Western blot analysis was used to determine the relative amount of internalized Lp(a). -actin was used as an internal standard. A representative blot is shown. Note the comparative inability of -ACA to compete for Lp(a) internalization (compare to Fig 2).(PDF) pone.0180869.s002.pdf (130K) GUID:?B6A2C130-D9C0-4E7F-B47D-8AE8CB0248B7 S3 Fig: Non-specific Lp(a) binding to collagen surfaces. A collagen matrix was prepared but seeding of hepatocytes was omitted. The matrix was then incubated with 200 nM Lp(a) for 4 hours. Wells were subjected to Bupranolol several different wash conditions as described below, at either 4C or 37C, Bupranolol and then lysed. Western blot analysis was used to determine IL4R the relative amount of internalized Lp(a). A representative blot is shown. Lane 1: 3 wash with PBS. Lane 2: 10 wash with PBS containing 0.5 M NaCl. Lane 3: 10 wash with PBS containing 0.5 M NaCl and 1% BSA. Lane 4: 10 wash with PBS containing 0.5 M NaCl, 1% BSA, and 200 mM -ACA. Lane 5: 10 wash with PBS containing 0.5 M NaCl, 1% BSA, and 200 mM -ACA, followed by an acid wash with 0.2 M acetic acid pH 2.5 containing 0.5 M NaCl. Lane 6: 3 with PBS, 0.8% BSA, 2 with PBS containing 10 g/ml heparin for 10 min, 1 with PBS, BSA, 0.2 M -ACA for 5 min; 2 with 0.2 M acetic acid, pH 2.5, containing 0.5 M NaCl for 10 min, 1 with 0.5 M HEPES, pH 7.5, 100 mM NaCl for 10 min, 1 with PBS (Lane 6 represents the normal washing conditions we employed elsewhere in this study). Note that progressively more extensive and harsh washing conditions appeared to actually Bupranolol promote binding to the collagen surfaces.(PDF) pone.0180869.s003.pdf (141K) GUID:?064DB4EA-3112-4F00-8055-23C9AF8BDE72 S4 Fig: Effect of isoform size on apo(a) internalization. HepG2 cells were treated with the indicated recombinant apo(a) variants (200 nM) for 4 hours. Cells were extensively washed to remove any bound apo(a) and lysed to determine the relative amount of internalized apo(a) compared to -actin using western blot analysis. The internalization values are expressed relative to that of 12K. The data represent the means s.e.m. of at least 7 independent experiments. No significant differences compared to 12K were observed (by one-sample t-test).(PDF) pone.0180869.s004.pdf (252K) GUID:?C48E51A8-A857-43A8-A08B-246B90BC4674 Data Availability StatementAll relevant data are within the paper. Abstract Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a causal risk factor for cardiovascular disease. The mechanisms underlying Lp(a) clearance from plasma remain unclear, which is an obvious barrier to the development of therapies to specifically lower levels of this lipoprotein. Recently, it has been documented that monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) can lower plasma Lp(a) levels by 30%. Since PCSK9 acts primarily through the low density lipoprotein receptor (LDLR), this result is in conflict with the prevailing view that the LDLR does not participate in Lp(a) clearance. To support our recent findings in HepG2 cells that the LDLR can act as a receptor for Lp(a) whose effects are sensitive to PCSK9, we undertook a series of Lp(a) internalization experiments using different hepatic cells, with different variants of PCSK9, and with different members of the LDLR family. We found that PCSK9 decreased Lp(a) and/or apo(a) internalization by Huh7 human hepatoma cells and by primary mouse and human hepatocytes. Overexpression of human LDLR appeared to enhance apo(a)/Lp(a) internalization in both types of primary cells. Importantly, internalization of Lp(a) by LDLR-deficient mouse hepatocytes was not affected by PCSK9, but the effect of PCSK9 was restored upon overexpression of human LDLR. In HepG2 cells, Lp(a) internalization was decreased by gain-of-function mutants of PCSK9 more than.
