Lipoprotein(a) Cellular Uptake Ex Vivo and Hepatic Capture In Vivo Is Insensitive to PCSK9 Inhibition With Alirocumab

Abstract

Lipoprotein(a) (Lp[a]) is the most common genetically inherited risk factor for cardiovascular disease. Many aspects of Lp(a) metabolism remain unknown. We assessed the uptake of fluorescent Lp(a) in primary human lymphocytes as well as Lp(a) hepatic capture in a mouse model in which endogenous hepatocytes have been ablated and replaced with human ones. Modulation of LDLR expression with the PCSK9 inhibitor alirocumab did not alter the cellular or the hepatic uptake of Lp(a), demonstrating that the LDL receptor is not a major route for Lp(a) plasma clearance. These results have clinical implications because they underpin why statins are not efficient at reducing Lp(a).

Keywords: 3D, 3-dimensional; AU, arbitrary unit; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; FCR, fractional catabolic rate; FRG, Fah(−/−)Rag2(−/−)Il2rg(−/−); HoFH, homozygous familial hypercholesterolemia; LC-MS/MS, liquid chromatography tandem mass spectrometry; LDL, low-density lipoprotein; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; Lp(a), lipoprotein(a); MFI, mean fluorescence intensity; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; PCSK9, proprotein convertase subtilisin/kexin type 9; apoB100, apolipoprotein B100; bodipy, boron dipyrromethene; lipoprotein(a); liver-humanized mice; low-density lipoprotein receptor; proprotein convertase subtilisin/kexin type 9; rPCSK9, recombinant proprotein convertase subtilisin/kexin type 9.

Graphical abstract

Figure 1

Lp(a) Cellular Uptake Is Not Modulated by Changes in LDLR Cell Surface Expression Ex Vivo Peripheral blood mononuclear cells were plated for 24 h in serum-deprived medium with or without mevastatin (10 μg/ml) and supplemented or not for the last 4 h of the incubation with recombinant proprotein convertase subtilisin/kexin type 9 (rPCSK9) (600 ng/ml) with or without alirocumab (19.2 μg/ml) before flow cytometry analysis. (A) Cell surface low-density lipoprotein receptor (LDLR) expression, (B) low-density lipoprotein (LDL)–boron dipyrromethene (bodipy) uptake, and (C) lipoprotein(a) (Lp[a])-bodipy uptake in primary lymphocytes from a control volunteer and a homozygous familial hypercholesterolemia (HoFH) patient. Data are expressed in Δ mean fluorescence intensity. Histograms represent mean ± SEM of a minimum of 3 independent experiments performed in duplicates. Comparisons were made by analysis of variance followed by a Tukey post hoc test. ∗p < 0.05. ∗∗p < 0.01.

Figure 2

Lp(a) Cellular Uptake Is Not Modulated by Recombinant PCSK9 Peripheral blood mononuclear cells (PBMCs) treated with (solid bars) or without (open bars) recombinant proprotein convertase subtilisin/kexin type 9 (600 ng/ml) were incubated with 10 μg/ml fluorescent lipoprotein(a) (Lp[a]) or native (unlabeled) Lp(a) for 3 h. (A) Cellular Lp(a) uptake was determined by measuring the content of apo(a) in the cellular extracts. (B) Lp(a) diluted in culture medium before and after 3 h of incubation with PBMCs was subjected to Western blot analysis for apolipoprotein B100 (apoB100) under reducing and nonreducing conditions; apoB100 association with apo(a) was evidenced in nonreducing conditions. (C) Lp(a)–boron dipyrromethene (bodipy) uptake in control lymphocytes was assessed in the presence of a 20-fold excess of unlabeled Lp(a) or in the presence of 0.2 mmol/l epsilon aminocaproic acid. Comparisons were made by analysis of variance followed by a Tukey post hoc test. ∗p < 0.05, ∗∗p < 0.01 vs. standard conditions.

Figure 3

Alirocumab Increases Fluorescent LDL But Not Fluorescent-Lp(a) Hepatic Uptake In Vivo After baseline imaging capture, Fah(−/−)Rag2 (−/−)Il2rg(−/−) (FRG) mice treated with alirocumab or immunoglobulin G1 were infused either with low-density lipoprotein (LDL)–boron dipyrromethene (bodipy) or lipoprotein(a) (Lp[a])-bodipy tracers and recordings of 3-dimensional (3D) transillumination fluorescence tomography imaging were performed 15, 30, and 45 min after tracer infusions. Fluorescence volumetric pixels were quantified in the region of interest and expressed in arbitrary units (AUs). (A) Representative recordings of 3D transillumination fluorescence tomography with fluorescence intensity scale bar. (B) Quantification of LDL-bodipy hepatic uptake in FRG mice treated with immunoglobulin G1 (plain line, n = 4) or alirocumab (dotted line, n = 6). (C) Quantification of Lp(a)-bodipy hepatic uptake in FRG mice treated with immunoglobulin G1 (plain line, n = 4) or alirocumab (dotted lines, n = 5). Comparisons between treatments were performed using the Mann-Whitney test. ∗p < 0.05 vs. immunoglobulin G1.