PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor

Abstract

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

Fig. 1. PCSK9 directly switched on pro-inflammatory genes and increased the expression of adhesion molecules.

a Immunoblot analysis demonstrated that proprotein convertase subtilisin/kexin type-9 (PCSK9) activated and phosphorylated nuclear factor (NF)-κB p65 in THP-1 (N = 5) and human umbilical vein endothelial cells (HUVECs, N = 3) in a dose-dependent manner (0, 50, 200, and 2000 ng/mL). b Luciferase assay demonstrating that the NF-κB gene promoter was significantly activated (P < 0.001) after 12-h treatment with PCSK9 (200, 2000 ng/mL, N = 5), resistin (10, 50 ng/mL, N = 5) or TNF-α (10, 20 ng/mL, N = 3) in HEK293T cells. c qPCR analysis demonstrating that PCSK9 significantly increased the expression of cytokines, including TNF-α (N = 6), IL-1β (N = 5), IL-6 (N = 6) and IL-10 (N = 5), and integrin-α4 (N = 3) and -β1(N = 3) in a dose-dependent manner (0, 50, 200, and 2000 ng/mL) in monocytes. d qPCR analysis demonstrating that PCSK9 significantly increased the expression of cytokines, including TNF-α (N = 7), IL-1β (N = 6), and IL-6 (N = 7) and C-reactive protein (CRP, N = 7) in a dose-dependent manner (0, 50, 200, and 2000 ng/mL), while albumin (N = 7) was not increased in hepatocytes. e qPCR analysis demonstrated that PCSK9 significantly increased the expression of pro-inflammatory cytokines, including TNF-α (N = 6), IL-1β (N = 5), and IL-6 (N = 5), as well as adhesion molecules (VCAM-1 (N = 6), ICAM-1 (N = 6), E-selectin [SELE, N = 6]) in HUVECs. f Immunoblot analysis demonstrating that PCSK9 increased the protein levels of integrin-α4 and -β1 in THP-1 cells (N = 4) and VCAM-1 and ICAM-1 in HUVECs (N = 3) in a dose-dependent manner (0, 50, 200, and 2000 ng/mL). g Fluorescence-activated cell sorting analysis reveals a 16% increase in VLA-4 activation on monocyte surfaces with PCSK9 treatment, compared to 2.7% in the vehicle group. h Immunoblot analysis demonstrating that PCSK9 treatment (2 µg/mL) for 40 min activated and phosphorylated NF-κB in BMDMs from Ldlr^−/− mice and BL6 control mice (N = 4). i qPCR analyses revealed PCSK9-induced cytokines (TNF-α, IL-1β, and IL-6) even in Ldlr^−/− BMDMs (N = 5) after 24-h treatment (2 µg/mL). The differences between the groups were compared using the unpaired t-test (two-tailed). All experiments were independently performed, and data are presented as mean values ± SD.

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.

Fig. 3. CAP1 is a receptor of PCSK9.

a Fluorescence-activated cell sorting analysis of THP-1 cells demonstrating that treatment of PCSK9 for 1 h changed CAP1 localization to the membrane surface of monocytes in a dose-dependent manner (0, 500, 1000, and 2000 ng/mL) (N = 3). b Immunofluorescent staining of THP-1 cells with CAP1 (green) and PCSK9 (red) demonstrating their colocalization (left panel) mainly to the membrane surface of monocytes (yellow). The colocalization between PCSK9 and CAP1 further increased 60 min after treatment with 2 µg/mL rhPCSK9 (right panel). Colocalization analysis within the membrane was performed using orthogonal views from different planes of confocal microscope images. Scale bar represents 5 μm. c A direct binding assay using the BLItz system showed that the binding affinity to PCSK9 was strongest for CAP1 (0.032 µM), intermediate for TLR4 (0.037 µM), and weakest for LOX1 (2.833 µM). d Immunoprecipitation analysis of THP-1 cells demonstrating the interaction between PCSK9 and CAP1 in monocytes. e Duolink Proximity Ligation Assay for detecting the interaction between PCSK9 and CAP1 after treatment with CTRL siRNA, TLR4 siRNA, or LOX1 siRNA. The interaction between PCSK9 and CAP1 was quantified by counting the red dots. The Proximity Ligation Assay showed that the interaction between PCSK9 and CAP1 was not affected by the presence or absence of TLR4 or LOX1 (N = 4). The scale bar represents 5 μm. f Duolink Proximity Ligation Assay for detecting the interaction between PCSK9 and TLR4 with CTRL or CAP1 siRNA. The extent of the interaction between PCSK9 and TLR4 was quantified by counting the red dots (N = 3). The scale bar represents 5 μm. g Immunofluorescence staining of CAP1 (green) and PCSK9 (red) in the arteries of AdV-CTRL or AdV-PCSK9-treated Ldlr^−/− mice under S-flow and D-flow. CAP1 and PCSK9 were colocalized in all groups. CAP1 and PCSK9 expression increased in the AdV-PCSK9 injection group compared with that in the AdV-CTRL group under D-flow. The scale bar represents 20 μm. 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 ± SD.

Fig. 4. CAP1 was required for PCSK9-mediated inflammation.

a Immunoblot demonstrating rhPCSK9 treatment (2 µg/mL for 20 min) resulted in NF-κB p65 phosphorylation, which was blocked in CAP1-deficient THP-1 cells, but not in TLR4-deficient THP-1 cells (N = 3). b rhPCSK9 treatment (2 µg/mL, 48 h) elevated TNF-α, IL-1β, and IL-6 protein levels in THP-1 cells treated with CTRL and TLR4 siRNA, but not in CAP1 siRNA (N = 3). c Luciferase assay demonstrating the activity of NF-κB gene promoter in CAP1^+/+ and CAP1^−/− 293 T cells. The activation of NF-κB signaling induced by rhPCSK9 treatment (2 µg/mL) was attenuated in CAP1^−/− 293 T cells (N = 3). d qPCR demonstrating rhPCSK9-induced pro-inflammatory cytokines in THP-1 cells transfected with CTRL or CAP1 shRNA. PCSK9 treatment increased TNF-α (N = 5), IL-1β (N = 5), IL-6 (N = 6), integrin-α4 (N = 6), and -β1 (N = 4) in CTRL group, not in CAP1-deficient cells. e Immunoblot to analyze the protein levels of rhPCSK9-induced adhesion molecules in THP-1 cells. PCSK9 treatment induced the integrin-α4 and -β1 expression in a time-dependent manner (0, 24, and 48 h) in THP-1 cells with CTRL siRNA, but not in with CAP1 siRNA (N = 3). f Fluorescence-activated cell sorting demonstrating VLA-4 activation in THP-1 cells with CTRL or CAP1 siRNA. THP-1 cells were transfected with CTRL or CAP1 siRNA and left untreated or treated with rhPCSK9 (2 µg/mL). PCSK9 treatment increased VLA-4 expression in THP-1 cells, which was reduced in CAP1-deficient THP-1 cells. g VCAM-1 and ICAM-1 expression increased after rhPCSK9 treatment in HUVECs transfected with CTRL shRNA, but not in with CAP1 shRNA (N = 3). h Cell adhesion assay of THP-1 and HUVECs with rhPCSK9 treatment demonstrating that adhesion to HUVECs was enhanced by PCSK9 in THP-1 cells with CTRL siRNA, which was blocked in CAP1-deficient THP-1 cells. Representative images of fluorescently labeled adherent THP-1 cells (upper-left panel), and fluorescence-positive cells were counted to quantify cell adhesion (upper-right panel) (N = 4). The schematic figure for adhesion assay (bottom panel). The scale bar represents 200 μm. The differences between the groups were compared using the unpaired t-test (two-tailed) or one-way analysis of variance. All experiments are independently performed, and data are presented as mean values ± SD.

Fig. 5. PCSK9 regulated the expression of scavenger receptors and ox-LDL uptake via CAP1.

a For the ox-LDL assay, THP-1 cells were transfected with CTRL or CAP1 siRNA and then treated with or without rhPCSK9. ox-LDL uptake decreased in CAP1-deficient THP-1 cells in response to PCSK9 (2 µg/mL). b qPCR analysis demonstrating the mRNA levels of several scavenger receptors in response to PCSK9 treatment in THP-1 cells transfected with CTRL or CAP1 siRNA. The mRNA levels of LOX1, CD36, SRA1, and TLR4 increased significantly with rhPCSK9 (2 µg/mL) treatment in monocytes with CAP1, but not in THP-1 cells with CAP1 knockdown (N = 4). c Immunoblot analysis demonstrating the protein levels of several scavenger receptors in response to PCSK9 treatment in THP-1 cells transfected with CTRL or CAP1 siRNA. Consistently, the protein levels of scavenger receptors were induced by PCSK9 treatment in control THP-1 cells, but not in CAP1-deficient THP-1 cells (N = 3). d Oil Red O staining of BMDMs from Cap1^+/+ and Cap1^+/− mice after differentiation into macrophages, after treatment with ox-LDL for 0, 24, 48, and 72 h. Ox-LDL treatment induced lipid formation and accumulation in Cap1^+/+ BMDMs (upper panel) because of ox-LDL uptake, which was significantly inhibited in CAP1-deficient BMDMs. The scale bar represents 20 μm (N = 3). e PCSK9 mRNA expression induced by pro-inflammatory cytokines was analyzed by qPCR. PCSK9 mRNA levels increased in response to various inflammatory stimuli in THP-1 cells (N = 8). f hPCSK9 levels in media were determined using ELISA in Cap1^+/+ and Cap1^+/− BMDMs. Positive feedback loop of PCSK9-induced PCSK9 secretion was attenuated in Cap1^+/− BMDMs (N = 3). g Immunoblot analysis of signal activation induced by PCSK9 in THP-1 cells transfected with CTRL or CAP1 siRNA demonstrating that PCSK9-induced SREBP-2 expression was attenuated when CAP1 was knocked down (N = 3). (P) and (N) denote the precursor and nuclear active forms of SREBP-2, respectively. The differences between the groups were compared using the unpaired t-test (two-tailed) or one-way analysis of variance. All experiments are independently performed and all data are presented as mean values ± SD.

Fig. 6. Syk and PKCδ were identified as binding partners of CAP1.

a Experimental scheme to generate an mFc-conjugated hCAP1 construct for the pull-down assay. Coomassie brilliant blue staining and immunoblotting to confirm the expression and homogeneity of hCAP1-mFc. Scheme of proteomic analysis for CAP1-binding proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The THP-1 lysate was incubated with either mFc or CAP1-mFc proteins and pulled down with mFc-specific beads. b Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional enrichment analysis was performed on differentially bound proteins. The graph displays the top-ranked significantly enriched Gene Ontology (GO) terms in biological process, cellular component, and molecular function. All the adjusted statistically significant values of the terms were transformed to their negative 10-base logarithm. The analysis was conducted using an EASE score threshold of <0.1. c List of nine proteins that bound to CAP1 with the most significant values of SAINT AvgP = 1 in the immune system process (GO.0002376). MS/MS spectral data for Syk (d) and PKCδ (e). f The BLItz system showed that binding affinity between CAP1 with Syk or PKCδ increased in a dose-dependent manner. The equilibrium dissociation constant for Syk or PKCδ from CAP1 was 5.957 nM or 322 nM, respectively. g Immunoprecipitation of endogenous CAP1, Syk, and PKCδ from THP-1 cell lysate, demonstrating that these proteins bind to each other.

Fig. 7. PCSK9 phosphorylated Syk and PKCδ, and their phosphorylation was dependent on CAP1, not on LDLR.

a Immunoblot analysis of signal activation of Syk, PKCδ, AKT, and NF-κB p65 after rhPCSK9 (2 µg/mL) treatment in a time-dependent manner (0, 20, 40, 60, and 120 min) in the THP-1 cell line. Syk and PKCδ were phosphorylated after 20 min of rhPCSK9 (2 µg/mL) treatment. AKT phosphorylation started at 40 min and lasted until 60 min (N = 3). b, c Multiplex ELISA of cAMP secretion to assess the involvement of Syk, PKCδ, and PKA in the PCSK9-induced inflammation pathway (N = 4). b R406, rottlerin, and H892HCl were used to inhibit Syk, PKCδ, and PKA, respectively. Rottlerin blocked PCSK9-mediated cAMP induction, whereas R405 and H892HCl did not (N = 3). c, d Immunoblot analysis demonstrating that PCSK9-induced the phosphorylation of Syk, PKCδ, and AKT in THP-1 cells. The phosphorylation was attenuated in cells transfected with CAP1 siRNA (N = 4 for p-Syk and p-AKT; N = 3 for p-PKCδ). e Immunoblot analysis of Ldlr^−/− BMDMs, demonstrating that PCSK9-induced phosphorylation of Syk, PKCδ, AKT, and NF-κB p65 was independent of LDLR (N = 3). f Ldlr^−/− mice arteries were partially ligated and treated with AdV-CTRL or AdV-PCSK9, followed by a comparison of arteries exposed to S-flow and D-flow. Immunofluorescence staining for analyzing the expression of PCSK9 (red, upper panels; N = 3), p-Syk (gray, middle panels; N = 4), and p-PKCδ (green, bottom panels; N = 4). The expression of PCSK9 significantly increased in the group treated with AdV-PCSK9 compared with that in the control group. Additionally, p-Syk and p-PKCδ increased more significantly under D-flow of AdV-PCSK9-treated mice than in the control group. The scale bar represents 10 μm. The differences between the groups were compared using the unpaired t-test (two-tailed). All experiments are independently performed. Data are presented as mean values ± SD and SEM (in (f) only).

Fig. 8. CAP1 deficiency attenuated PCSK9-induced atherosclerosis in CAP1-heterozygous knockout mice.

a Experimental scheme demonstrating that PCSK9 aggravated atherosclerosis in Cap1^+/+ versus Cap1^+/− mice. AdV-PCSK9 (1 × 10¹¹ infectious units/mouse) was intravenously administered to mice on a high-fat diet. b Plasma hPCSK9 levels were measured using ELISA in Cap1^+/+ (AdV-CTRL N = 4; AdV-PCSK9 N = 9) and Cap1^+/− (AdV-CTRL N = 4; AdV-PCSK9 N = 12) mice with or without PCSK9 overexpression. The plasma hPCSK9 concentration in the AdV-CTRL group was 12–14 ng/mL, whereas it was 30–50% higher in the AdV-PCSK9 group. c Oil Red O staining of carotid arteries after AdV-CTRL or AdV-PCSK9 injection into Cap1^+/+ (AdV-CTRL N = 7; AdV-PCSK9 N = 18) and Cap1^+/− (AdV-CTRL N = 8; AdV-PCSK9 N = 14) mice. The atherosclerotic plaque area in the D-flow arteries increased after PCSK9 administration in Cap1^+/+ mice, which was prevented in Cap1^+/− mice. The scale bar represents 2 mm. d qPCR analysis demonstrating pro-inflammatory molecules in S-flow or D-flow arteries from Cap1^+/+ versus Cap1^+/− mice. Expression of TNF-α, IL-1β, and IL-6 was higher in D-flow than in S-flow arteries (P < 0.001, P = 0.004, and P = 0.023, respectively) in Cap1^+/+ mice with a high serum level of PCSK9. However, induction of inflammatory cytokines under D-flow was prevented in Cap1^+/− mice (P = 0.002, 0.007, 0.029, respectively) (N = 3). e H&E staining of S-flow or D-flow arteries (from the aortic root at 0.3, 0.6, and 1 mm, respectively) from Cap1^+/+ versus Cap1^+/− mice with AdV-CTRL or AdV-PCSK9. In the presence of a high serum level of PCSK9, significant atherosclerotic plaques developed under D-flow in Cap1^+/+ mice, which was prevented in Cap1^+/− mice. The scale bar represents 200 μm. f Masson’s trichrome and Oil red O staining. Each scale bar represents 200 μm, and 50 μm (magnified fields). g Immunofluorescence images stained with TUNEL (green). The bottom panel displays a magnified view, specifically highlighting the arteries exposed to low shear stress from the AdV-PCSK9 group. The scale bar represents 100 μm (top), 25 μm (mid) and 20 μm (enlarged). The differences between the groups were compared using the unpaired t-test (two-tailed). All experiments are independently performed. Data are presented as mean values ± SEM and SD (in (b, d) only).

Fig. 9. PCSK9 colocalized with CAP1 in atherosclerotic plaques, and PCSK9-induced phosphorylation was inhibited in CAP1-heterozygous knockout mice.

a Immunofluorescence staining for PCSK9 (red) and F4/80 (green) in arteries under D-flow in Cap1^+/+ and Cap1^+/− mice injected with AdV-CTRL or AdV-PCSK9. PCSK9 and F4/8 expression significantly increased in Cap1^+/+ mice with AdV-PCSK9 compared with that in AdV-CTRL. However, in Cap1^+/− mice, the increase in PCSK9 was less significant and F4/80 showed no significant change (N = 3). The scale bar represents 10 μm. b Partially ligated Cap1^+/+ and Cap1^+/− mice with only arteries exposed to D-flow were compared after AdV-CTRL or AdV-PCSK9 injection. Immunofluorescence staining of PCSK9 (red, upper panels; N = 3), p-Syk (gray, middle panels; N = 4), and p-PKCδ (green, bottom panels; N = 3). AdV-PCSK9 increased PCSK9 expression in Cap1^+/+ mice, lesser than in Cap1^+/− mice. Additionally, p-Syk and p-PKCδ increased more significantly in Cap1^+/+ mice than in Cap1^+/− mice. The scale bar represents 10 μm. c Immunofluorescence images of arteries under D-flow from Cap1^+/+ mice injected with AdV-PCSK9 showing colocalization of PCSK9 (red), CAP1 (gray), and F4/80 (green). The scale bar represents 5 μm. d Immunofluorescence images of arteries under D-flow from Cap1^+/+ mice injected with AdV-PCSK9 showing colocalization of PCSK9 (red), CAP1 (gray), and CD31 (green). The scale bar represents 5 μm. e Immunofluorescence images of arteries under D-flow from Cap1^+/+ mice injected with AdV-PCSK9 showing colocalization of PCSK9 (red), CAP1 (gray), and αSMA (green). The scale bar represents 10 μm. f Immunofluorescence staining for PCSK9 (red), αSMA (green), and F4/80 (gray) in arteries under D-flow from Cap1^+/+ mice injected with AdV-PCSK9. White arrow indicates PCSK9, αSMA, and F4/80 colocalization. The yellow arrow denotes PCSK9 and αSMA colocalization (top middle panel). The three bottom panels show the colocalization of PCSK9 and αSMA (bottom left), PCSK9 and F4/80 (bottom middle), and αSMA and F4/80 (bottom right), respectively. The scale bar represents 10 μm. g Schematic model showing PCSK9-mediated inflammation in monocytes mediated by CAP1 recruiting PKCδ and Syk and modulating PCSK9-mediated inflammatory signal transduction. Group differences were compared using the unpaired t-test (two-tailed). All data are presented as mean values ± SEM.

Fig. 10. PCSK9 levels in the serum of patients with coronary artery disease (CAD) correlated with Syk, PKC, and NF-κB phosphorylation, and PCSK9-mediated phosphorylation was blocked by CAP1-hFc.

a Simple linear regression analysis of Pearson correlation by distance. This figure illustrates the relationship between serum PCSK9 concentration and the quantified levels of phosphorylated proteins in a single individual. Serum PCSK9 concentration displayed a positive correlation with the phosphorylation of Syk, PKCδ, p65(S276), and p65(S536) in human peripheral blood mononuclear cells (PBMCs). The black line represents an asymptotic regression line fitted to the raw data (N = 19). b Average serum PCSK9 concentration and lipid profile results from 13 hyperlipidemia patients and five healthy donors. T-Chol total cholesterol, TG triglyceride, HDL high-density lipoprotein, LDL low-density lipoprotein, AST aspartate aminotransferase, ALT alanine aminotransferase. c Competitive ELISA binding assay showed that PCSK9-His bound to CAP1, and its interaction was competitively inhibited by CAP1-hFc, whereas evolocumab did not hinder the PCSK9-CAP1 interaction (N = 3). d Immunoblot analysis of NF-κB p65 signal activation after treatment with rhPCSK9 (2 µg/mL) along with human IgG, evolocumab, or CAP1-hFc in THP-1 cells revealed that the phosphorylation of p65 induced by PCSK9 was notably reduced by CAP1-hFc (N = 5). e Immunoblot analysis demonstrated that PCSK9-induced phosphorylation of Syk, PKCδ, and NF-κB p65 in human PBMC-derived macrophages was attenuated by CAP1-hFc. After 1-h treatment of rhPCSK9 with human IgG, evolocumab, or CAP1-hFc, the PCSK9-induced phosphorylation of Syk, PKCδ, and NF-κB p65 was significantly decreased by CAP1-hFc (N = 8). f Schematic diagram depicting CAP1 as the binding partner of PCSK9, which mediates not only caveolae-dependent endocytosis and lysosomal degradation of LDLR, but also recruits Syk and PKCδ and modulates PCSK9-mediated inflammatory signal transduction. CAP1-hFc inhibits the binding of CAP1 and PCSK9, which sequentially block LDLR degradation and the inflammatory signaling pathway, whereas the PCSK9 inhibitor evolocumab can only block the LDLR degradation pathway. 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 ± SD.