Chitinase inhibition promotes atherosclerosis in hyperlipidemic mice

Abstract

Chitinase 1 (CHIT1) is secreted by activated macrophages. Chitinase activity is raised in atherosclerotic patient sera and is present in atherosclerotic plaque. However, the role of CHIT1 in atherosclerosis is unknown. Preliminary studies of atherosclerosis in cynomolgous monkeys revealed CHIT1 to be closely correlated with areas of macrophage infiltration. Thus, we investigated the effects of a chitinase inhibitor, allosamidin, on macrophage function in vitro and on atherosclerotic development in vivo. In RAW264.7 cells, allosamidin elevated monocyte chemoattractant protein 1 and tumor necrosis factor alpha expression, and increased activator protein 1 and nuclear factor-κB transcriptional activity. Although inducible nitric oxide synthase, IL-6, and IL-1β expression were increased, Arg1 expression was decreased by chitinase inhibition, suggesting that suppression of CHIT1 activity polarizes macrophages into a M1 phenotype. Allosamidin decreased scavenger receptor AI, CD36, ABCA1, and ABCG1 expression which led to suppression of cholesterol uptake and apolipoprotein AI-mediated cholesterol efflux in macrophages. These effects were confirmed with CHIT1 siRNA transfection and CHIT1 plasmid transfection experiments in primary macrophages. Apolipoprotein E-deficient hyperlipidemic mice treated for 6 weeks with constant administration of allosamidin and fed an atherogenic diet showed aggravated atherosclerotic lesion formation. These data suggest that CHIT1 exerts protective effects against atherosclerosis by suppressing inflammatory responses and polarizing macrophages toward an M2 phenotype, and promoting lipid uptake and cholesterol efflux in macrophages.

Figure 1

A: Chitinase activity increased in BMDM treated with Ac-LDL, AGE-BSA, LPS, and IFN-γ. BMDM from C57BL/6 mice were isolated and stimulated with 50 μg/mL AcLDL, 100 ng/mL MCP-1, 100 ng/mL LPS, 100 μg/mL AGE-BSA, or 100 units/mL IFN-γ. After 24 hours of stimulation, chitotriosidase activity in the media was measured as described in Materials and Methods. B: Chitinase inhibitor modifies expression of pro-inflammatory cytokines and polarizes macrophages into M1 phenotype. RAW264.7 macrophages were treated with medium alone or 10 μmol/L allosamidin for 24 hours before RNA isolation. Transcripts for TNFα, MCP-1, CCR2, inducible nitric oxide synthase, IL-6, IL-12p35, IL-1β, Arg1, FIZZ1, and IL-10 were quantified by real-time PCR. Results are representative of at least three independent experiments and values are expressed as fold change in abundance (means ± SEM). ^∗P < 0.05, ^∗∗P < 0.01 versus control.

Figure 2

Chitinase inhibitor increases AP-1 and NF-κB transcriptional activity. A: RAW264.7 macrophages were treated with medium alone or 10 μmol/L allosamidin overnight. Nuclear protein was extracted and transcriptional activity of AP-1 and NF-κB were accessed by electromobility shift assay. B: RAW264.7 macrophages were transfected with AP-1 or NF-κB promoter–luciferase plasmid and 2 μg of β-galactosidase gene driven by SV40 promoter-enhancer sequence. The cells were then treated with medium alone or 10 μmol/L allosamidin overnight, and luciferase activity and β-galactosidase were measured. ^∗P < 0.05 versus control.

Figure 3

Chitinase inhibitor modifies expression of genes related to cholesterol metabolism and decreases cholesteryl ester uptake and cholesterol efflux in macrophage. A: RAW264.7 macrophages were treated with medium alone or 10 μmol/L allosamidin for 24 hours before RNA isolation. Transcripts for ABCA1, ABCG1, CD36, SR-AI, PPARγ, and LXRα were quantified by real-time PCR. ^∗P < 0.05 versus control. B: Chitinase inhibitor lowers cholesteryl ester uptake by RAW264.7 cells. Cells were treated with or without allosamidin overnight followed by incubation of Dil-AcLDL at 10 μg/mL in medium for 2 hours at 37°C. Intensity of fluorescence was determined by either confocal microscopy or flow cytometry. C: Chitinase inhibitor decreases apoAI and HDL-mediated cholesterol transport. RAW264.7 cells were labeled with ³H-cholesterol for 36 hours in the presence or absence of 10 μmol/L allosamidin. ApoAI- or HDL-dependent or independent cholesterol efflux was measured by incubating [³H] cholesterol-labeled RAW264.7 cells with or without apoAI or HDL. Radioactivity in the media was determined as a percentage of total radioactivity in the cells and media (n = 3; means ± SEM). ^∗P < 0.05, ^∗∗P < 0.01 versus control.

Figure 4

Chitinase inhibition, CHIT1 inhibition, and CHIT1 overexpression modify pro-inflammatory cytokines and polarize primary macrophages toward a M1 phenotype. BMDM were treated with medium alone or 10 μmol/L allosamidin for 24 hours and were stimulated with or without 10 units/mL IFN-γ for 6 hours before RNA isolation. Thioglycollate-elicited peritoneal macrophages were transfected with siRNA against mouse CHIT1 or nontargeting siRNA using lipofectamine siRNA maximum. After 48 hours of transfection, the cells were harvested for mRNA isolation. BMDM were also transfected with control pcDNA3.1 or pcDNA3.1-CHIT1 plasmid by electroporation (MicroPorator MP-100). After 48 hours of transfection, the cells were stimulated with or without 10 units/mL IFN-γ for 6 hours and harvested for mRNA isolation. Transcripts for TNFα, MCP-1, inducible nitric oxide synthase, IL-6, IL-12p35, IL-1β, Arg1, FIZZ1, and IL-10 were quantified by real-time PCR. The results are representative of at least three independent experiments and values are expressed as fold change in abundance (means ± SEM). PMφ, thioglycollate-elicited peritoneal macrophages. ^∗P < 0.05, ^∗∗P < 0.01 versus control.

Figure 5

Chitinase inhibition, CHIT1 inhibition, and CHIT1 overexpression modify expression of genes related to cholesterol metabolism in primary macrophages. Transcripts for ABCA1, ABCG1, CD36, SR-AI, PPARγ, and LXRα were quantified by real-time PCR. The results are representative of at least three independent experiments and values are expressed as fold change in abundance (means ± SEM). PMφ, thioglycollate-elicited peritoneal macrophages. ^∗P < 0.05, ^∗∗P < 0.01 versus control.

Figure 6

Chitinase inhibitor reduces atherosclerotic lesions in spontaneous ApoE-deficient hyperlipidemic mice fed a Western diet. A: CHIT1 mRNA expression in atherosclerotic aortas of ApoE-deficient mice fed a chow diet or high-fat diet with 1.9 mg/kg per day infusion of angiotensin II for 4 weeks. The descending aortas were harvested and total mRNA was extracted. CHIT1 mRNA expression was evaluated by real-time RT-qPCR (n = 7 in control group; n = 8 in high-fat diet + angiotensin II group; means ± SEM). B: The 12-week-old mice were implanted with an osmotic mini pump model 2006 (ALZET) containing 10 mg/kg per day allosamidin or control vehicle, and fed a Western diet for 6 weeks. Serum chitinase activity was measured before and after 6 weeks of allosamidin treatment. Allosamidin administration suppressed serum chitinase activity by approximately 40%. C: Aortas (aortic arch to the iliac bifurcation) from control and allosamidin-treated mice fed the atherogenic diet for 6 weeks were opened and stained en face with Oil Red O (Sigma-Aldrich). Representative images of the aortas showing the surface plaque (top) and quantification of surface area occupied by lesions in the aortic arch (bottom left) and the descending aortas (bottom right) are shown. Data (percentage of area occupied by lesions) are expressed as means ± SEM for each group. D: Aortic sinus atherosclerosis in control and allosamidin-treated mice. Atherosclerotic lesion in the aortic sinus from cryosections was stained with Oil Red O and counterstained with H&E. Sections were also stained with Mac-3 or CD4 antibody coupled with horseradish peroxidase-labeled secondary antibody and counterstained with hematoxylin to identify the macrophages and T lymphocytes present in the aortic sinus lesions. Representative images for each staining are shown. The atherosclerotic lesion area stained with Oil Red O and the macrophage infiltration area stained with Mac-3 antibody were quantitated using a computer-assisted imaging system. ^∗P < 0.05.