Figure 6.

Vascular smooth muscle cell (VSMC)-specific Txnip (thioredoxin-interacting protein) ablation recapitulates the calcification-rich plaque phenotype of whole-body Txnip knockout mice (Txnip KO) mice. A, Schematic illustration of the experiment. Tagln-cre; Txnip^flox/flox (SMC^KO) mice and their littermate controls that did not have Tagln-cre (SMC^WT) were subjected to atherosclerosis. The mice were injected with PCSK9-AAV (adeno-associated virus serotype 8 encoding mouse proprotein convertase subtilisin/kexin type 9) and fed high fat diet (HFD) for 16 weeks. n of SMC^WT/SMC^KO mice=12/15. B and C, Validation of SMC-specific ablation of Txnip. B, qRT-PCR data showing Txnip mRNA expression in the aortic media, adventitia, liver, and quadriceps muscle (n=4 mice for both groups). Txnip expression was normalized to that of β-actin. C, Western blot showing TXNIP level in the aortic media and quadriceps muscle. The representative blot images of 2 mice per group (a total of 4 mice per group were analyzed). D, The plasma concentrations of the total cholesterol (CHO)‚ triglyceride (TG)‚ high-density lipoprotein (HDL)‚ and low-density lipoprotein (LDL). E, Comparison of the Oil Red O (ORO)-positive areas between SMC^WT and SMC^KO on en-face aorta. F–J, Characterization of atherosclerotic lesions using the 7 μm of perpendicular serial sections (total 70–80 sections) prepared from the aortic sinus. Representative serial sections and quantification results of ORO staining (F, for lesion size), Moma-2 immunostaining (G, for monocyte/macrophage), Alizarin red staining (H, for calcium), Masson trichrome staining (I, for collagen), Alcian Blue (J, for glycosaminoglycan ECM). The applied statistical tests and results are summarized in the Table S11. The error bars denote standard deviation. The exact P values are specified.