Apoe-KO/Nos3-KO

Fig.1 Detection of Apoe/Nos3 expression in mammary gland by RT-PCR. 

Wild type: one band at 987 bp with primers F1/R1(mApoe), one band at 113 bp with primers F2/R2(mNos3) and one band at 123 bp with primers F/R(mGapdh).

Homozygous: one band at ~150bp with primers F1/R1(mApoe) and one band at 123 bp with primers F/R(mGapdh).

Heterozygous: two bands at 987 bp/~150bp with primers F1/R1(mApoe) and one band at 123 bp with primers F/R(mGapdh).

Abbr. M, DNA marker; HE, heterozygous; HO, homozygous; WT, wild type.

Fig.2 Detection of mouse APOE expression in Apoe-KO/Nos3-KO mice by WB. Various tissues lysates were collected from wild-type C57BL/6 mice (WT, male, 6 weeks old) and heterozygous (HE/HO, male, 6 weeks old) and homozygous Apoe-KO/Nos3-KO mice (HO/HO, female, 5 weeks old), and then analyzed by western blot with anti-APOE antibody (Abcam, ab183596). 20 μg of total proteins were loaded for western blotting analysis. 

· Chow conditions

Fig.3 The body weight curve of Apoe-KO/Nos3-KO mice under CD conditions (n=3-10 males).

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

Fig.4 The blood pressure of male C57BL/6, Apoe-KO, Nos3-KO, and Apoe-KO/Nos3-KO mice under CD conditions. Blood pressure measurements were performed in mice fed a chow diet when they reached 16 and 24 weeks of age. The results demonstrated that under a standard chow diet, both C57BL/6 and Apoe-KO mice maintained blood pressure within the normal range, whereas Nos3-KO and Apoe-KO/Nos3-KO mice exhibited a characteristic hypertensive phenotype of the same age.(n=3-10 males, Mean±SEM, Unpaired t-test, *P < 0.05, ***P < 0.001).

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

Fig.5 Lipid profile biochemical markers of male C57BL/6, Apoe-KO, NOS3-KO, and Apoe-KO/Nos3-KO mice under CD conditions. Blood lipids were measured and analyzed in mice fed a chow diet when they reached 16 and 24 weeks of age (n=3-10 males, Mean±SEM, Unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

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

Fig.6 Oil Red O staining of aortic valve of male C57BL/6, Apoe-KO, and Apoe-KO/Nos3-KO mice under CD conditions. To assess lipid deposition, aortic roots were harvested from mice at 16 and 24 weeks of age, followed by fixation, paraffin embedding, and Oil Red O staining (n=3 males). The Oil Red O staining results indicated that no lipid accumulation was observed in the aortic valves of WT C57BL/6 mice, while all Apoe-KO mice (3/3) and Apoe-KO/Nos3-KO mice (3/3) exhibited lipid accumulation in the aortic valves at 16 and 24 weeks of age, with the condition being more pronounced in the Apoe-KO/Nos3-KO mice.

Fig.7 Oil Red O staining of aorta in male C57BL/6, Apoe-KO, and Apoe-KO/Nos3-KO mice under CD conditions. To assess lipid deposition, aortic were harvested from mice at 16 and 24 weeks of age, followed by fixation and Oil Red O staining. The Oil Red O staining results indicated that WT C57BL/6 mice exhibit no significant lesions in the aorta, while Apoe-KO mice (3/3) and Apoe-KO/Nos3-KO mice (3/3) show fat accumulation near the aortic arch, with the condition being more pronounced in the Apoe-KO/Nos3-KO mice (n=3 males).

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

Fig.8 Histopathological Characteristics of aortic valve in C57BL/6, Apoe-KO, and Apoe-KO/Nos3-KO Mice under CD conditions. The black box indicates the location of the magnified view. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in Apoe-KO and Apoe-KO/Nos3-KO mice, the aortic wall exhibits marked heterogeneity in thickness and irregular morphology. Subendothelial regions display focal accumulations of foam cells (blue arrows) that protrude into the lumen. There is a substantial increase in fibrous connective tissue, and a large number of cholesterol crystals (yellow arrows) are observed, appearing as needle-shaped voids. Occasional necrotic cellular debris is also noted (green arrows). Additionally, the smooth muscle cells in the tunica media are arranged in a disordered pattern (n=3 males, 16 weeks old).

Fig.9 Histopathological Characteristics of aortic valve in Apoe-KO and Apoe-KO/Nos3-KO Mice under CD conditions. The black box indicates the location of the magnified view. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in Apoe-KO and Apoe-KO/Nos3-KO mice, the surface of the blood vessels within the cardiac tissue is uneven and irregular in thickness. Medium-sized plaques protrude into the lumen, with a small amount of fibrous connective tissue proliferation observed on the plaque surface (brown arrows). Small accumulations of foam cells (green arrows) are present, and the plaque contains occasional needle-like fissures (blue arrows) that are suspected to be cholesterol crystals. A few necrotic cellular fragments (purple arrows) are also visible. Minor brownish-yellow pigment deposition (orange arrows) can be observed. The surrounding myocardial cells are loosely arranged, with a small number of infiltrating lymphocytes and granulocytes (blue arrows) (n=3 males, 24 weeks old).

Fig.10 H&E staining of liver in Apoe-KO and Apoe-KO/Nos3-KO Mice under CD conditions. HE staining results revealed that under a normal chow diet, no evident features of steatosis, lobular inflammation, hepatocyte ballooning, or fibrosis stage were observed in the livers of Nos3-KO and Apoe-KO/Nos3-KO mice at 16 and 24 weeks of age.

Fig.11 H&E staining of kidney tissue under CD conditions. At 16 and 24 weeks of age, kidney tissues were collected, fixed, and subsequently subjected to HE staining. HE staining results revealed that at 16 weeks of age, compared with WT C57BL/6 mice, Nos3-KO and Apoe-KO/Nos3-KO mice all exhibited a marked phenotype of hypertensive nephropathy. This phenotype was characterized by glomerular vacuolation, renal interstitial fibrosis, and increased inflammatory cell infiltration. At 24 weeks of age, however, the differences among groups were not statistically significant.

· HFD conditions

Fig.12 The body weight curve of Apoe-KO/Nos3-KO mice under HFD conditions. A high-fat diet was initiated in mice at 8 weeks of age (n=3-10 males).

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

Fig.13 The blood pressure of male C57BL/6, Apoe-KO, Nos3-KO, and Apoe-KO/Nos3-KO mice under HFD conditions. Blood pressure measurements were performed in mice fed a high-fat diet when they reached 16 and 24 weeks of age. A high-fat diet was initiated in mice at 8 weeks of age. The results demonstrated that under a high-fat diet, the blood pressures of Nos3-KO and Apoe-KO/Nos3-KO mice had a typical hypertensive phenotype than those of WT mice of the same age (n=3-10, Mean±SEM, Unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001). 

Fig.14 Lipid profile biochemical markers of male C57BL/6, Apoe-KO, NOS3-KO, and Apoe-KO/Nos3-KO mice under HFD conditions. Analysis of serum lipid profiles revealed that under a high-fat diet, the TC and LDL levels of Apoe-KO mice and Apoe-KO/Nos3-KO mice at 16 and 24 weeks of age were significantly higher than those of WT mice in the same period. Blood Lipid profile measurements were performed in mice fed a high-fat diet when they reached 16 and 24 weeks of age. A high-fat diet was initiated in mice at 8 weeks of age (n=3-10, Mean±SEM, Unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig.15 Oil Red O staining of aortic valve of male C57BL/6, Apoe-KO, and Apoe-KO/Nos3-KO mice under HFD conditions. The Oil Red O staining results indicated that under a high-fat diet, no lipid accumulation was observed in the aortic valves of WT C57BL/6 mice, while all Apoe-KO mice (3/3) and Apoe-KO/Nos3-KO mice (3/3) exhibited marked lipid accumulation in the aortic valves at 8 and 16 weeks of HFD, with the condition being more pronounced in the Apoe-KO/Nos3-KO mice. To assess lipid deposition, aortic roots were harvested from 8 and 16 weeks of HFD mice, followed by fixation, paraffin embedding, and Oil Red O staining. A high-fat diet was initiated in mice at 8 weeks of age (n=3 males).

Fig.16 Oil Red O staining of aorta in male C57BL/6, Apoe-KO, and Apoe-KO/Nos3-KO mice under HFD conditions. The Oil Red O staining results indicated that WT C57BL/6 mice exhibit no significant lesions in the aorta, while Apoe-KO mice (3/3) and Apoe-KO/Nos3-KO mice (3/3) show fat accumulation near the aortic arch, with the condition being more pronounced in the Apoe-KO/Nos3-KO mice. To assess lipid deposition, aortic were harvested from 8 and 16 weeks of HFD mice, followed by fixation and Oil Red O staining. A high-fat diet was initiated in mice at 8 weeks of age (n=3 males).

Fig.17 Histopathological Characteristics of aortic valve in Apoe-KO and Apoe-KO/Nos3-KO Mice under HFD conditions. The black box indicates the location of the magnified view. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in Apoe-KO and Apoe-KO/Nos3-KO mice, the aortic wall exhibits marked heterogeneity in thickness and irregular morphology. Extensive atherosclerotic plaque formation is observed beneath the endothelial cells, protruding into the lumen. Within the plaques, large aggregations of foam cells (blue arrows) are present, accompanied by substantial proliferation of fibrous connective tissue. Numerous cholesterol crystals (yellow arrows) are visible, appearing as needle-shaped voids. Small amounts of necrotic cellular debris (green arrows) are also noted. Additionally, the smooth muscle cells in the tunica media are arranged in a disordered pattern (n=3 males, 16 weeks old). A high-fat diet was initiated in mice at 8 weeks of age. 

Fig.18 Histopathological Characteristics of aortic valve in Apoe-KO and Apoe-KO/Nos3-KO Mice under HFD conditions. The black box indicates the location of the magnified view. In WT C57BL/6 mice, no significant plaque formation, necrosis, or inflammatory cell infiltration was observed. However, in Apoe-KO and Apoe-KO/Nos3-KO mice, The vascular surfaces within the myocardial tissue are markedly irregular, displaying variable thickness and extensive atheromatous plaque formation protruding into the lumen. The plaque surface exhibits a modest degree of fibrous connective tissue hyperplasia (indicated by brown arrows). Notably, there is a substantial accumulation of foam cells (green arrows) and numerous pinhole-like fissures (blue arrows), which are presumed to represent cholesterol crystals. Additionally, scant necrotic cellular debris (purple arrows) is observed within the plaque. The adjacent myocardial cells appear loosely arranged, accompanied by a mild infiltration of lymphocytes (blue arrows) (n=3 males, 24 weeks old). A high-fat diet was initiated in mice at 8 weeks of age.