A small-molecule inhibitor of PCSK9 transcription ameliorates atherosclerosis through the modulation of FoxO1/3 and HNF1α

Abstract

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates hepatic low-density lipoprotein receptor (LDLR) by binding and shuttling LDLR to lysosomes for degradation. The development of therapy that inhibits PCSK9 has attracted considerable attention for the management of cardiovascular disease risk. However, only monoclonal antibodies of PCSK9 have reached the clinic use. Oral administration of small-molecule transcriptional inhibitors has the potential to become a therapeutic option. Methods: Here, we developed a cell-based small molecule screening platform to identify transcriptional inhibitors of PCSK9. Through high-throughput screening and a series of evaluation, we found several active compounds. After detailed investigation on the pharmacological effect and molecular mechanistic characterization, 7030B-C5 was identified as a potential small-molecule PCSK9 inhibitor. Findings: Our data showed that 7030B-C5 down-regulated PCSK9 expression and increased the total cellular LDLR protein and its mediated LDL-C uptake by HepG2 cells. In both C57BL/6 J and ApoE KO mice, oral administration of 7030B-C5 reduced hepatic and plasma PCSK9 level and increased hepatic LDLR expression. Most importantly, 7030B-C5 inhibited lesions in en face aortas and aortic root in ApoE KO mice with a slight amelioration of lipid profiles. We further provide evidences suggesting that transcriptional regulation of PCSK9 by 7030B-C5 mostly depend on the transcriptional factor HNF1a and FoxO3. Furthermore, FoxO1 was found to play an important role in 7030B-C5 mediated integration of hepatic glucose and lipid metabolism. Interpretation: 7030B-C5 with potential suppressive effect of PCSK9 expression may serve as a promising lead compound for drug development of cholesterol/glucose homeostasis and cardiovascular disease therapy.

Pterostilbene Increases LDL Metabolism in HL-1 Cardiomyocytes by Modulating the PCSK9/HNF1α/SREBP2/LDLR Signaling Cascade, Upregulating Epigenetic hsa-miR-335 and hsa-miR-6825, and LDL Receptor Expression

“…To investigate whether pterostilbene regulated PCSK9 promoter activity as previously described [ 31 ], we amplified the human PCSK9 gene promoter by polymerase chain reaction (PCR), with HL-1 genomic DNA serving as our template, to generate pGL3-Basic vector clones (Promega, Madison, WI, USA) at the Xho I and Hind III sites, resulting in the pGL3-PCSK9-P plasmid, which was used as a template for PCR amplification of the PCSK9 gene promoter fragments (P1–P6) ( Figure 4 A). Using the constructed luciferase reporter plasmid containing fragments of the 5′ flanking 1455-bp promoter region (P1–P6) of PCSK9, the initial post-transfection amplification of PCSK9 promoter-luciferase activity (10-fold, p < 0.01) in HL-1 cardiomyocytes was significantly and dose-dependently suppressed after treatment with 2.5 (5.04-fold, 5.04-fold, p < 0.01) and 5 μM (2.85-fold, p < 0.01) pterostilbene as shown in Figure 4 A.…”

“…Transfection of reported plasmids and the establishment of stable PCSK9 promoter –cells, and the luciferase report assay were conducted strictly following already established protocols [ 31 ] and according to the reagent manufacturers’ instructions. Transfected cells were then treated with DMSO- or pterostilbene for 24 h. Luciferase activity was evaluated using the Luciferase Reporter Assay System (#E1500; Promega) and all the luminescence was Renilla luciferase activity-normalized.…”

PCSK9 inhibitor and atorvastatin reduce cardiac impairment in ovariectomized prediabetic rats via improved mitochondrial function and Ca2+ regulation

“…The protective effects of atorvastatin and PCSK9 inhibitor are likely to be through the same mechanisms. Previous studies reported that PCSK9 inhibitor and atorvastatin could effectively reduce LDL levels in obese rats 47,48 . In addition, it has been shown that PCSK9 inhibitor effectively reduced inflammatory markers including the number of monocytes adhering and the number of T cells in the aortic root area in atherosclerotic model rats 49 .…”

“…Previous studies reported that PCSK9 inhibitor and atorvastatin could effectively reduce LDL levels in obese rats. 47,48 In addition, it has been shown that PCSK9 inhibitor effectively reduced inflammatory markers including the number of monocytes adhering and the number of T cells in the aortic root area in atherosclerotic model rats. 49 Also, atorvastatin could reduce inflammation in the heart as indicated by increased AMPK activity and decreased NF-κB activity in obese rats.…”

Regulation of PCSK9 Expression and Function: Mechanisms and Therapeutic Implications

“…Huh7 cells), Pinostrobin (HepG2 cells), and tanshinone IIA (HepG2 cells) reduce PCSK9 expression in a FoxO3a-dependent manner, probably via attenuating HNF1α-mediated activation of PCSK9 expression and/or methylation in the promoter region of PCSK9 (79-82). A small molecular, 7030B-C5, also reduces PCSK9 expression in HepG2 cells and mice mainly through the FoxO1 and HNF1α pathway (83). In addition, Berberine reduces PCSK9 expression mainly through attenuating SREBP2 and HNF1α-mediated upregulation of PCSK9 transcription in HepG2 cells (73), which may account for its cholesterol-lowering effect.…”