Fig. 5.
Fig. 5. Gene therapy of ApoE−/− mice using a CD68S retroviral vector. / (A) ApoE expression by thioglycollate-elicited macrophages from wild-type mice (ApoE+/+) orApoE−/− mice that had received transplants of HSCs transduced with either CD68S–HA-EGFP or CD68s-ApoE retroviruses was assessed by immunoblotting of lysates (CELL) or supernatants (SUP) from macrophages cultured ex vivo for 24 hours using an antibody recognizing ApoE (α-ApoE). A similar blot probed with an antibody against macrosialin (α-mCD68) served as loading control, and blotting with an antibody recognizing the HA epitope (α-HA) showed expression of HA-EGFP only in macrophages fromApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP retrovirus. (B) Serum cholesterol was measured in samples obtained from bleeds performed 6 and 12 weeks after HSC transplantation. Graphs show the mean serum cholesterol ± SD (n = 10). *P < .0001 as measured by the Mann-Whitney nonparametric test. (C) ApoE levels in sera fromApoE−/− mice that had received transplants of HSCs transduced with CD68S-ApoE retrovirus were measured by densitometric analyses of immunoblots performed with an antibody against ApoE. The graphs show mean ApoE expression ± SD (n = 10) expressed as a percentage of ApoE present in serum from anApoE+/+ control mouse. (D) A plot of serum cholesterol and ApoE values from individual mice reveals an inverse, linear relationship (R = −0.86, p = .003, by Spearman rank correlation test). (E) Quantitative measurement of atherosclerosis in the aortic root inApoE−/− mice 12 weeks after transplantation. Each point (open circle) represents the mean lesional area (μm2 × 103 per section) from an individual mouse. A horizontal line indicates the average lesion area for each group of mice. **P < .0001 as measured by the Mann-Whitney nonparametric test (n = 10). (F-G) Representative Oil Red O–stained sections from the aortic root ofApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP (F) or CD68S-ApoE (G) retroviruses. (H-K) Immunohistochemical analysis of similar lesions fromApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP (H-I) or CD68S-ApoE (J-K) retroviruses reveals comparable levels of macrophage staining with an antibody recognizing MAC-2 (H,J), but only macrophages in lesions from mice receiving CD68S–HA-EGFP–transduced HSCs show reactivity with an antibody against the HA epitope (I,K). Original magnifications: × 10, F-G; × 40, H-K.

Gene therapy of ApoE−/− mice using a CD68S retroviral vector.

(A) ApoE expression by thioglycollate-elicited macrophages from wild-type mice (ApoE+/+) orApoE−/− mice that had received transplants of HSCs transduced with either CD68S–HA-EGFP or CD68s-ApoE retroviruses was assessed by immunoblotting of lysates (CELL) or supernatants (SUP) from macrophages cultured ex vivo for 24 hours using an antibody recognizing ApoE (α-ApoE). A similar blot probed with an antibody against macrosialin (α-mCD68) served as loading control, and blotting with an antibody recognizing the HA epitope (α-HA) showed expression of HA-EGFP only in macrophages fromApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP retrovirus. (B) Serum cholesterol was measured in samples obtained from bleeds performed 6 and 12 weeks after HSC transplantation. Graphs show the mean serum cholesterol ± SD (n = 10). *P < .0001 as measured by the Mann-Whitney nonparametric test. (C) ApoE levels in sera fromApoE−/− mice that had received transplants of HSCs transduced with CD68S-ApoE retrovirus were measured by densitometric analyses of immunoblots performed with an antibody against ApoE. The graphs show mean ApoE expression ± SD (n = 10) expressed as a percentage of ApoE present in serum from anApoE+/+ control mouse. (D) A plot of serum cholesterol and ApoE values from individual mice reveals an inverse, linear relationship (R = −0.86, p = .003, by Spearman rank correlation test). (E) Quantitative measurement of atherosclerosis in the aortic root inApoE−/− mice 12 weeks after transplantation. Each point (open circle) represents the mean lesional area (μm2 × 103 per section) from an individual mouse. A horizontal line indicates the average lesion area for each group of mice. **P < .0001 as measured by the Mann-Whitney nonparametric test (n = 10). (F-G) Representative Oil Red O–stained sections from the aortic root ofApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP (F) or CD68S-ApoE (G) retroviruses. (H-K) Immunohistochemical analysis of similar lesions fromApoE−/− mice that had received transplants of HSCs transduced with CD68S–HA-EGFP (H-I) or CD68S-ApoE (J-K) retroviruses reveals comparable levels of macrophage staining with an antibody recognizing MAC-2 (H,J), but only macrophages in lesions from mice receiving CD68S–HA-EGFP–transduced HSCs show reactivity with an antibody against the HA epitope (I,K). Original magnifications: × 10, F-G; × 40, H-K.

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