Figure 7. Plasma cholesterol levels and atherosclerotic lesion progression/regression regulated by Dox in ApoE iKO mice.

(A) Plasma cholesterol levels in the ApoE iKO, KO and WT group mice in the absence and presence of Dox. (Littermate mice of various genotypes other than homozygous KO or iKO, i.e. ApoE+/+, ApoE+/−, ApoE+/+;TRE-ApoE and ApoE+/−;TRE-ApoE, all displayed normal and indistinguishable blood cholesterol levels, and never developed lesions under any treatment regime used in our study. Consequently they were lumped together as the “WT group”.) Plasma cholesterol levels were first measured in mice fed with normal food without Dox (−Dox) and both ApoE iKO and KO mice showed significantly higher cholesterol levels compared to WT group mice (p = 0.35 between iKO and KO, p<0.0001 between iKO and WT or between KO and WT, Student's t-test). The mice were then switched to Dox-containing food, and plasma cholesterol levels were measured again 4 days (+Dox 4d) and 7 days (+Dox 7d) later. Cholesterol level of ApoE iKO mice dropped to WT levels in less than 4 days while that of ApoE KO remained high (p<0.0001 between iKO and KO or between KO and WT, p = 0.86 between iKO and WT). Sometime later, some Dox-treated mice were switched back to normal food, and plasma cholesterol levels were measured again 4 days (−Dox 4d) and 7 days (−Dox 7d) after Dox withdrawal. Cholesterol level of ApoE iKO mice significantly elevated by day 4 (p<0.001 between iKO and WT, p<0.01 between iKO and KO) and approached pre-Dox treatment level by day 7 (p<0.0001 between iKO and WT, p = 0.13 between iKO and KO). ***P<0.001. (B) Atherosclerotic lesion progression. Aortas were stained with Sudan IV to visualize the lesions in red. The arch region of the aorta contains the most extensive areas of lesions and is shown here. A group of iKO, KO and WT mice were treated with Dox-containing food starting before the onset of lesions, and were compared with mice fed with normal food. At 7 months of age, aortas were dissected from these mice and lesions were examined. ApoE iKO mice showed extensive aortic lesions as the KO mice in the absence of Dox, and yet no lesions at all as the WT mice in the presence of Dox. (C) Atherosclerotic lesion regression. ApoE iKO and KO mice of 5 months of age were switched from normal food to Dox-containing food. Aortic lesions were examined before (at 5 months) and after (at 9 months) the Dox treatment. After 4 months of Dox treatment, the lesions in KO mice continued to grow, whereas in the iKO mice the lesions had regressed. (D) Quantification of the aortic atherosclerotic lesion areas in the arch region above the first intercostal artery, as expressed by the percentage of lesion areas versus the whole aortic area in this segment. All genotypes are matched with ages for different Dox treatment. Dox-treated groups are: iKO+Dox: Dox food started at 2–4 months of age (before the onset of atherosclerosis); iKO+Dox regression: Dox food started at 5–6 months of age (after the onset of atherosclerosis); KO+Dox: Dox food started at 2–4 months of age (before the onset of atherosclerosis). The results are compared using one-way ANOVA followed by Neuman-Keul's post hoc test. Both iKO+Dox and iKO+Dox regression groups had significantly reduced atherosclerotic areas compared to the remaining groups. *P<0.05, **P<0.01. The iKO mice without Dox, as well as KO mice either with or without Dox, all developed comparable areas of lesions (p>0.05 in all pair-wise comparisons). The iKO mice treated with Dox before the onset of atherosclerosis had nearly no lesions and were significantly different from the above groups (p<0.01 iKO+Dox versus iKO−Dox; p<0.01 iKO+Dox versus KO−Dox; p<0.05 iKO+Dox versus KO+Dox). The iKO mice treated with Dox after the onset of atherosclerosis had significantly reduced atherosclerotic areas compared to iKO mice without Dox or KO mice either with or without Dox (p<0.05 in all comparisons between iKO+Dox regression versus iKO−Dox, KO−Dox or KO+Dox).