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. Author manuscript; available in PMC: 2021 Aug 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2020 May 28;40(8):1838–1853. doi: 10.1161/ATVBAHA.118.314087

Figure 5. S100A9 expressed by macrophages regulates vascular calcification in diabetic Apoe−/− mice.

Figure 5.

(A) Macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9) were injected via tail vein, and F4/80 positive splenic macrophages were isolated. S100A9 mRNA was quantified by real-time PCR. (left panel, n = 3 per each group). Fluorescent in situ hybridization shows less S100A9 mRNA expression localized to macrophage-positive cells in Apoe−/− mice injected with macrophage-targeted LNP containing siS100a9. White arrows show the co-localization of S100A9 mRNA in macrophage-positive cells. One of 4 animals per group is shown (right panel). Scale bars: 20 μm. (B) mRNA expression of S100A9, inflammatory cytokines, and osteogenic factors was measured in splenic macrophages from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl (No diabetes and Diabetes) and siS100a9 (Diabetes with siS100a9) (n = 13, No diabetes and Diabetes with siS100a9; n = 16, Diabetes). (C) Immunohistochemical evaluation of ALP activity in aortic plaques from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 14, No diabetes and Diabetes with siControl; n = 13, No diabetes with siControl; n = 11, Diabetes with siS100a9). Scale bars: 100 μm. (D) In vivo molecular imaging of carotid arteries from diabetic Apoe−/− mice. Vascular calcification assessed by osteogenic activity, OsteoSense680 (red) and vascular inflammation assessed by proteolytic activity, ProSense750 (green). White arrow shows the co-localization of calcification and inflammation, indicating the formation of microcalcification (yellow). The image represents 7–10 mice per group. Scale bar = 200 μm. (E) Molecular imaging of aortic vascular calcification assessed by OsteoSense680 and vascular inflammation assessed by ProSense750. (n= 13–18). The graph shows the SUM intensity related to total aortic area for FRI in diabetic and non-diabetic Apoe−/− mice treated with macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9). (F) 3D micro-CT scanning of aorta from diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 4, per each group). The graph shows the mean number of microcalcifications assessed by size contribution. P value was calculated by paired student’s t-test or two-way ANOVA followed by Bonferroni test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.