Pcsk9

The levels of total cholesterol, triglyceride, high-density lipoprotein-cholesterol (HDL-C), and LDL-C were measured in all the subjects. The subjects fasted and avoided alcohol for at least 12 hours before blood sampling. Samples were analyzed within 4 hours by a laboratory that was certified by the Korean Society of Laboratory Medicine. Circulating apoB (Roche, Basel, Switzerland) and proprotein convertase subtilisin/kexin type 9 (PCSK9) levels (R&D Systems, Minneapolis, MN, USA) were measured using ELISA assays.

Before performing ProtoArray microarrays, recombinant (r) PCSK9 (carrier-free; R&D Systems; Minneapolis, MN) was biotinylated using the EZ Link Sulfo-NHS-SS Biotinylation kit (Pierce Thermo Scientific; Rockford, IL). First, to exchange buffers, 10 μg of rPCSK9 was added to a Zeba™ spin desalting column that has been pre-equilibrated according to the manufacturer’s protocol. Centrifugation was performed at 1000 × g for 2 minutes, and the resulting flow-through that contained the rPCSK9 protein in PBS was used in the next step. Sulfo-NHS-SS-Biotin was prepared by dissolving in ultrapure water at the concentration of 10 mM. Biotinylation of rPCSK9 was done with an at least 40-fold molar excess of Sulfo-NHS-SS-Biotin for 1 hour at room temperature. Filtering through another pre-equilibrated Zeba™ spin desalting column was performed as described above to remove excess biotin. Alternatively, excess biotin was removed by filtering through a 3 kDa Amicon ultra-2 centrifugal unit (Fisher Scientific; Pittsburg, PA). Protein concentrations were measured using a NanoDrop 2000 (Fisher Scientific). A HABA assay (reagents provided with the Biotinylation kit) was used to measure biotinylation levels. Calculations for the HABA assay were performed according to the protocol provided with the assay.

Conditioned media was centrifuged (13,000 rpm for 10 min.) and stored at −20 °C. The amount of PCSK9 (R&D System, Minneapolis, MN, USA) was quantified according to manufacturer’s instructions. The minimum detectable concentration was 0.219 ng/mL. Specifically, HepG2 cells were cultured in 12-well plates (6 × 10⁵ cells/well) and after 24-h treatment with YVVNPDNDEN peptide, YVVNPDNNEN peptide, and simvastatin, the medium was collected and PCSK9 dosed. Samples were not diluted, and results normalized for the total amount of proteins.

Levels of fibrosis biomarkers were measured on EDTA plasma by ELISA or multiplex assay using commercially available kits. HA (Echelon Biosciences, Salt Lake City, UT), LOXL2 (R&D Systems, Minneapolis, Minnesota), and CICP and CIC C1Q (Quidel, San Diego, CA) were measured by ELISA. PAI-1, PCSK9, TGF-ß₁-₃, MMP-2, MMP-9, TIMP-1, CXCL4, Chi3L1 (all R&D Systems, Minneapolis, MN) were measured using Luminex xMAP technology. All analytes except LOXL2 were measured according to the manufacturers' instructions. For LOXL2, the ELISA plates were coated overnight with capture antibody at room temperature. The plate was blocked with reagent diluent 1 (R&D Systems, Minneapolis, MN) for 1 hour and washed. Plasma was diluted 1:10 in PBS-Tween 10 and added to the plate. After incubating for 2 hours at 37° C and washing, detection antibody was added for 2 hours at 37° C. The plate was washed, streptavidin was added and the plate was incubated for 20 minutes at room temperature. After washing, TMB was added (Sigma-Aldrich Corp., St. Louis, MO), the plate was incubated for 20 minutes at room temperature, and sulfuric acid was added to stop the reaction. The plate was read at 450 nm.