Fig 1: Survival rates and possible reasons for the death of hApoE2-PD and ApoE KO-PD rats. (A,B) The survival rates of hApoE2 and ApoE KO rats fed with the ND or PD are plotted and compared (hApoE, n = 8 each group; ApoE KO, n = 6 each group). (C) White color fatty embolus was detected in formalin-fixed hApoE2-PD aorta from autopsy (left gross image). Such fatty embolus was HE stained after paraffin preparation, shown in the right photomicrograph image. Black arrow indicates the fatty embolus in the aorta. (D) ApoE KO-PD aorta full of blood clot was HE stained. Blood cells were red stained, and small emboli were pink stained (black arrow). (E1,E2) Representative right ventricles of hApoE2-PD rats were HE stained. (F) Representative right ventricle of ApoE KO-PD rat was HE stained. Representative HE-stained slices of the hApoE2-PD (G1,G2) and ApoE KO-PD (H1,H2) lungs are shown. Neutrophils (blue), monocytes (purple), type II pneumocytes or alveolar macrophages (orange), and cell debris (gray).
Fig 2: Mild atherosclerotic plaque formation in hApoE2 and ApoE KO rats. (A) Representative photographs of Oil Red O-stained en face aortas from rats aged 6 months are shown. (B) Plaque lesion areas in cross-sectional aortic roots of different rats were quantified at age 6 months. (n = 6 per group, One-way ANOVA followed by a Tukey post hoc test. a vs. b, significant difference p < 0.05). (C) Representative photomicrographs of Oil Red O-stained cross-sectional aortic roots are shown.
Fig 3: Generation and analysis of cell-specific ApoE knockout mice in vivo(A) Cell-specific ApoE−/− mice were generated using the cre/lox system. For CD11c+-specific knockout mice, ApoEfl/fl mice were bred to CD11ccre+ mice. For obtaining mice with liver-knocked out ApoE, Albumincre+ mice were bred to ApoEfl/fl mice.(Band C) Western blot (n = 5 animals per group. ∗p<0.05.) and (C) ELISA (n = 5–7 animals per group. ∗p<0.05.) analysis of sera for ApoE showed that ApoE levels in CD11ccre+ApoEfl/fl mice were reduced by about 25%, whereas in Albcre+ApoEfl/fl mice we observed an even more pronounced and significant reduction of ApoE reflecting the bulk ApoE production in the liver.(D and E) CD11ccre+ApoEfl/fl, Albcre+ApoEfl/fl, and ApoE−/− mice were fed with HC diet for a period of 12 weeks and aortae were perfused with PBS, fixed with 4%PFA and dissected vessels, free from adventitia and surrounding fat tissue, were stained with Oil Red O. (D) Plaque area was measured and the analysis showed significantly reduced plaque development in Albcre+ApoEfl/fl compared with ApoE−/− mice. n = 5 animals per group. ∗p<0.05. (E) In CD11ccre+ApoEfl/flmice, plaque development was significantly increased compared to cre− littermates. ApoE−/− mice served as positive ctrl. n = 5 animals per group. ∗p<0.05.(F) IL-1β levels in sera of CD11ccre+ApoEfl/fl and Albumincre+ApoEfl/fl were analyzed by ELISA. IL-1β levels appeared significantly enhanced after cell-specific knockdown in both CD11c+ and liver cells compared to cre− animals. Data are the mean ± SD.
Fig 4: Increased lipid deposition and hepatic nodular cirrhosis in hApoE2 rats. (A) Oil Red O staining was used to assess the neutral fat deposit in rat lungs with 8 µm frozen sectioning. Increased Oil Red O-stained cells were detected in the lung tissue of hApoE2 and ApoE KO rats. Anti-CD68 antibody stained these lipid-laden cells. (B) Fat deposit in renal medulla was stained by Oil Red O staining. Red blood cells differentiate vessels from tubules in the renal medulla. (C) The hepatic nodular tissue of hApoE2 rats was located on the top of the liver and clinging to the diaphragm. Some of the hepatic nodular tissue even bumped into the chest. White arrow indicates the hepatic nodular tissue. (D) Incidence of hepatic nodular change in hApoE2 rats is indicated. (E) The histological abnormalities in hepatic nodular tissue of hApoE2 showed changes in hepatic nodular cirrhosis, including hepatic cords without the classical radial structure, bile duct hyperplasia, increased plasma cells (blue arrow), vacuole-like droplets (lipid droplets after staining processing), and hepatic fibrosis. The hepatic structure in areas other than nodular change is presented in Figure S1E. Masson staining was used to assess fibrosis, and HE staining was used for the rest of the slides.
Fig 5: CD11c+ cells showed increased cholesterol efflux and were able to secrete ApoE after exposure to acLDL(A–C) BM-derived CD11c+ cells were screened for expression of different genes relevant for cholesterol export using qPCR. n = 5 per group. ∗p<0.05 vs ctrl. In WT BM-derived CD11c+ cells, LXR was significantly upregulated upon treatment with acLDL (A) as well as its downstream targets ABCA1 and ABCG1 (B) and ApoE itself (C).(D) In BM-derived CD11c+ cells isolated from LXR−/− mice, ApoE mRNA was significantly reduced compared with WT BM-derived CD11c+ cells.(E) BM-derived CD11c+ cells of WT mice were screened on day 7 of culture for their ability to secrete ApoE upon loading with acLDL. Culture supernatant was objected to western blotting 24 h after treatment with acLDL and screened for ApoE. AcLDL-treated BM-derived CD11c+ cells showed significantly increased levels of secreted ApoE. Blots were quantified by densitometry. n = 5, ∗p<0.05.(F) Cholesterol efflux analysis of BM-derived CD11c+ cells revealed that cholesterol efflux was significantly enhanced if these cells were exposed to an atherosclerotic environment (loading with acLDL). (C–F) Data are the mean ± SD.
Supplier Page from Abcam for Anti-Apolipoprotein E antibody [EPR19378]