Alirocumab, a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes.

Proprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.Flow cytometry and Western blotting of human hepatocyte Huh-7 cells showed that, although LDLR levels were reduced when cells were exposed to increasing PCSK9 concentrations, there was no correlation between total or surface CD81 levels and the presence and amount of soluble PCSK9. Moreover, inhibiting PCSK9 with the monoclonal antibody alirocumab did not affect expression levels of CD81. In an in vitro model of HCV entry, addition of soluble PCSK9 or treatment with alirocumab had no effect on the ability of either lentiviral particles bearing the HCV glycoproteins or JFH-1 based cell culture virus to enter hepatocytes. Consistent with these in vitro findings, no differences were observed in hepatic CD81 levels using in vivo mouse models, including Pcsk9-/- mice compared with wild-type controls and hyperlipidemic mice homozygous for human Pcsk9 and heterozygous for Ldlr deletion, treated with either alirocumab or isotype control antibody.These results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry

ANGPTL8 has both endocrine and autocrine effects on substrate utilization

The angiopoietin-like protein ANGPTL8 (A8) is one of 3 ANGPTLs (A8, A3, A4) that coordinate changes in triglyceride (TG) delivery to tissues by inhibiting lipoprotein lipase (LPL), an enzyme that hydrolyzes TG. Previously we showed that A8, which is expressed in liver and adipose tissue, is required to redirect dietary TG from oxidative to storage tissues following food intake. Here we show that A8 from liver and adipose tissue have different roles in this process. Mice lacking hepatic A8 have no circulating A8, high intravascular LPL activity, low plasma TG levels, and evidence of decreased delivery of dietary lipids to adipose tissue. In contrast, mice lacking A8 in adipose tissue have higher postprandial TG levels and similar intravascular LPL activity and plasma A8 levels and higher levels of plasma TG. Expression of A8, together with A4, in cultured cells reduced A4 secretion and A4-mediated LPL inhibition. Thus, hepatic A8 (with A3) acts in an endocrine fashion to inhibit intravascular LPL in oxidative tissues, whereas A8 in adipose tissue enhances LPL activity by autocrine/paracrine inhibition of A4. These combined actions of A8 ensure that TG stores are rapidly replenished and sufficient energy is available until the next meal

Flow cytometry quantification of surface levels of LDLR (parts A and C) and CD81 (parts B and D) on Huh-7 cells, following incubation with the indicated concentrations of wild-type PCSK9 (parts A and B) or the gain-of-function PCSK9 D374Y mutant (parts C and D) and 300 nM alirocumab (monoclonal antibody to PCSK9; right panels) or isotype control monoclonal antibody (left panels) for 6 hours.

<p>LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.</p

Effect of <i>Pcsk9</i> deletion on LDLR and CD81 levels in age- and sex-matched <i>Pcsk9</i>^<i>-/-</i> mice (n = 5) and their wild type littermates (n = 4).

<p>Shown are (A) mean ± SE serum LDL-C levels and (B) Western blot analysis of CD81 and LDLR levels in liver extracts with TfR as loading control, in animals sacrificed after a 4-hour fast. Lanes in panel B represent samples from individual mice. LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SE, standard error; TfR, transferrin receptor; wt, wild type.</p

Effect of PCSK9 and alirocumab on the HCVcc full replication cycle (A) or HCV RNA replication cycle (B).

<p>Effect of PCSK9 and alirocumab on the HCVcc full replication cycle (A) or HCV RNA replication cycle (B).</p

Effect of soluble PCSK9, gain-of-function PCSK9 D374Y and alirocumab on HCVpp entry.

<p>Huh-7 cells were incubated for 6 hours with the indicated concentrations of wild-type PCSK9 or the gain-of-function PCSK9 D374Y mutant and alirocumab (monoclonal antibody to PCSK9) or isotype control monoclonal antibody (n = 3 replicates per treatment). Cells were then infected with HCVpp, incubated for 48–72 hours and intracellular luciferase activity was measured. Luciferase activity is shown as relative light units. Ab, antibody; HCVpp, hepatitis C virus pseudoparticles; PCSK9, proprotein convertase subtilisin/kexin type 9; RLU, relative light units; w/o, without.</p

Patients with post-baseline positive HCV test according to baseline status—data from alirocumab Phase 3 placebo- and ezetimibe-controlled trials.

<p>Patients with post-baseline positive HCV test according to baseline status—data from alirocumab Phase 3 placebo- and ezetimibe-controlled trials.</p

Western blot analysis on Huh-7 cells following incubation with decreasing concentrations of wild-type PCSK9 (500 nM, 50 nM and 5 nM) or gain-of-function PCSK9 D374Y mutant (20 nM, 2 nM and 0.2 nM) and 200 nM of alirocumab (monoclonal antibody to PCSK9) or isotype control monoclonal antibody for 6 hours.

<p>Lane 1 represents control cells incubated without PCSK9 protein or alirocumab/control Ab. Ab, antibody; LDLR, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9.</p