(A) Male C57BL/6 WT mice were injected twice with PCSK9-AAV8 (D377Y) virus and fed a WD for 16 weeks and followed by normal diet feeding with the treatment of S2PNP-siCtrl or S2PNP-siEpsin1/2 for 4 weeks (2 doses per week). (B) En face ORO staining of aortas from above mice. Lesion area was analyzed using NIH ImageJ, scale bar=5mm. (C) Immunostaining of CD68 (green) and αSMA (red) in the above groups. White arrows indicate the luminal αSMA. For (B) and (C), n=6/group, scale bar: B (aorta)=5mm, B (aortic root) =500μm, C=500μm. (D) Statistics for ORO staining (B) and IF staining (C). (E-H) Co-staining of CD31 (endothelial cell marker, green) with vascular cell adhesion molecule-1 (VCAM-1, red, E), intercellular adhesion molecule-1 (ICAM-1, red, F), P-selectin (red, G) or E-selectin (red, H) and DAPI on the luminal surface of aortic root sections. White arrows indicate CD31/ICAM-1, CD31/VCAM-1, CD31/E-selectin or CD31/P-selectin colocalization. Scale bars=20μm. (I) Statistical analysis of E-H, n=6/group. (J) Isolated macrophages from PCSK9-mice were treated with S2PNP-siCtrl or S2PNP-siEpsin1/2 for 24h, RNA was isolated, and qRT-PCR was performed (n=4). B-J are presented as mean ± SD. and were analyzed using one-way ANOVA (B and C) and the unpaired Student’s t-test (E-J). One-way ANOVA followed by Tukey’s post hoc multiple comparisons test was conducted in D; Unpaired t test was conducted in I; Mann-Whitney U test was conducted in J.