A robust and aggressive mouse model of aortic stenosis. (A) Ultrasound B-mode images (upper panels) and histological pictures (lower panels) of aortic valves in WT and LDLR mice on HFD. Yellow lines and black lines outline the AVs; arrows indicate AV leaflets. (B) Total valve area measured from echocardiographic images (n = 14 WT; n = 9 LDLR) and from histological images (n = 14 WT; n = 19 LDLR) of AV from mice at 6 months on HFD. (C) Pearson correlation of AV areas measured from echocardiography and histology (r = 0.8626; P < .0001; n = 16). (D-E) Immunohistochemistry for α-1 type I collagen (n = 4 WT; n = 5 LDLR) and picrosirius red staining for collagen (n = 8 WT; n = 17 LDLR) in AV from WT and LDLR mice after 6 months on HFD. Quantification of collagen staining in the valves by immunohistochemical (left) and picrosirius red (right) staining. (F) Pearson correlation of collagen positive area measured from immunohistochemical and picrosirius red-stained images of AV in LDLR mice after 6 months on HFD (r = 0.87; P = .055; n = 5). (G) Total TGF-β1 levels in the plasma of LDLR and WT mice at 0, 3, and 6 months on HFD, as measured by ELISA. (H) Pearson correlation of WSS across aortic valves and plasma TGF-β1 levels in LDLR mice at 6 months on HFD (r = 0.63; P = .002; n = 20). (I) Representative images of immunofluorescent staining for p-Smad2 in aortic valves from WT and LDLR mice after 6 months on HFD and quantification of p-Smad2–positive nuclei in the aortic valves of WT (n = 5) and LDLR (n = 10) mice after 6 months of HFD (see supplemental Figure 6 for individual valves with p-Smad2 staining). Two-tailed unpaired Student t test with Welch’s correction was used to compare 2 groups with different sample sizes. Data represented as mean ± SEM throughout.