Ldlr-KO/Apoe-KO

Fig.1 Representative images of aortic atherosclerotic plaque in male Ldlr-KO/Apoe-KO mice and WT mice under CD conditions. 

Abbr. WT, wild type; CD, chow diet; M: month.

Fig.2 Phenotype of Ldlr-KO/Apoe-KO male mice under chow diet conditions.

(A) The body weight (n=8-10); 

(B) Blood lipid profile (n=6-10); 

(C) Masson staining of aortic valve; 

(D) Oil Red O staining of aortic valve and aorta. In aortic valve, the results indicated that no lipid accumulation was observed in the aortic valves of WT mice at 7 months of age. In contrast, all Ldlr-KO/Apoe-KO mice (2/2) exhibited significant lipid accumulation in the aortic valves as early as 3 months of age, with the severity of this phenotype progressively worsening with age (n=2); In aorta, the results indicated that at 7 months of age, WT mice exhibit no significant lesions in the aorta, while some Ldlr-KO/Apoe-KO mice (2/2) show fat accumulation near the aortic arch, with the severity of this phenotype progressively worsening with age (n=2); 

(E) Representative pictures of aortic valve. In WT mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in male Ldlr-KO/Apoe-KO mice, extensive plaque formation was observed in the aortic valve, protruding into the lumen. Numerous interwoven collagen fibers are present, along with abundant infiltration of foam cells (yellow arrows). A small amount of cholesterol crystals (green arrows), appearing as needle-shaped voids, are visible, accompanied by a small amount of necrotic tissue (cyan arrows). A small number of infiltrating lymphocytes (blue arrows) and macrophage (purple arrows) were also noted. Myocardial cells display loose and irregular arrangement. The black box indicates the location of the magnified view (n=2).

Abbr. WT, wild type; CD, chow diet; M: month. (Mean±SEM, Unpaired t-test, *p < 0.05, **p < 0.01，***p < 0.001).

Fig.3 Phenotype of Ldlr-KO/Apoe-KO female mice under chow diet conditions.

(A) The body weight (n=8-10); 

(B) Blood lipid profile (n=6-10); 

(C) Masson staining of aortic valve; 

(D) Oil Red O staining of aortic valve and aorta. In aortic valve. The results indicated that no lipid accumulation was observed in the aortic valves of WT mice at 7 months of age. In contrast, all Ldlr-KO/Apoe-KO mice (2/2) exhibited significant lipid accumulation in the aortic valves as early as 3 months of age, with the severity of this phenotype progressively worsening with age (n=2); In aorta, the results indicated that at 7 months of age, WT mice exhibit no significant lesions in the aorta, while some Ldlr-KO/Apoe-KO mice (2/2) show fat accumulation near the aortic arch, with the severity of this phenotype progressively worsening with age (n=2); 

(E) Representative pictures of aortic valve. In WT mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in female Ldlr-KO/Apoe-KO mice, extensive plaque formation（green polygon）was observed in the aortic valve, protruding into the lumen. Numerous interwoven collagen fibers are present (orange arrows), along with abundant infiltration of foam cells (yellow arrows). A small amount of cholesterol crystals (green arrows), appearing as needle-shaped voids, are visible, accompanied by a small amount of necrotic tissue (cyan arrows). A small number of infiltrating lymphocytes (blue arrows) and macrophage (purple arrows) were also noted. Myocardial cells display loose and irregular arrangement. The black box indicates the location of the magnified view (n=2). 

Abbr. WT, wild type; CD, chow diet; M: month. (Mean±SEM, Unpaired t-test, *p<0.05, **p<0.01，***p<0.001).

Fig.4 Phenotype of Ldlr-KO/Apoe-KO male mice under high fatty diet conditions. 

(A) The body weight (n=6-10); 

(B) Blood lipid profile (n=6-10). Under HFD conditions, male Ldlr-KO/Apoe-KO mice exhibited elevated levels of triglycerides (TG), total cholesterol (T-CHO), and low-density lipoprotein cholesterol (LDL-C) compared to WT mice, while high-density lipoprotein cholesterol (HDL-C) levels were decreased;

(C) Oil Red O staining of aortic valve. The results indicated that no lipid accumulation was observed in the aortic valves of WT mice at 7 and 17 weeks of HFD. In contrast, all Ldlr-KO/Apoe-KO mice (2/2) exhibited significant lipid accumulation in the aortic valves as early as 7 weeks of HFD, with the severity of this phenotype progressively worsened with age (n=2);

(D) Oil Red O staining of aortic valve and aorta. In aortic valve (n=2-3);

(E) Masson staining of aortic valve in male Ldlr-KO/Apoe-KO mice and WT mice under HFD conditions. Muscle fibers appear red, while collagen fibers are stained blue. Ldlr-KO/Apoe-KO mice exhibited increased collagen content, suggesting enhanced plaque stability (n=2).

(F) Representative pictures of aortic valve. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed at 17 weeks of HFD. However, in homozygous Ldlr-KO/Apoe-KO mice, extensive plaque formation was observed in the aortic valve, protruding into the lumen. A few interwoven collagen fibers are present (brown arrows), along with a scant accumulation of foam cells (yellow arrows). A small amount of cholesterol crystals, appearing as needle-shaped voids (blue arrows), are visible, accompanied by a small amount of necrotic tissue (purple arrows). A small number of infiltrating lymphocytes (cyan arrows) were also noted. Myocardial cells display loose and irregular arrangement. The black box indicates the location of the magnified view (n=2).

Abbr. WT, wild type; HO, homozygous; HFD, high-fat diet; W: week.

Note. HFD started at 6 weeks of age. 

Fig.5 Phenotype of Ldlr-KO/Apoe-KO female mice under high fatty diet conditions. 

(A) The body weight (n=6-10); 

(B) Blood lipid profile (n=6-10);

(C) Oil Red O staining of aortic valve. The results indicated that no lipid accumulation was observed in the aortic valves of WT mice at 7 and 17 weeks of HFD. In contrast, all Ldlr-KO/Apoe-KO mice (2/2) exhibited significant lipid accumulation in the aortic valves as early as 7 weeks of HFD, with the severity of this phenotype progressively worsened with age (n=2);

(D) Oil Red O staining of aortic valve and aorta. In aortic valve (n=2-3).

(E) Masson staining of aortic valve in female Ldlr-KO/Apoe-KO mice and WT mice under HFD conditions. Muscle fibers appear red, while collagen fibers are stained blue. Ldlr-KO/Apoe-KO mice exhibited increased collagen content, suggesting enhanced plaque stability (n=2).

(F) 10. Representative pictures of aortic valve. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed at 28 weeks of HFD. However, in homozygous Ldlr-KO/Apoe-KO mice, extensive plaque formation was observed in the aortic valve, protruding into the lumen. A few interwoven collagen fibers are present (green arrows), along with a scant accumulation of foam cells (orange arrows). A small amount of cholesterol crystals, appearing as needle-shaped voids (blue arrows), are visible, accompanied by a small amount of necrotic tissue (purple arrows). A small number of infiltrating lymphocytes (brown arrows) were also noted. Myocardial cells display loose and irregular arrangement. The black box indicates the location of the magnified view (n=2).

Abbr. WT, wild type; HO, homozygous; HFD, high-fat diet; W: week.

Note. HFD started at 6 weeks of age.