Fig. 2. PCSK9 directly activated the pro-inflammatory genes independently of LDLR in vitro and in vivo.

a Experimental scheme demonstrating that PCSK9 aggravated atherosclerosis in Ldlr^−/− mice. AdV-PCSK9 at 1 × 10¹¹ infectious units/mouse was intravenously administered to mice under a high-fat diet. Partial ligation at the distal end exposed the right common carotid artery (RCA) to disturbed blood flow (D-flow), accelerating atherosclerosis. The left common carotid artery (LCA) remained under stable flow (S-flow) (AdV-CTRL N = 8; AdV-PCSK9 N = 12). b Oil red O staining of whole carotid arteries shows atherosclerotic plaque in the partial ligation-induced carotid atherosclerosis in Ldlr^−/− mice. The lesion area was significantly broader in AdV-PCSK9-treated Ldlr^−/− mice (44.4% of total RCA) than in AdV-control mice (CTRL) (22.5%). The scale bar represents 2 mm. c–g Carotid artery cross-section staining shows atherosclerotic plaque development in partial ligation-induced atherosclerosis in Ldlr^−/− mice. Enlarged atherosclerotic plaques were observed in the arteries of AdV-PCSK9-treated mice under D-flow compared with those of AdV-CTRL, indicating the significant impact of PCSK9 on atherosclerosis. c Hematoxylin and eosin staining of serial sections from the aortic root at 0.3, 0.6, and 1 mm. The scale bar represents 200 μm. d Masson’s trichrome staining. The scale bar represents 200 μm, and scale bars of magnified fields represent 50 μm. e Oil red O staining. The scale bar represents 200 μm, and scale bars of magnified fields represent 50 μm. f Immunofluorescence images stained with TUNEL (green). The scale bar represents 100 μm, and scale bars of magnified fields represent 50 μm. g Immunofluorescence images stained with F4/80 (green) and PCSK9 (red) in Ldlr^−/−, demonstrating significantly elevated PCSK9 expression in AdV-PCSK9-injected mice and increased F4/80 expression under D-flow. Each scale bar represents 20 μm (N = 3). h qPCR analysis of the carotid artery from Ldlr^−/− mice revealed significantly higher expression of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in AdV-PCSK9 compared with AdV-CTRL (N = 3). The differences between the groups were compared using the unpaired t-test (two-tailed). All experiments are independently performed and all data are presented as mean values ± SEM.