Roles of the low density lipoprotein receptor and related receptors in inhibition of lipoprotein(a) internalization by proprotein convertase subtilisin/kexin type 9.

Rocco Romagnuolo,Nabil G Seidah,Santica M Marcovina,Michael B Boffa,Marlys L Koschinsky,Matthew Gemin,Corey A Scipione,Maria Cristina Vinci

doi:10.1371/journal.pone.0180869

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 bona fide 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 by wild-type PCSK9, and a loss-of function variant had a reduced ability to influence Lp(a) internalization. Apo(a) internalization by HepG2 cells was not affected by apo(a) isoform size. Finally, we showed that very low density lipoprotein receptor (VLDLR), LDR-related protein (LRP)-8, and LRP-1 do not play a role in Lp(a) internalization or the effect of PCSK9 on Lp(a) internalization. Our findings are consistent with the idea that PCSK9 inhibits Lp(a) clearance through the LDLR, but do not exclude other effects of PCSK9 such as on Lp(a) biosynthesis.

Highlights

Genetic studies performed within the last decade have provided conclusive evidence that elevated plasma lipoprotein(a) [Lp(a)] concentrations are a causal risk factor for coronary heart disease [1, 2]
The discovery that antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) lower plasma Lp(a) concentrations triggered excitement in the Lp(a) field, and presented a paradox: given the apparent lack of a role for the low density lipoprotein receptor (LDLR) in Lp(a) catabolism, what could be the mechanism behind the effect of these therapies? Our own studies, and those of others, using in vitro internalization assays with HepG2 cells indicated that Lp(a) was a bona fide ligand for the LDLR, and that PCSK9 inhibited internalization of Lp(a) through this route by decreasing LDLR abundance [34, 37]
In the setting of PCSK9 inhibitors, a situation is set up where LDLR levels in the liver are supraphysiological and low density lipoprotein (LDL) plasma concentrations are markedly reduced; in this circumstance, LDLR could play a greater role in Lp(a) clearance

Summary

Introduction

Genetic studies performed within the last decade have provided conclusive evidence that elevated plasma lipoprotein(a) [Lp(a)] concentrations are a causal risk factor for coronary heart disease [1, 2]. Apo(a) can contain from as few as 3 to greater than 40 identically repeated KIV2 domains which accounts for the phenomenon of Lp(a) isoform size heterogeneity, a hallmark of this lipoprotein [6, 7]. A general inverse relationship between apo(a) size and Lp(a) plasma concentration has been well-established, with Lp(a) levels varying widely in the population [8]. It has been reported that the inverse correlation between apo(a) isoform size and plasma Lp(a) levels is primarily dictated by the level of production rather than catabolism of the particle [9, 10]. Up to 90% of the variation in Lp(a) levels is genetically determined based on variation in the apo(a) gene including its size heterogeneity [11]; this has presented challenges in the development of therapeutic strategies to lower Lp(a) [12]

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