Development of a Novel Murine Model of In‐Stent Neoatherosclerosis

Figure 1. Stented aorta‐carotid interposition grafting procedure. A, Stainless‐steel stent (2.5×0.7 mm) is deployed into the thoracic aorta of a donor mouse, then excised and interposition grafted into the carotid region of a recipient mouse. B, Under full isoflurane anesthesia, the stented vessel is fully exposed. C, A small incision is made at the proximal end. D, The imaging catheter is inserted.

Figure 2. Visualization of in‐stent neoatherosclerosis using bimodal high‐resolution OCT and fluorescence imaging with 3D reconstruction. A, OCT (gray) and fluorescence (color) cross‐sections of a stented vessel from the black dashed region of (G). B, OCT (gray) and fluorescence (color) cross‐sections of the stented vessel from the red dashed region of (G). C, Coregistered histological section of (A, scale bars: 250 μm); zoomed images in black frame (scale bar: 50 μm). D, Coregistered histological section of (B, scale bar: 250 μm); zoomed image in black frame (scale bar: 50 μm). E, CD68+ immunofluorescently‐stained coregistered section of (A, scale bar: 200 μm); zoomed image in red frame (scale bar: 50 μm). F, CD68+ immunofluorescently‐stained co‐registered section of (B, scale bar: 200 μm); zoomed images in red frame (scale bar: 50 μm). G, The 3D reconstruction of OCT + fluorescence images of the stented vessel obtained from a mouse fed HCD for 4 weeks post stenting (also see Video S2), red/yellow channel: OCT (intravascular structure), light green channel: ICG fluorescence (inflammation/instability). Red arrowheads mark stent struts, the blue dashed line outlines neointimal edges. 3D…

Figure 3. Distinct morphology of in‐stent neoatherosclerosis in murine model. A, Representative multiple‐stained cross‐sections of stented vessels grafted into Apoe −/− mice, and hematoxylin‐ and eosin‐stained cross‐sections of nonsurgical descending aortas of Apoe −/− mice (scale bars: 250 μm). B, Corresponding zoomed images in the black/red frame, black arrowheads mark extracellular lipids (white acellular regions), red arrowheads mark SMCs (scale bars: 50 μm). Quantitative histomorphometry of vessel sections showing the neointimal area (C), percentage of stenosis (D), and extracellular lipid content in neointimas (E). Data expressed as mean±SD. **P<0.01, ****P<0.0001 by 1‐way ANOVA with Bonferroni’s multiple comparisons or Brown–Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons (unequal variance). Apoe indicates apolipoprotein E; DA, descending aorta; HCD, high‐cholesterol diet; and SMCs, smooth muscle cells.

Figure 4. Compositional changes between in‐stent neoatherosclerosis and neointimal hyperplasia. A, Representative Masson’s trichrome‐stained cross‐sections of stented vessels grafted into Apoe −/− mice (scale bars: 250 μm). B, Corresponding zoomed images in the yellow frame, yellow arrowheads mark leukocytes (scale bars: 50 μm). C, Quantitative histomorphometry of vessel sections showing the surface‐adherent leukocyte count. D, Corresponding zoomed images in the black frame, blue arrowheads mark collagen (blue region), black arrowheads mark extracellular lipids (white regions) (scale bars: 50 μm). E, Quantitative histomorphometry of vessel sections showing the neointimal collagen content. F, Higher magnification images in red frames showing medias, red arrowheads mark vessel walls, yellow arrowheads mark leukocytes (scale bars: 50 μm). Quantitative histomorphometry of vessel sections showing the media area (G) and media lipid content (H). Data expressed as mean±SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by 1‐way ANOVA with Bonferroni’s multiple comparisons (parametric data) or Kruskal–Wallis test with Dunn’s multiple comparisons (nonparametric data) or Mann–Whitney U test for…

Figure 5. Reduced content of SMCs in neoatherosclerotic neointimas and medias. A, Representative α‐SMA‐stained cross‐sections of stented vessels and nonsurgical descending aortas of Apoe −/− mice (scale bars: 250 μm). B, Higher magnification images in black frames, black arrowheads mark α‐actin+ SMCs (red regions) in the neointima (scale bars: 50 μm). C, Higher magnification images in red frames, red arrowheads mark the vessel wall with α‐actin+ SMCs (scale bars: 50 μm). Quantitative histomorphometry of vessel sections showing the α‐actin+ SMC content in the neointima (D) and in the media (vessel wall) (E), respectively. Data expressed as mean±SD. *P<0.05, **P<0.01, ****P<0.0001 by 1‐way ANOVA with Bonferroni’s multiple comparisons (parametric data) or Kruskal–Wallis test with Dunn’s multiple comparisons (nonparametric data). Apoe indicates apolipoprotein E; α‐SMA, alpha‐smooth muscle actin; DA, descending aorta; HCD, high‐cholesterol diet; and SMCs, smooth muscle cells.

Figure 6. Increased macrophage content in in‐stent neoatherosclerotic neointimas and medias. A, Representative CD68‐stained cross‐sections of stented vessels grafted into Apoe −/− mice (scale bars: 200 μm). B, Higher magnification images in yellow frames, CD68+ cells (green) in the neointima (scale bars: 50 μm). C, Quantitative assessment of neointimal CD68+ cell content. D, Higher magnification images in red frames, red arrowheads mark the vessel wall with CD68+ cells inside (scale bars: 50 μm). E, Quantitative assessment of medial CD68+ cell content. Data expressed as mean±SD. *P<0.05, **P<0.01 by 1‐way ANOVA with Bonferroni’s multiple comparisons. Apoe indicates apolipoprotein E; and HCD, high‐cholesterol diet.

Figure 7. Altered cellular compositions of aortas after stenting. A, Monocytes, CD45+CD11b+Ly6C+. B, Macrophages, CD45+CD11b+F4/80+. C, M1 macrophages, CD45+CD11b+F4/80+CD86+CD206−. D, M2 macrophages, CD45+CD11b+F4/80+CD86−CD206+. E, CD36+ macrophages, CD45+CD11b+F4/80+CD36+. F, TREM2+ macrophages, CD45+CD11b+F4/80+TREM2+. G, CD47+ macrophages, CD45+CD11b+F4/80+CD47+. H, CCR7+ Macrophages, CD45+CD11b+F4/80+CCR7+. I, Endothelial cells, CD45−CD11b−F4/80−CD31+. J, SMC α‐actin, CD45−CD11b−F4/80−α‐SMA+. K, Fibroblasts, CD45−CD90+. Data expressed as mean±SD. *P<0.05, **P<0.01 by Kruskal–Wallis test with Dunn’s multiple comparisons or Mann–Whitney U test for comparisons between 2 groups (nonparametric data). α‐SMA indicates alpha‐smooth muscle actin; DA, descending aorta; HCD, high‐cholesterol diet; SMC, smooth muscle cell; and TREM, triggering receptor expressed on myeloid cells 2.