In-vitro experiments have provided conflicting evidence regarding the role of the LDLR in Lp(a) cellular uptake. Thus, PCSK9 was shown to reduce the binding and the cellular uptake of Lp(a) via the LDLR in human hepatoma cells, in primary human fibroblasts, and in primary murine hepatocytes, whereas the LDLR-related protein 1 (LRP1) was ruled out as a putative receptor for Lp(a) [17–19]. In another study, the LDLR did not seem to play any significant role in mediating Lp(a) cellular uptake in primary human hepatocytes, in primary human fibroblasts, as well as in human hepatoma cells [14,20,21]. Neither did we observe any significant difference in Lp(a) cellular uptake in primary lymphocytes isolated from controls and homozygous familial hypercholesterolemia patients who totally lack LDLR function (G. Lambert, unpublished observation). Other receptors have been proposed to mediate Lp(a) hepatic uptake, including the scavenger receptor BI (SR-BI), and the plasminogen receptor PlgRKT . In that study conducted in hepatoma cells, Lp(a) is taken up by the plasminogen receptor PlgRKT, and apo(a) is to some extent re-secreted.
In-vivo experiments have also provided conflicting evidence regarding the role of the LDLR and the effects of PCSK9 inhibitors on Lp(a) turnover, production and catabolism. Thus, PCSK9 inhibition with alirocumab increased, although not significantly, the fractional catabolic rate of Lp(a) in human volunteers . In contrast, PCSK9 neutralization with evolocumab in monotherapy did not alter the fractional catabolic rate but rather enhanced the production rate of Lp(a) in another human study [23▪]. Discrepancies between these studies in terms of Lp(a) kinetic parameters might stem from the facts that the degree of Lp(a) reduction induced by PCSK9 inhibitors substantially differed (−19 vs. −35%, respectively), and that the demographical characteristics of the patients were quite different (mixed race and sex vs. caucasian men only, respectively) [22,24]. On top of statin treatment, however, evolocumab enhanced the catabolism of Lp(a) [23▪]. In addition, alirocumab was recently shown in nonhuman primates to efficiently reduce apolipoprotein B100 primarily by enhancing its catabolism and apolipoprotein (a) primarily by lowering its production [25▪], an observation in line with a previous study in which PCSK9 enhanced apo(a) secretion from human primary hepatocytes .
The studies mentioned above have not provided a consensus regarding the mechanism(s) governing Lp(a) clearance. In favor of a role of the LDLR in mediating Lp(a) clearance: familial hypercholesterolemia patients presenting with LDLR defects usually display higher Lp(a) level than their nonaffected relatives, the correlations between changes in LDL (−50/60%) and changes in Lp(a) (−20/30%) following PCSK9 inhibitors treatments are weak but consistently significant [5,25▪,26] and appear independent of apo(a) polymorphisms . In favor of a non-LDLR mediated pathway: PCSK9 inhibitors lower Lp(a) even in receptor-negative homozygous familial hypercholesterolemia patients without modulating LDL-C, up-regulating the LDLR even in the most potent statins does not reduce Lp(a), there is no correlation between the levels of LDLR expression assessed ex vivo and the levels of circulating Lp(a) in familial hypercholesterolemia patients [13,28▪]. More work is, therefore, required to elucidate the exact mechanisms governing Lp(a) production and clearance and their modulation by PCSK9 and PCSK9 inhibitors.
From a clinical point of view, it has not been established whether the reduction in Lp(a) induced by PCSK9 inhibitors per se confers cardiovascular protection. Clinical trials with evolocumab or alirocumab have not shown any incremental benefits in terms of cardiovascular risk (CVD) risk reduction compared with what would have been anticipated from the sole reduction in LDL-C induced by these drugs [29,30]. Nevertheless, it has recently been shown that patients with high baseline Lp(a) levels benefit more from these treatments in terms of absolute cardiovascular risk reduction than patients with low-baseline Lp(a) levels (Dr M O’Donoghue presentation at EAS meeting 2018 and Dr V Bittner presentation at ISA meeting 2018).
Despite their undisputable beneficial effects in lowering LDL-C and CVD risk, statins have shown to slightly but significantly increase the risk of new-onset diabetes among predisposed individuals. As both statins and PCSK9 inhibitors reduce LDL-C by increasing the abundance of the LDLR at the surface of many cell types, most importantly hepatocytes, a very logical question is to determine whether PCSK9 inhibitors might also increase the risk of diabetes. Whereas enhancing the LDLR pathway in the liver is antiatherogenic and clearly beneficial, activating the same pathway in pancreatic β-cells may lead to LDL cholesterol overload and be harmful to those cells. This hypothesis is underpinned by Mendelian randomization studies showing that genetic variants in HMGCR and PCSK9 that confer lifelong reductions in LDL-C levels and are thus cardioprotective, also additively associate with an increased risk of developing diabetes [31–33]. Further advocating for a role of the LDLR in the development of diabetes, the observation that diabetes prevalence is twice lower in patients with familial hypercholesterolemia compared with their nonaffected relatives (1.75 vs. 2.93%, respectively) . Diabetes risk was further reduced in carriers of the most severe mutations (i.e. negative vs. defective LDLR genetic defects at 1.41 vs. 1.80%, respectively), but also in carriers of familial hypercholesterolemia causing APOB mutations (at 2.42%) .
The LDLR is expressed abundantly at the surface of pancreatic β-cells where it promotes LDL uptake [35▪]. However, β-cell lines exposed to LDL in culture often display increased necrosis or apoptosis as well as reduced insulin secretion ability in response to glucose, indicating that excessive uptake of LDL cholesterol could be detrimental to β-cells [36,37]. Fully in line with its role in most tissues, PCSK9 is involved in the regulation of the LDLR in pancreatic islets. Thus, islet cells from PCSK9 knockout mice show increased LDLR expression, whereas human islets incubated with recombinant PCSK9 display reduced LDLR expression levels . Experiments conducted in rodents have, however, yielded opposite conclusions about the effect of PCSK9-deficiency on glucose homeostasis. There was no alteration in glucose homeostasis in PCSK9 knockout mice, including glucose-stimulated insulin secretion measured in one study , whereas another study showed that PCSK9 knockout male mice are glucose intolerant, with an increased rate of β-cell apoptosis . In a more recent study, PCSK9 knockout mice were shown to present with glucose intolerance without any sign of insulin resistance [35▪]. The secretion of insulin appeared impaired in PCSK9 knockout mice, an effect associated with an increased presence of islets of large sizes, and dependent on the presence of a functional LDLR. In contrast, liver-specific PCSK9-knockout mice, a model in which there is no detectable circulating PCSK9, have normal pancreatic LDLR expression and do not show any sign of glucose intolerance [35▪]. In mice, PCSK9 was found to be expressed in Langherans islets δ-cells and proposed to contribute to the limitation of cholesterol overload in neighbouring β-cells [35▪,38]. In humans, however, PCSK9 gene expression appears to occur preferentially in β-cells (Fig. 2). Beyond its role on β-cell function, the role of PCSK9 on insulin sensitivity remains largely unknown. In epidemiological studies, plasma PCSK9 concentrations have been found to be positively correlated with the index of insulin resistance HOMA-IR.
The successful developments of evolocumab and alirocumab unambiguously demonstrate that PCSK9 is an extremely valuable therapeutic target, given that these novel therapies clearly improve clinical outcomes with extremely limited side effects. The cellular trafficking pathways by which PCSK9 enhance LDLR degradation either via the endolysosomal extracellular route or via the Golgi-lysosomal intracellular route still need to be elucidated. Likewise, the mechanisms by which PCSK9 inhibitors reduce lipoprotein (a) remain a point of controversy and certainly merit additional research efforts. Additional work on the putative role of PCSK9 in the overall metabolism of glucose are also needed, given the paucity of data from long-term follow-up studies with PCSK9 inhibitors. No doubt that these gaps in knowledge will be filled in the coming years.
Papers of particular interest, published within the annual period of review, have been highlighted as:
1. Seidah NG, Chrétien M, Mbikay M. The ever-expanding saga of the proprotein convertases and their roles in body homeostasis: emphasis on novel proprotein convertase subtilisin kexin number 9 functions and regulation. Curr Opin Lipidol 2018; 29:144–150.
2▪. Lai Q, Giralt A, Le May C, et al. E2F1 inhibits circulating cholesterol clearance by regulating Pcsk9
expression in the liver. JCI Insight 2017; 2: doi: 10.1172/jci.insight.89729.
An elegant study showing that the transcription factor E2F1 maintains cellular cholesterol homeostasis through the positive regulation of PCSK9 gene expression.
3. Naeli P, Mirzadeh Azad F, Malakootian M, et al. Posttranscriptional Regulation of PCSK9
by miR-191, miR-222, and miR-224. Front Genet 2017; 8:189.
4▪. Gustafsen C, Olsen D, Vilstrup J, et al. Heparan sulfate proteoglycans present PCSK9
to the LDL receptor
. Nat Commun 2017; 8:503.
A provocative demonstration that the N terminal domain of PCSK9 contains a cluster of basic residues that interact with HSPGs, which is essential for PCSK9 function in vitro and in vivo.
5. Shapiro MD, Tavori H, Fazio S. PCSK9
: from basic science discoveries to clinical trials. Circ Res 2018; 122:1420–1438.
6. Susan-Resiga D, Girard E, Kiss RS, et al. The proprotein convertase subtilisin/kexin type 9-resistant R410S low density lipoprotein receptor mutation: a novel mechanism causing familial hypercholesterolemia. J Biol Chem 2017; 292:1573–1590.
7. Poirier S, Mamarbachi M, Chen W-T, et al. GRP94 regulates circulating cholesterol levels through blockade of PCSK9
-induced LDLR degradation. Cell Rep 2015; 13:2064–2071.
8. Poirier S, Hamouda HA, Villeneuve L, et al. Trafficking dynamics of PCSK9
-induced LDLR degradation: focus on human PCSK9
mutations and C-terminal domain. PloS One 2016; 11:e0157230.
9▪. Lebeau P, Platko K, Al-Hashimi AA, et al. Loss-of-function PCSK9
mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained. J Biol Chem 2018; 293:7329–7343.
An elegant study showing that the PCSK9-Q152H loss of function variant retained in the ER does not cause ER stress as it is efficiently degraded in the proteasome.
10. Di Taranto MD, Benito-Vicente A, Giacobbe C, et al. Identification and in vitro characterization of two new PCSK9
gain of function variants found in patients with familial hypercholesterolemia. Sci Rep 2017; 7:15282.
11▪. Dron JS, Hegele RA. Complexity of mechanisms among human proprotein convertase subtilisin-kexin type 9 variants. Curr Opin Lipidol 2017; 28:161–169.
A comprehensive review on the mode of action of PCSK9 gain-of-function and PCSK9 loss of-function mutants
12. Chorba JS, Galvan AM, Shokat KM. Stepwise processing analyses of the single-turnover PCSK9
protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype. J Biol Chem 2018; 293:1875–1886.
13. Arsenault BJ, Petrides F, Tabet F, et al. Effect of atorvastatin, cholesterol ester transfer protein inhibition, and diabetes
mellitus on circulating proprotein subtilisin kexin type 9 and lipoprotein(a) levels in patients at high cardiovascular risk. J Clin Lipidol 2018; 12:130–136.
14. Lambert G, Thedrez A, Croyal M, et al. The complexity of lipoprotein (a) lowering by PCSK9
monoclonal antibodies. Clin Sci (Lond) 2017; 131:261–268.
15▪▪. Tsimikas S, Fazio S, Ferdinand KC, et al. NHLBI Working Group recommendations to reduce lipoprotein(a)-mediated risk of cardiovascular disease and aortic stenosis. J Am Coll Cardiol 2018; 71:177–192.
An excellent expert recommendation article on lipoprotein(a) listing the main gaps in knowledge on this understudied lipoprotein.
16. Boffa MB, Koschinsky ML. The journey towards understanding lipoprotein(a) and cardiovascular disease risk: are we there yet? Curr Opin Lipidol 2018; 29:259–267.
17. Romagnuolo R, Scipione CA, Marcovina SM, et al. Roles of the low density lipoprotein receptor and related receptors in inhibition of lipoprotein(a) internalization by proprotein convertase subtilisin/kexin type 9. PloS One 2017; 12:e0180869.
18. Raal FJ, Giugliano RP, Sabatine MS, et al. PCSK9
inhibition-mediated reduction in Lp(a) with evolocumab: an analysis of 10 clinical trials and the LDL receptor
's role. J Lipid Res 2016; 57:1086–1096.
19. Romagnuolo R, Scipione CA, Boffa MB, et al. Lipoprotein(a) catabolism is regulated by proprotein convertase subtilisin/kexin type 9 through the low density lipoprotein receptor. J Biol Chem 2015; 290:11649–11662.
20. Sharma M, Redpath GM, Williams MJA, McCormick SPA. Recycling of apolipoprotein(a) after PlgRKT-mediated endocytosis of lipoprotein(a). Circ Res 2017; 120:1091–1102.
21. Villard EF, Thedrez A, Blankenstein J, et al. PCSK9
modulates the secretion but not the cellular uptake of lipoprotein(a) ex vivo: an effect blunted by alirocumab. JACC Basic Transl Sci 2016; 1:419–427.
22. Reyes-Soffer G, Pavlyha M, Ngai C, et al. Effects of PCSK9
inhibition with alirocumab on lipoprotein metabolism in healthy humans. Circulation 2017; 135:352–362.
23▪. Watts GF, Chan DC, Somaratne R, et al. Controlled study of the effect of proprotein convertase subtilisin-kexin type 9 inhibition with evolocumab on lipoprotein(a) particle kinetics. Eur Heart J 2018; 39:2577–2585.
A rigorous demonstration showing how PCSK9 inhibition reduces Lp(a) by a series of in-vivo human kinetic studies.
24. Watts GF, Chan DC, Dent R, et al. Factorial effects of evolocumab and atorvastatin on lipoprotein metabolism. Circulation 2017; 135:338–351.
25▪. Croyal M, Tran T-T-T, Blanchard RH, et al. PCSK9
inhibition with alirocumab reduces lipoprotein(a) levels in nonhuman primates by lowering apolipoprotein(a) production rate. Clin Sci (Lond) 2018; 132:1075–1083.
This cross over study demonstrates that the PCSK9 inhibitor alirocumab reduces Lp(a) production and augments LDL catabolism in vivo.
26. Edmiston JB, Brooks N, Tavori H, et al. Discordant response of low-density lipoprotein cholesterol and lipoprotein(a) levels to monoclonal antibodies targeting proprotein convertase subtilisin/kexin type 9. J Clin Lipidol 2017; 11:667–673.
27. Enkhmaa B, Anuurad E, Zhang W, et al. The roles of apo(a) size, phenotype, and dominance pattern in PCSK9
-inhibition-induced reduction in Lp(a) with alirocumab. J Lipid Res 2017; 58:2008–2016.
28▪. Thedrez A, Blom DJ, Ramin-Mangata S, et al. Homozygous familial hypercholesterolemia patients with identical mutations variably express the LDLR (low-density lipoprotein receptor): implications for the efficacy of evolocumab. Arterioscler Thromb Vasc Biol 2018; 38:592–598.
A comprehensive study showing that residual LDLR activity is key for PCSK9i-mediated reductions in LDL-C among homozygote familial hypercholesterolemia.
29. Sabatine MS, Giugliano RP, Keech AC, et al. Evolocumab and clinical outcomes in patients with cardiovascular disease. N Engl J Med 2017; 376:1713–1722.
30. Ridker PM, Revkin J, Amarenco P, et al. SPIRE Cardiovascular Outcome Investigators. Cardiovascular efficacy and safety of bococizumab in high-risk patients. N Engl J Med 2017; 376:1527–1539.
31. Lotta LA, Sharp SJ, Burgess S, et al. Association between low-density lipoprotein cholesterol-lowering genetic variants and risk of type 2 diabetes
: a meta-analysis. JAMA 2016; 316:1383–1391.
32. Schmidt AF, Swerdlow DI, Holmes MV, et al. PCSK9
genetic variants and risk of type 2 diabetes
: a Mendelian randomisation study. Lancet Diabetes
Endocrinol 2017; 5:97–105.
33. Ference BA, Robinson JG, Brook RD, et al. Variation in PCSK9
and HMGCR and risk of cardiovascular disease and diabetes
. N Engl J Med 2016; 375:2144–2153.
34. Besseling J, Kastelein JJP, Defesche JC, et al. Association between familial hypercholesterolemia and prevalence of type 2 diabetes
mellitus. JAMA 2015; 313:1029–1036.
35▪. Da Dalt L, Ruscica M, Bonacina F, et al. PCSK9
deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor. Eur Heart J 2018; doi: 10.1093/eurheartj/ehy357.
An elegant study showing that PCSK9 deficiency in mouse models causes impairment in glucose homeostasis as a result of defective pancreatic islets.
36. Roehrich M-E, Mooser V, Lenain V, et al. Insulin-secreting beta-cell dysfunction induced by human lipoproteins. J Biol Chem 2003; 278:18368–18375.
37. Cnop M, Hannaert JC, Grupping AY, Pipeleers DG. Low density lipoprotein can cause death of islet beta-cells by its cellular uptake and oxidative modification. Endocrinology 2002; 143:3449–3453.
38. Langhi C, Le May C, Gmyr V, et al. PCSK9
is expressed in pancreatic delta-cells and does not alter insulin secretion. Biochem Biophys Res Commun 2009; 390:1288–1293.
39. Mbikay M, Sirois F, Mayne J, et al. PCSK9
-deficient mice exhibit impaired glucose tolerance and pancreatic islet abnormalities. FEBS Lett 2010; 584:701–706.
40▪. Sabatine MS, Leiter LA, Wiviott SD, et al. Cardiovascular safety and efficacy of the PCSK9
inhibitor evolocumab in patients with and without diabetes
and the effect of evolocumab on glycaemia and risk of new-onset diabetes
: a prespecified analysis of the FOURIER randomised controlled trial. Lancet Diabetes
Endocrinol 2017; 5:941–950.
This study demonstrates that the PCSK9 inhibitor evolocumab does not worsen diabetes or cause new onset diabetes.
* Stéphane Ramin-Mangata and Valentin Blanchard Equal contributions.