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[Preprint]. 2025 Jul 16:rs.3.rs-6979508.
doi: 10.21203/rs.3.rs-6979508/v1.

Elevated apolipoprotein C3 heightens atherosclerosis risk by mediating arterial accumulation of free cholesterol and local inflammation in diabetes

Jenny E Kanter  1   2 , Cheng-Chieh Hsu  1   2 , Farah Kramer  1   2 , Baohai Shao  1   2 , Tomas Vaisar  1   2 , Laura J den Hartigh  1   2 , Abigail Reed  1   2 , Jason Luo  1   2 , Alan Tran  1   2 , Jingjing Tang  1   2 , Henry Mangalapalli  1   2 , Jocelyn Cervantes  1   2 , Masami Shimizu-Albergine  1   2 , Peter D Reaven  3 , Juraj Koska  3 , Majken K Jensen  4 , Brandon S J Davies  5   6 , Edward A Fisher  7 , Nicholas O Davidson  8 , Nathan O Stitziel  9   10 , Adam E Mullick  11 , Ira J Goldberg  12 , Karin E Bornfeldt  1   2   13
Affiliations

Elevated apolipoprotein C3 heightens atherosclerosis risk by mediating arterial accumulation of free cholesterol and local inflammation in diabetes

Jenny E Kanter et al. Res Sq. .

Abstract

Cardiovascular outcome trials are being considered for therapeutics that silence apolipoprotein C3 (APOC3) or angiopoietin-like 3 (ANGPTL3) because of their abilities to lower triglyceride-rich lipoproteins (TRLs) and their remnants in individuals with increased cardiovascular disease (CVD) risk1-4. Here we demonstrate that plasma APOC3 predicts CVD events in individuals with diabetes more strongly than in those without diabetes. Accordingly, plasma APOC3 levels are elevated, clearance of TRLs/remnants is slowed, and plasma TRL remnants are increased in two mouse models of diabetes-accelerated atherosclerosis. Silencing mouse APOC3 by a liver-targeted antisense oligonucleotide lowers both cholesterol and triglycerides carried by TRL/remnants and LDL and prevents aortic free cholesterol accumulation in diabetes, while ANGPTL3 silencing reduces triglycerides. Single-cell RNA-sequencing revealed that APOC3 silencing prevents a majority of diabetes-induced pathways in macrophages, endothelial cells, and smooth muscle cells, with inflammation as a major predicted upstream regulator, adding promise to APOC3 as a CVD target in diabetes.

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Conflict of interest statement

Competing interest. A.E.M. is employed by Ionis Pharmaceuticals. K.E.B. serves on the scientific advisory board of Esperion Therapeutics, Inc.

Figures

Figure 1.
Figure 1.. Hepatic APOC3 silencing prevents elevated plasma levels of TRL/RLP and LDL in diabetes.
Aortic lipophilic area at indicated time points in mouse models of STZ-diabetes (a) and LCMV-induced diabetes (b) fed a low-fat semipurified diet at the onset of diabetes (week 0). c-n. Diabetic (D, LCMV model) and non-diabetic (ND) littermates were injected weekly with control GalNAc ASO (cASO) or APOC3 GalNAc ASO. c. Plasma APOC3 at 12 weeks. d. Blood glucose over 12-weeks. e-h. Plasma triglycerides (TG), cholesterol-, and triglyceride- and cholesterol lipoprotein profiles. i-k. Isolated TRLs analyzed for APOB content, triglycerides, and cholesterol. l-n. Isolated LDL analyzed for APOB content, cholesterol, and triglycerides. Data are expressed as mean ± SEM, two-way ANOVA (see Original Data supplement for details on number of mice/group and statistical analyses).
Figure 2.
Figure 2.. Hepatic APOC3 silencing prevents elevated levels of aortic free cholesterol in diabetes.
Diabetic (D, LCMV model) and non-diabetic (ND) littermates were injected weekly with control GalNAc ASO (cASO) or APOC3 GalNAc ASO. a. Aortic Sudan IV-positive lipophilic en face area at the end of the 12-week study. b. Aortic total cholesterol. c. Aortic cholesteryl esters (CE). d. Aortic free cholesterol. e. Aortic triglycerides (TG). Data are expressed as mean ± SEM, two-way ANOVA (see Original Data supplement for details on number of mice/group and statistical analyses).
Figure 3.
Figure 3.. A majority of diabetes-induced transcriptional signatures in arterial cells are mediated by hepatic APOC3.
a. Lesioned aortic arches from 3 biological replicates from the two diabetes models and cASO- and APOC3 ASO-treated diabetic (D) mice and non-diabetic (ND) cASO controls analyzed by scRNA-seq. b-c. UMAPs of identified cells. d. UMAP of reclustered leukocytes in the LCMV diabetes model. e-g. IPA using DEGs (adjusted p<0.05) in (e) the resident macrophage cluster (f) EC cluster and (g) SMC cluster from D (STZ) versus ND, D (LCMV) versus ND, and APOC3 ASO (LCMV D) versus cASO (LCMV D). The top 30 pathways based on Z-scores and p-values are shown in g. h. Schematic representation of the effects of hepatic APOC3 and ANGPTL3 silencing.
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