Apoe-KO(2)/hAPOC3

1. Chow diet conditions：

Fig.1 hAPOC3 levels in Apoe-KO(2)/hAPOC3 mice under CD conditions. Under CD conditions, hAPOC3 was detectable in homozygous Apoe-KO(2)/hAPOC3 mice. Serum hAPOC3 levels in Apoe-KO(2)/hAPOC3 mice slightly decreased with age. Serum samples were collected following a 6-hour fast at different ages to measure levels of serum hAPOC3 (n=4-12, Mean ± SEM).

 Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.2 Body weight change of Apoe-KO(2)/hAPOC3 mice under CD conditions. Apoe-KO(2)/hAPOC3 mice exhibited comparable body weight gain to both Apoe-KO(2) and hAPOC3 mice within the same sex. WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice were maintained on CD feeding. Body weight and body weight gain were recorded weekly (n=4-12, Mean ± SEM). 

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.3 Blood lipid profiles of Apoe-KO(2)/hAPOC3 mice after ARO-APOC3 treatment under CD conditions. Male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice were maintained on CD feeding. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C) (n=4-12, Mean ± SEM, t-test, Apoe-KO(2) mice versus Apoe-KO(2)/hAPOC3 mice: #p<0.05, ##p<0.01; hAPOC3 mice versus Apoe-KO(2)/hAPOC mice: **p<0.01, ***p<0.001, ****p<0.0001). 

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.4 Blood lipid profiles of Apoe-KO(2)/hAPOC3 mice after ARO-APOC3 treatment under CD conditions. Under CD conditions, female Apoe-KO(2)/hAPOC3 mice exhibited significantly elevated levels of TG, T-CHO, and LDL-C, along with decreased levels of HDL-C, compared to hAPOC3 mice. Female WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice were maintained on CD feeding. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C) (n=5-12, Mean ± SEM, t-test, Apoe-KO(2) mice versus Apoe-KO(2)/hAPOC3 mice: #p<0.05, ##p<0.01; hAPOC3 mice versus Apoe-KO(2)/hAPOC mice: **p<0.01, ***p<0.001, ****p<0.0001). 

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.5 Oil Red O staining of aortas of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 in each group, 3 months old). No significant lipid accumulation was observed in aortas in WT mice, Apoe-KO(2) mice and hAPOC3 mice at 3 months of age under chow diet conditions. In contrast, Apoe-KO(2)/hAPOC3 mice exhibited slight lipid accumulation near the aortic arch at 3 months of age under chow diet conditions.

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.6 Oil Red O staining of aortas of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 in each group, 5 months old). No significant lipid accumulation was observed in the aortas of WT mice and hAPOC3 mice at 5 months of age under chow diet conditions. In contrast, slight lipid deposition was detected in Apoe-KO(2) mice, whereas Apoe-KO(2)/hAPOC3 mice showed significant lipid accumulation near the aortic arch at 5 months of age under chow diet conditions.

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.7 Oil Red O staining of aortas of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 or 4 in each group, 7 months old). No significant lipid accumulation was observed in the aortas of WT mice and hAPOC3 mice at 7 months of age under chow diet conditions. In contrast, Apoe-KO(2) mice and Apoe-KO(2)/hAPOC3 mice showed significant lipid accumulation near the aortic arch at 7 months of age under chow diet conditions.

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.8 Oil Red O staining of aortic valves of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice at 3, 5 and 7 months of age under CD conditions (n=3 or 4 in each group). The sacle bar is 0.5 mm. No lipid accumulation was observed in the aortic valves of WT or hAPOC3 mice at any age. In contrast, both Apoe-KO(2) and Apoe-KO(2)/hAPOC3 mice showed slight lipid accumulation in the aortic valves at 3 months of age, with a significant increase observed at 5 and 7 months.

Abbr. WT, wild-type; HO, homozygous; CD chow diet.

Fig.9 Oil Red O staining of aortas of female WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 in each group, 3 months old). No significant lipid accumulation was observed in aortas in all mice at 3 months of age under chow diet conditions. 

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.10 Oil Red O staining of aortas of female WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 in each group, 5 months old). No significant lipid accumulation was observed in the aortas of WT mice and hAPOC3 mice at 5 months of age under chow diet conditions. In contrast, slight lipid deposition was detected in Apoe-KO(2) mice and Apoe-KO(2)/hAPOC3 mice near the aortic arch at 5 months of age under chow diet conditions.

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.11 Oil Red O staining of aortas of female WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice under CD conditions (n=3 in each group, 7 months old). No significant lipid accumulation was observed in the aortas of hAPOC3 mice at 7 months of age under chow diet conditions. In contrast, Apoe-KO(2) mice and Apoe-KO(2)/hAPOC3 mice showed significant lipid accumulation near the aortic arch at 7 months of age under chow diet conditions.

Abbr. WT, wild-type; HO, homozygous; CD, chow diet.

Fig.12 Oil Red O staining of aortic valves of female WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice at 3, 5 and 7 months of age under CD conditions (n=2 or 3 in each group). No lipid accumulation was observed in the aortic valves of WT or hAPOC3 mice at any age. In contrast, both Apoe-KO(2) and Apoe-KO(2)/hAPOC3 mice showed slight lipid accumulation in the aortic valves at 3 months of age, with a significant increase observed at 5 and 7 months.

Abbr. WT, wild-type; HO, homozygous; CD chow diet.

2. Western diet conditions：

Fig.13 hAPOC3 levels in male Apoe-KO(2)/hAPOC3 mice under WD conditions. Under WD conditions, hAPOC3 was detectable in homozygous Apoe-KO(2)/hAPOC3 mice. Serum hAPOC3 levels in Apoe-KO(2)/hAPOC3 mice were comparable across 8 weeks to 18 weeks of age. Serum samples were collected following a 6-hour fast at different ages to measure levels of serum hAPOC3 (n=4-6, Mean ± SEM).

Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.14 Blood lipid profiles of Apoe-KO(2)/hAPOC3 mice after ARO-APOC3 treatment under WD conditions. Under WD conditions, male Apoe-KO(2)/hAPOC3 mice exhibited significantly elevated levels of TG, T-CHO, and LDL-C, along with decreased levels of HDL-C, compared to hAPOC3 mice. Male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice were maintained on WD feeding from 6 weeks of age. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A) triglycerides (TG), (B) total cholesterol (T-CHO), (C) high-density lipoprotein cholesterol (HDL-C) and (D) low-density lipoprotein cholesterol (LDL-C) (n=4-6, Mean ± SEM, t-test, Apoe-KO(2) mice versus Apoe-KO(2)/hAPOC3 mice: #p<0.05; hAPOC3 mice versus Apoe-KO(2)/hAPOC mice: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). 

Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.15 Oil Red O staining of aortas of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice after 7 weeks of WD feeding (n=2 in each group, 13 weeks old). No significant lipid accumulation was observed in the aortas of all mice after 7 weeks of WD feeding. WD feeding was start at 6 weeks of age.

Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.16 Oil Red O staining of aortas of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice after 12 weeks of WD feeding (n=2 in each group, 18 weeks old). After 12 weeks of WD feeding, no significant lipid accumulation was observed in the aortas of WT mice and hAPOC3 mice. Conversely, Apoe-KO(2) mice and Apoe-KO(2)/hAPOC3 mice showed significant lipid accumulation near the aortic arch. WD feeding was start at 6 weeks of age.

Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.17 Oil Red O staining of aortic valves of male WT C57BL/6 mice, HO Apoe-KO(2) mice, HO hAPOC3 mice and HO/HO Apoe-KO(2)/hAPOC3 mice after 7 weeks or 12 weeks of Western diet feeding (n=2 or 4 in each group). WD feeding was start at 6 weeks of age. No lipid accumulation was observed in the aortic valves of WT or hAPOC3 mice at any timepoint. In contrast, both Apoe-KO(2) and Apoe-KO(2)/hAPOC3 mice showed slight lipid accumulation in the aortic valves after 7 weeks of Western diet feeding, with a significant increase observed after 12 weeks of Western diet feeding.

Abbr. WT, wild-type; HO, homozygous; WD, Western diet.

Fig.18 Body weight of Apoe-KO(2)/hAPOC3 mice treated with ARO-APOC3 and atorvastatin. Apoe-KO(2)/hAPOC3 mice exhibited comparable body weight gain in each group. Male HO/HO Apoe-KO(2)/hAPOC3 mice were fed a Western diet (WD; Dyets, ASHF3) starting at 6 weeks of age. ARO-APOC3 was administered via subcutaneous injection at 13 and 16 weeks. Concurrently, atorvastatin was administered by oral gavage twice daily from 13 weeks of age until the time of euthanasia. Body weight and body weight gain were recorded weekly (n=7-10, Mean ± SEM). 

Abbr. WT, wild-type; HO, homozygous.

Fig.19 Blood lipid profiles of Apoe-KO(2)/hAPOC3 mice treated with ARO-APOC3 and atorvastatin. Treatment with ARO-APOC3 and atorvastatin significantly reduced serum TG, TC, and LDL-C levels. The combination therapy achieved a greater reduction in LDL-C compared with ARO-APOC3 monotherapy. Male HO/HO Apoe-KO(2)/hAPOC3 mice were fed a Western diet starting at 6 weeks of age. ARO-APOC3 was administered via subcutaneous injection at 13 and 16 weeks. Concurrently, atorvastatin was administered by oral gavage twice daily from 13 weeks of age until the time of euthanasia. Serum samples were collected following a 6-hour fast at different ages to measure levels of (A, E) triglycerides (TG), (B, F) total cholesterol (T-CHO), (C, G) high-density lipoprotein cholesterol (HDL-C) and (D, H) low-density lipoprotein cholesterol (LDL-C) (n=7-10, Mean ± SEM, t-test, G1 versus G2: *p<0.05, **p<0.01; G1 versus G3: #p<0.05, ##p<0.01, ####p<0.0001; G2 versus G4: †p<0.05; G3 versus G4: §p<0.05). 

Abbr. WT, wild-type; HO, homozygous.