Recent advances in demystifying the metabolism of lipoprotein(a).

Lipoprotein (a) (Lp(a)) is a strange lipoprotein species causatively independently and significantly associated with cardiovascular diseases and calcified aortic valve stenosis. Elevated plasma Lp(a) levels increase the rate of cardiovascular events at any achieved low-density lipoprotein (LDL) level. The major structural difference between Lp(a) and LDL is that Lp(a) has a second large protein, apolipoprotein (a) (apo(a)), bound to the apolipoprotein B100 moiety of an LDL sized particle by a single disulfide bond. Over the past decades, several investigators have tried to elucidate the molecular, cellular and metabolic pathways governing the production of Lp(a), the contribution of Lp(a) to lipid transport in the plasma, and the catabolic fate of Lp(a). The metabolism of this enigmatic lipoprotein nevertheless still remains poorly understood. The objectives of the present manuscript are to comprehensively review the knowns and unknowns of the complexities of Lp(a) metabolism with a focus on apo(a) biosynthesis in hepatocytes, Lp(a) assembly, and Lp(a) plasma clearance and catabolism. We also discuss the controversy surrounding the exact role of the LDL receptor in mediating Lp(a) cellular uptake by reviewing seminal in vitro and in vivo data, the metabolism of Lp(a) in familial hypercholesterolemia, as well as the divergent effects of statins and proprotein convertase subtilisin kexin type 9 inhibitors in modulating Lp(a) plasma concentrations. We also provide new insights into the physiology and pathophysiology of Lp(a) metabolism from human kinetic studies in the context of contemporary molecular and cell biological investigations.

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants.

BACKGROUND: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. OBJECTIVE: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. METHODS: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. RESULTS: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. CONCLUSIONS: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.

Novel PCSK9 (Proprotein Convertase Subtilisin Kexin Type 9) Variants in Patients With Familial Hypercholesterolemia From Cape Town.

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by elevated low-density lipoprotein-cholesterol and markedly increased cardiovascular risk. In patients with a genetic diagnosis, low-density lipoprotein receptor (LDLR) mutations account for >90% of cases, apolipoprotein B (APOB) mutations for ≈5% of cases, while proprotein convertase subtilisin kexin type 9 (PCSK9) gain of function mutations are rare (<1% of cases). We aimed to evaluate the functional impact of several novel PCSK9 variants in a cohort of patients with FH by genetic cascade screening and in vitro functionality assays. Approach and Results: Patients with clinically diagnosed FH underwent genetic analysis of LDLR, and if negative, sequential testing of APOB and PCSK9. We analyzed cosegregation of hypercholesterolemia with novel PCSK9 variants. Gain of function status was determined by in silico analyses and validated by in vitro functionality assays. Among 1055 persons with clinical FH, we identified nonsynonymous PCSK9 variants in 27 (2.6%) patients and 7 of these carried one of the 4 previously reported gain of function variants. In the remaining 20 patients with FH, we identified 7 novel PCSK9 variants. The G516V variant (c.1547G>T) was found in 5 index patients and cascade screening identified 15 additional carriers. Low-density lipoprotein-cholesterol levels were higher in these 15 carriers compared with the 27 noncarriers (236±73 versus 124±35 mg/dL; P<0.001). In vitro studies demonstrated the pathogenicity of the G516V variant. CONCLUSIONS: In our study, 1.14% of cases with clinical FH were clearly attributable to pathogenic variants in PCSK9. Pathogenicity is established beyond doubt for the G516V variant.

A small molecule inhibitor of PCSK9 that antagonizes LDL receptor binding via interaction with a cryptic PCSK9 binding groove.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Homozygous Familial Hypercholesterolemia Patients With Identical Mutations Variably Express the LDLR (Low-Density Lipoprotein Receptor): Implications for the Efficacy of Evolocumab.

OBJECTIVE: Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)-neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. APPROACH AND RESULTS: Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. CONCLUSIONS: Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.