showed that ox-LDL markedly suppressed SIRT1 protein expression and the autophagy level, while it elevated the MCP-1 production and lipid uptake. Additionally, the application of the SIRT1 inhibitor EX-527 or SIRT1 siRNA further attenuated ox-LDL-induced autophagy inhibition. In conclusion, our results show that the inhibition of SIRT1 promoted atherosclerotic plaque development in ApoE^-/- mice by increasing the MCP-1 expression and macrophage accumulation. In particular, we demonstrate that blocking SIRT1 can exacerbate the acetylation of key autophagy machinery, the Atg5 protein, which further regulates the THP-1 macrophage-derived foam cell formation that is triggered by ox-LDL.

Keywords: Atg5; SIRT1; atherosclerosis; autophagy; deacetylation.

Figure 1. EX-527 administration promoted AS and enhanced macrophage infiltration in ApoE^-/- mice

In both administration schedules, ApoE^-/- mice were treated with EX-527 at 10 mg/kg by i.p. for 4 wks (5 days per wk). The development of atherosclerotic plaques was monitored in the collared carotid artery using H&E (A) and ORO staining methods (B). The effect of EX-527 on macrophage accumulation in carotid atherosclerotic plaques was detected by Mac-2 immunostaining method (C). Scale bar: 25 μm. Arrows are indicative of representative Mac-2 immunostaining positively regions.

Figure 2. EX-527 administration inhibited SIRT1 expression in the collared carotid artery of ApoE^-/- mice

In both treatment protocols, ApoE^-/- mice that underwent collar placement surgeries were treated with EX-527 at 10 mg/kg by i.p. for 4 wks (5 days per wk). Serial cross sections of the collared carotid arteries were immunostained with SIRT1 antibody (brown) and observed in a light microscope (A). Western blot for SIRT1 protein was analyzed from the collared carotid arteries of the ApoE^-/- mice. β-actin was used as loading control (B) and (C). Scale bar: 25 μm. Arrows are indicative of representative SIRT1 immunostaining positively regions. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. Sham group; ^#P < 0.05 and ^##P < 0.01 vs. Control group; ^&P < 0.05 and ^&&P < 0.01 represent significant differences between EX-527 (8 wks) group and EX-527 (12 wks) group.

Figure 3. EX-527 treatment did not further change LC3 expression in the collared carotid artery of ApoE^-/- mice

In both treatment schedules, ApoE^-/- mice with collar placement surgeries were treated with EX-527 at 10 mg/kg by i.p. for 4 wks (5 days per wk). Serial cross sections of the collared carotid arteries were immunostained with LC3 antibody (brown) and observed in a light microscope. Scale bar: 25 μm. Arrows are indicative of representative LC3 immunostaining positively regions.

Figure 4. Ox-LDL induced macrophage foam cell formation, SIRT1 inhibition, autophagy impairment, and MCP-1 production in THP-1 cells

Human THP-1 macrophages were exposed to 0, 20, 40, 60, and 80 μg/mL of ox-LDL for 24 hrs. Treated cells were photographed using light microscopy (A). The THP-1 macrophage-derived foam cell formation was determined using ORO staining method (B) and (C). Western blot for SIRT1, LC3, Beclin1, p62, and MCP-1 proteins were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (D-I). Scale bar: 20 μm. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. Cont group (0 μg/mL of ox-LDL).

Figure 5. Inhibition of SIRT1 using EX-527 or SIRT1 siRNA transfection enhanced MCP-1 expression and foam cell formation

Human THP-1 macrophages were pretreated with EX-527 (2 μM, for 2 hrs) or SIRT1 siRNA (20 μM, for 24 hrs), and then exposed to 80 μg/mL of ox-LDL for an additional 24 hrs. Western blot for SIRT1 and MCP-1 proteins were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (A-C) and (F-H). THP-1 macrophage-derived foam cell formation was determined using ORO staining method (D-E) and (I-J). Scale bar: 40 μm. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. Cont group (NC siRNA group); ^#P < 0.05 and ^##P < 0.01 vs. EX-527 group (SIRT1 siRNA group); ^&P < 0.05 and ^&&P < 0.01 represent significant differences between ox-LDL group (NC siRNA+ox-LDL group) and EX-527+ox-LDL group (SIRT1 siRNA+ox-LDL group).

Figure 6. Inhibition of autophagy with 3-MA led to increased foam cell formation and MCP-1 production

Human THP-1 macrophages were pretreated with 3-MA (5 μM) for 2 hrs, and then exposed to 80 μg/mL of ox-LDL for an additional 24 hrs. Western blot for LC3, Beclin1, p62, and MCP-1 proteins were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (A-E). THP-1 macrophage-derived foam cell formation was determined using ORO staining method (F-G). Scale bar: 40 μm. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. Cont group; ^#P < 0.05 and ^##P < 0.01 vs. 3-MA group; ^&P < 0.05 and ^&&P < 0.01 represent significant differences between ox-LDL group and 3-MA+ox-LDL group.

Figure 7. Inhibition of autophagy using Atg5 siRNA aggravated foam cell formation and MCP-1 expression

Human THP-1 macrophages were pretreated with Atg5 siRNA (20 μM) for 24 hrs, and then exposed to 80 μg/mL of ox-LDL for an additional 24 hrs. Western blot forAtg5, LC3, Beclin1, p62, and MCP-1 proteins were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (A-F) and (I). THP-1 macrophage-derived foam cell formation was determined using ORO staining method (G) and (H). Scale bar: 40 μm. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. NC siRNA group; ^#P < 0.05 and ^##P < 0.01 vs. Atg5 siRNA group; ^&P < 0.05 and ^&&P < 0.01 represent significant differences between NC siRNA+ox-LDL group and Atg5 siRNA+ox-LDL group.

Figure 8. Inhibiting SIRT1 using EX-527 and SIRT1 siRNA further blocked autophagy that was down-regulated by ox-LDL exposure

THP-1 macrophages were pretreated with EX-527 (2 μM, for 2 hrs), SIRT1 siRNA (20 μM, for 24 hrs), RSV (10 μM, for 2 hrs), or RAP (100 nM, for 2 hrs) and then sexposed to 80 μg/mL of ox-LDL for an additional 24 hrs. Western blot for LC3, Beclin1, p62, and Atg5 proteins and immunoprecipitation for acetyl-Lys Atg5 were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (A-G). Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. Cont group; ^#P < 0.05 and ^##P < 0.01 vs. ox-LDL group; ^&P < 0.05 and ^&&P < 0.01 represent significant differences between RSV+ox-LDL group and RAP+ox-LDL group.

Figure 9. Overexpression of SIRT1 using adenoviral transfection reversed ox-LDL-induced macrophage foam cell formation and autophagy impairment in THP-1 cells

Human THP-1 macrophages were transfected by SIRT1 over-expressing adenovirus (HBAD-SIRT1) or NC adenovirus (HBAD-GFP) for 24 hrs and then exposed to 80 μg/mL of ox-LDL for an additional 24 hrs. The transfected THP-1 cells were observed using an inverted fluorescence microscope (A) and then were harvested for transfection efficiency analysis by Western blot method (B). THP-1 macrophage-derived foam cell formation was determined using ORO staining method (C) and (D). Western blot for LC3, Beclin1, p62, and Atg5 proteins and immunoprecipitation for acetyl-Lys Atg5 were analyzed from the ox-LDL-stimulated THP-1 cells. β-actin was used as loading control (E-L). Scale bar: 40 μm. Bar graph indicates the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 vs. HBAD-GFP group; ^#P < 0.05 and ^##P < 0.01 vs. HBAD-SIRT1 group; ^&P < 0.05 and ^&&P < 0.01 represent significant differences between HBAD-GFP+ox-LDL group and HBAD-SIRT1+ox-LDL group.