REVIEW published: 23 March 2021 doi: 10.3389/fcvm.2021.639727 PCSK9 Functions in Atherosclerosis Are Not Limited to Plasmatic LDL-Cholesterol Regulation Aureli Luquero 1 , Lina Badimon 1,2,3 and Maria Borrell-Pages 1,2* 1 Cardiovascular Program ICCC, IR-Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain, 2 Centro de Investigación en Red- Área Cardiovascular, Instituto de Salud Carlos III, Madrid, Spain, 3 Cardiovascular Research Chair, Universitat Autònoma de Barcelona, Barcelona, Spain The relevance of PCSK9 in atherosclerosis progression is demonstrated by the benefits observed in patients that have followed PCSK9-targeted therapies. The impact of these therapies is attributed to the plasma lipid-lowering effect induced when LDLR hepatic expression levels are recovered after the suppression of soluble PCSK9. Different studies show that PCSK9 is involved in other mechanisms that take place at different stages Edited by: during atherosclerosis development. Indeed, PCSK9 regulates the expression of key Gabrielle Fredman, receptors expressed in macrophages that contribute to lipid-loading, foam cell formation Albany Medical College, United States and atherosclerotic plaque formation. PCSK9 is also a regulator of vascular inflammation Reviewed by: Marit Westerterp, and its expression correlates with pro-inflammatory cytokines release, inflammatory University of Groningen, Netherlands cell recruitment and plaque destabilization. Furthermore, anti-PCSK9 approaches have Jürgen Bernhagen, demonstrated that by inhibiting PCSK9 activity, the progression of atherosclerotic Ludwig Maximilian University of Munich, Germany disease is diminished. PCSK9 also modulates thrombosis by modifying platelets steady- *Correspondence: state, leukocyte recruitment and clot formation. In this review we evaluate recent Maria Borrell-Pages findings on PCSK9 functions in cardiovascular diseases beyond LDL-cholesterol plasma mborrellpa@santpau.cat levels regulation. Specialty section: Keywords: atherosclerosis, PCSK9 (proprotein convertase subtilisin kexin type 9), lipoprotein receptors, This article was submitted to inflammation, lipid loading, LDL—cholesterol Atherosclerosis and Vascular Medicine, a section of the journal Frontiers in Cardiovascular Medicine INTRODUCTION Received: 10 December 2020 Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a soluble protein synthesized as a Accepted: 01 March 2021 zymogen that undergoes autocatalytic cleavage in the endoplasmic reticulum (1). In 2007, PCSK9 Published: 23 March 2021 was found to be a ligand for Low Density Lipoprotein Receptor (LDLR) a key cell membrane Citation: receptor in cholesterol homeostasis regulation (2). LDLRs bind and internalize low density Luquero A, Badimon L and lipoproteins (LDL) from the bloodstream, clearing the blood from highly-enriched cholesterol Borrell-Pages M (2021) PCSK9 lipoproteins. The LDL-LDLR complex is guided to the lysosome where LDLs are digested and Functions in Atherosclerosis Are Not Limited to Plasmatic LDL-Cholesterol LDLRs are recycled to the cell surface to keep clearing LDL particles from the circulation. PCSK9 Regulation. inhibits LDLR recircularization by promoting its degradation in the lysosomes along with LDLs (3). Front. Cardiovasc. Med. 8:639727. This effect highly reduces the presence of LDLR at the hepatocyte’s cell surface and consequently, doi: 10.3389/fcvm.2021.639727 there is an increase in LDL particles in the bloodstream. Frontiers in Cardiovascular Medicine | www.frontiersin.org 1 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation Although PCSK9 is known since 2003 (4) and was almost with placebo in patients that suffered previous cardiovascular immediately associated with hypercholesterolemia (5), the events. These studies did not show benefits from bococizumab knowledge of its potential role on LDL metabolism regulation treatment despite showing significant improvements for patients and its associated diseases is increasing over the years (6, 7). It with a high cardiovascular risk. There was reduced clinical was first described that mutations on PCSK9 that lead to gain- efficacy because half of patients receiving bococizumab therapy of-function variants of the protein were responsible for different developed antidrug antibodies probably because bococizumab is cases of human familial hypercholesterolemia [FH; (8, 9)]. FH is a murine humanized antibody containing approximately a 3% an inherited disease where patients have LDL plasma levels above of murine sequence. Contrarily, alirocumab and evolocumab 190 mg/dL, contributing to an elevated risk of atherosclerotic are antibodies with full human sequence. The negative results plaque formation and coronary adverse events. Contrarily, concluded in a premature stop of the trial by the sponsor (25). PCSK9 loss-of-function mutations are associated with very low Besides monoclonal antibodies, other approaches that target levels of LDL in blood reducing the cardiovascular associated PCSK9 have been developed. Inclisiran (a small interference risk (10, 11). These data encouraged studies to test if PCSK9 RNA) and statin administration in patients with atherosclerotic was a good target for clinical trials to treat hypercholesterolemia. cardiovascular disease or heterozygous hypercholesterolemia Hypercholesterolemia is commonly treated with statins, which patients reduced PCSK9 levels in blood and LDL-cholesterol are drugs that inhibit HMG-CoA reductase, a key enzyme for levels in ORION-9 and ORION-10/ORION-11 phase 3 clinical cholesterol biosynthesis that reduces cholesterol production and trials (26, 27). lowers LDL concentration in plasma. However, some patients Regulation of cholesterol-rich LDL blood levels is not the present statin intolerance which hampers the treatment (12). only role that PCSK9 drives in atherosclerosis pathogenesis. Both PCSK9 and LDLR gene expression are regulated by There is a strong background suggesting alternative roles for SREBP2 [Sterol Regulatory Element-Binding Protein 2; (13–15)]. PCSK9 in the development of atherosclerosis (28, 29). Indeed, When intracellular levels of cholesterol are low (as after statin a prospective cohort of 4.232 sixty-year-old men and women treatments), there is activation of SREBP2 that promotes PCSK9 living in Stockholm County showed that independently of LDL and LDLR transcription. Therefore, both LDLR and LDLR’s plasma levels, PCSK9 levels correlate with elevated probability of inhibitor protein levels increase resulting in an intrinsic loop that future cardiovascular events (30). In another study that included limits statin therapy efficacy (16–18). SREBP2 transcriptional 643 participants, high plasma PCSK9 levels correlated with activity is regulated upstream by AMPK (AMP-activated protein enhanced atherosclerosis progression independently of LDL, as Kinase). Activation of AMPK leads to SREBP2 phosphorylation measured by carotid plaque formation and total plaque area and its inability to promote transcription of target genes (19, 20). (31). However, prospective studies have failed to demonstrate Anti-PCSK9 drugs started to be developed as a a relationship between PCSK9 expression levels and future risk secondary approach to reduce LDL cholesterol levels in of cardiovascular events despite revealing correlations between hypercholesterolemic patients. To date, only two monoclonal PCSK9 plasma levels and atherosclerotic markers including LDL- antibodies targeting PCSK9 are available for treating cholesterol, blood triglycerides or insulin (32, 33). hypercholesterolemia: evolocumab and alirocumab. They This review will discuss the role of PCSK9 in modulating were tested in the OSLER trial (21) and in the ODYSSEY LONG the activity of different cell lineages involved in atherosclerosis TERM trial (22), respectively (Table 1). Both studies showed progression including macrophages, vascular smooth muscle a ∼60% decrease of LDL particles in blood and a decrease in cells (VSMC), endothelial cells (EC), lymphocytes and platelets cardiovascular events including myocardial infarction, unstable and its associated cardiovascular risk. We will comment on angina or stroke (2.18% in placebo and 0.95% in evolocumab- PCSK9 effector roles showing that they extend far beyond treated patients in the OSLER trial and 5.1% in placebo and the regulation of LDL particles and reveal new insights 4.6% in alirocumab-treated patients in the ODYSSEY LONG by which PCSK9 inhibitors may lower the incidence of TERM study). However, the cardiovascular events reported atherosclerosis progression. during these studies were too low to demonstrate clinical relevance in this area. The FOURIER trial enrolled patients with previous atherosclerotic cardiovascular disease that were PCSK9 MODULATES MACROPHAGE’S on statin therapy (23). Results showed a 59% reduction of LDL LIPID-UPTAKE RECEPTORS content in bloodstream and decreased cardiovascular events including cardiovascular mortality, myocardial infarction, stroke, Atherosclerosis is commonly described as a chronic hospitalization for unstable angina or coronary revascularization inflammatory disease that starts with an excess of cholesterol by more than 15% in evolocumab treated patients after 26 months accumulation in the vascular wall triggering inflammation follow-up. The ODYSSEY OUTCOMES study in patients with (34). Macrophages are inflammatory cells that play a key role recent acute coronary syndrome at maximum tolerated dose of in lipid uptake and atherosclerosis progression. In 2012, it statins showed that alirocumab administration was associated was shown that LDLR expressed in the surface of human with a reduced risk of recurrent ischemic cardiovascular events macrophages were downregulated by PCSK9 produced by and also with a reduced mortality (24). Finally, the SPIRE VSMC, reducing the ability of macrophages to internalize 1 and SPIRE 2 trials were randomized trials that compared native LDL molecules and avoiding the formation of foam the efficacy of bococizumab, another anti-PCSK9 antibody, cells, indicating that PCSK9-stimulated macrophages reduce Frontiers in Cardiovascular Medicine | www.frontiersin.org 2 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation TABLE 1 | Study characteristics and outcomes of clinical trials with monoclonal antibodies against PCSK9. Dosing Patients/ Inclusion criteria Results Study limitations Treatment ratio OSLER TRIAL • OSLER-1: open-label, 420 mg/month 2:1 • No adverse events in previous • 61% reduction in • Open-label design randomized and controlled or 2,976 patients evolocumab studies. LDL levels • Low number of adverse study of patients from Phase II 140 on Evolocumab • Not having unstable medical • 56% reduction in CVE Evolocumab trials mg/2 weeks : condition. adverse CVE • Only patients who did not • OSLER-2: open-label, 1,489 patients • Not expected to need suffer CVE during previous randomized and controlled on previous adjustments of background Evolocumab therapy were study of patients from Phase III treatment lipid-regulating therapy. accepted Evolocumab trials (± statins) • High variability in patients’ cardiovascular risk and use of statins ODYSSEY LONG TERM A Phase III, randomized, 150 mg/2 2: 1 • Heterozygous FH, coronary • 62% reduction in • Short follow-up period double-blind, placebo-controlled, weeks 1,553 patients heart disease or equivalent risk LDL levels for a chronic disease parallel-group and multinational on Alirocumab • LDL-cholesterol levels above 70 • 48% reduction evaluation (20 months). study : mg/dL at screening adverse CVE • Low number of CVE, 788 patients on • Patients under high-dose statin limiting the robustness of placebo therapy or the data. maximum-tolerated dose FOURIER TRIAL Randomized, double-blinded, 140 1: 1 • ≥40 and ≤ 85 years-old • 59% reduction in Median of 2,2 years placebo-controlled, multicenter mg/2 weeks 13,784 patients • Clinical evidence of LDL cholesterol after trial or on Evolocumab atherosclerotic cardiovascular 42 weeks 420 : disease • 15% reduction in mg/month 13,780 patients • LDL cholesterol ≥ 70 mg/dL, CVE after 26 months on placebo non-HDL cholesterol ≥ 100 mg/dL while on lipid lowering therapy ODYSSEY OUTCOMES Randomized, double-blinded, 75 mg/2 1: 1 • ≥40 years old • 54,7% reduction in Median of 2,8 years placebo-controlled, multicenter weeks 9,462 Patients • Hospitalization 1 ≤ and ≥ 12 LDL cholesterol after trial on Alirocumab months with acute coronary 48 months : syndrome • 15% reduction of 9,462 patients • LDL cholesterol ≥ 70 mg/dL, CVE and 15% on placebo non-HDL cholesterol ≥ 100 reduction of death mg/dL and apoB ≥ 80 mg/dL SPIRE-1 and SPIRE-2 • Spire-1 patients were eligible 150 mg/2 1: 1 • Men ≥ 50/Women ≥ 60, in case • 59% reduction in Median of 10 months (the with at least 70 mg/dL of LDL weeks 13,720 Patients of FH Men ≥35/Women ≥ 45 LDL cholesterol after study was not finished) cholesterol at screening on Bococizumab • Previous CVE or a history of 14 weeks • Spire-2 patients were eligible : diabetes, chronic kidney disease • 12% reduction of with at least 100 mg/dL of LDL 13,718 patients or peripheral vascular disease CVE incidence cholesterol at screening on placebo with cardiovascular risk or familial hypercholesterolemia • Additional risk factors • On statin-therapy unless completely intolerance to statins is presented. LDL, low density lipoprotein; HDL, high density lipoprotein; FH, familial hypercholesterolaemia; apoB, apolipoprotein B; CVE, cardiovascular event. foam cells formation and hence, reduce atherosclerosis facilitate their modification by several secreted enzymes progression (35). However, native LDL molecules are not the including secretory phospholipase A2, sphingomyelinase, major source of cholesterol accumulation in macrophages. lipoxygenase or myeloperoxidase (38–40). Modified LDL Upon vascular extravasation, LDL molecules undergo several particles generate aggregated (agLDL) and oxidized (oxLDL) modifications including aggregation and oxidation. LDL LDLs, which are the major source of cholesterol ester aggregation and oxidation occur after extracellular matrix accumulation in macrophages and VSMCs [Figure 1; (41– components such as glycosaminoglycans (36) or chondroitin 44)]. Macrophages do not internalize agLDL or oxLDL sulfate proteoglycans (37) retain native LDL particles and through LDLR but through a different group of receptors Frontiers in Cardiovascular Medicine | www.frontiersin.org 3 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation FIGURE 1 | PCSK9 in atherosclerosis progression. Schematic showing the role of PCSK9 in different stages of atherosclerosis progression. called scavenger receptors (45) and LDLR related proteins reduce PCSK9 expression while ROS inducers pyocyanin and [LRPs; (46–48)]. antimycin A increase PCSK9 release showing that PCSK9 is Scavenger receptors including scavenger receptor A (SRA), expressed during macrophage proinflammatory procedures cluster of differentiation 36 (CD36) and lectin-like oxidized low- (50). ROS production is dependent on NADPH oxidase. density lipoprotein receptor 1 (LOX-1) promote the endocytosis Upon TNFα-stimulation lack of different NADPH oxidase of oxLDL particles in monocytes and macrophages and their complex subunits reduces the amount of scavenger receptors expression is highly increased under different inflammatory in the surface of macrophages (50). Recombinant PCSK9 stimulus including lipopolysaccharide (LPS) or tumor necrosis administration increases SRA, CD36, and LOX-1 both at factor-α [TNFα; Figure 1; (49, 50)]. Main features of scavenger gene and protein levels in cultured mouse macrophages. receptors are summarized in Table 2. LPS are major components Concomitantly, oxLDL uptake is increased (Figure 1). This of the outer membrane of Gram-negative bacteria that are increased lipid uptake is abolished in macrophages that lack recognized by Toll-Like Receptor 4 (TLR4) expressed in SRA, CD36, or LOX-1 suggesting that all three receptors macrophage’s cell surface. Binding of LPS to TLR4 triggers are involved in oxLDL uptake and consequently, in the an intracellular response that activates both MAPK and NFκB generation of foam cells in atherosclerosis (50). Table 2 pathways triggering inflammation. PCSK9 expression levels summarizes the involvement and regulation of scavenger are increased in mouse macrophages after LPS stimulation receptors and LDLR in different processes associated as a result of the activation of the NLRP3 (NOD-Like with atherosclerosis. Receptor Protein 3) inflammasome. Indeed, NLRP3 and its Other cell surface receptors expressed in macrophages downstream signals IL-1β, IL-18, and caspase 1 all participate modulated by PCSK9 are LRP1, LRP5, and LRP8. These in PCSK9 secretion as confirmed by specific gene deletion receptors belong to the LRP subfamily of the LDLR superfamily experiments (51). of receptors and conserve the characteristic EGF domain that PCSK9 expression in macrophages after TNFα stimulation allows PCSK9 binding (52). LRP1 surface levels, together with relies on the generation of reactive oxygen species (ROS). LDLR, are downregulated by human PCSK9 in atherosclerotic ROS inhibitors diphenyleneiodonium (DPI) and apocynin mouse macrophages inducing increased gene expression of Frontiers in Cardiovascular Medicine | www.frontiersin.org 4 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation TABLE 2 | Scavenger Receptors and LDLR main features. LOX-1 SRA CD36 LDLR Cell expression • VSMCs, endothelial cells, • Macrophages, VSMCs, • Macrophages, monocytes, • Particularly elevated macrophages, endothelial cells platelets, endothelial in hepatocytes platelets, fibroblasts cells, erythrocytes • Ubiquitous expression Upon PCSK9 ↑ expression ↑ expression ↑ expression ↓ expression stimulation Deficiency • Reduced oxLDL uptake in • Reduced oxLDL uptake in • Reduced oxLDL uptake in • Responsible for FH macrophages macrophages macrophages • Atheroprotective • Reduced inflammatory response • Atheroprotective and anti-inflammatory • Macrophage apoptosis Functions • Pro-atherogenic • Pro-atherogenic • Pro-atherogenic • Atheroprotective • Pro-inflammatory • Pro-inflammatory • Pro-inflammatory • Endocytosis of nLDL • Pro-thrombotic • Endocytosis of oxLDL • Pro-thrombotic • Induces PCSK9 expression in • In antigen presenting cells, • Endocytosis of oxLDL VSMCs mediates pathogen phagocytosis • Inhibits macrophage migration • Endocytosis of oxLDL • Promotes • Endothelial dysfunction platelet activation/aggregation • Foam cell formation • Macrophages, VSMC, endothelial cell apoptosis Other Upregulated in VSMCs, Upregulated in VSMCs and Upregulated in macrophages by regulations macrophages and monocytes endothelial cells during oxidative fat-rich diets, inflammation and during oxidative stress and stress oxidative stress inflammation Upregulated in macrophages and monocytes during inflammation nLDL, native low density lipoproteins; oxLDL, oxidized low density lipoproteins; VSMCs, vascular smooth muscle cells; FH, familial hypercholesterolemia. the proinflammatory markers TNFα and IL-1β and decreased PCSK9 MODULATES VASCULAR gene expression of the anti-inflammatory markers IL-10 and INFLAMMATION arginase-1 indicating enhanced macrophage polarization toward a pro-inflammatory phenotype (53). LRP8 (aka PCSK9 is mainly produced in liver, kidney and small intestine apoER2) a receptor known for recognizing ApoE protein (4). However, it is also expressed in vascular cells including is also downregulated upon recombinant PCSK9 binding endothelial cells (ECs) and vascular smooth muscle cells [VSMCs; in different cell lines including HEK293, 3T3 fibroblasts, (35, 57, 58)]. Vascular cells are affected by hemodynamic factors CHO, NeuroA2 and HuH7 (54). We have recently described like blood flow that, by inducing wall shear stress, play a that LRP5 is required for lipid internalization in human critical role in atherosclerosis development and progression (59). macrophages as in the absence of PCSK9 and/or LRP5, Human ECs and VSMCs under low-blood flow have higher macrophages show reduced cholesterol ester accumulation PCSK9 protein expression than cells under high blood flow, (55). Both proteins form a complex at the perinuclear area an effect conserved even after LPS stimulation. Indeed, aortas of human macrophages that immunoprecipitate together. from Wt mice showed significantly higher PCSK9 expression Their interaction is stronger in lipid loaded macrophages (55). in high shear stress regions, an effect further potentiated by In addition, macrophages silenced for LRP5 show reduced LPS administration (60). Also, in rabbits fed at high-fat diet, release of PCSK9, indicating that LRP5 is involved in soluble low-flow aortic regions had higher PCSK9 expression while PCSK9 release, probably by participating in the intracellular regions with high flow such the aortic arch showed lower transport of PCSK9 to the plasma membrane (55). Furthermore, vascular PCSK9 expression [Figure 1; (61)]. Therefore, there is we also show that the complex LRP5-PCSK9 up-regulates a negative correlation between PCSK9 vascular expression levels TLR4/NFκB signaling to favor macrophage inflammation. and blood flow. Interestingly, LRP5 surface levels remain unaltered by secreted PCSK9 has been shown to promote vascular inflammation. PCSK9 (55). Binding of PCSK9 to the inflammatory receptor TLR4 was Finally, VLDLR, a receptor that also belongs to the LDLR first hypothesized by the structural homology of the C- superfamily and displays a similar structure to that of LDLR terminal domain of PCSK9 and the TLR4 ligand resistin in in is also downregulated by PCSK9 binding. Indeed, treatment of silico simulations (62). TLRs are cell receptors that recognize HEK293 cells or 3T3 fibroblasts cells with human recombinant pathogens and regulate the expression of pro-inflammatory PCSK9 shows a downregulation of VLDLR expression levels (54). cytokines and also the early immune responses to infection (63). Both VLDLR and LRP8 are known to generate anti-inflammatory Among TLRs, TLR4 acts as a receptor for LPS and activates signaling in macrophages (56). NF-κB to promote an inflammatory response (64). PCSK9 Frontiers in Cardiovascular Medicine | www.frontiersin.org 5 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation expression in ECs and VSMCs is dependent on the TLR4/NFκB peptide (8–13 amino acids) that mimics the N-terminal domain signaling pathway as inhibition of different components of the of mature PCSK9 to a carrier protein that confers immunogenic activation cascade show that PCSK9 expression relies on the properties to activate the immune system. Syntheses of TLR4-MyD88-NFkB axis and is independent of the TLR4/TRIF host specific antibodies against PCSK9 generating long-term signaling, postulating the MyD88 pathway as a possible target inhibition are obtained. Vaccination therapy aims to overcome for future therapies to prevent excessive PCSK9 production monoclonal antibody therapy disadvantages including short in in the vasculature (61). Hence, PCSK9 synthesis is regulated vivo half-lives, frequent dosage administration and high costs by the TLR4 receptor signaling pathway through MyD88 and (80). In atherosclerosis mice models, inhibition of PCSK9 activity NFκB activation, and soluble PCSK9 can act as an inflammatory through vaccination decreased the expression of intercellular mediator by TLR4 binding and recognition as demonstrated in adhesion molecule 1 (ICAM-1) in the diseased aortic root and ApoE knockout mice (65). consequently there was a reduction in monocyte adhesion and Vascular stability depends on cellular apoptosis. PCSK9 migration to the endothelium that contributed to a reduction modulates the expression of the apoptosis inducer Bax and in atherosclerotic lesions (81). The anti-PCSK9 vaccine AT04A, the apoptosis inhibitor Bcl-2. The balance between these two which generates persistent humoral immune response against proteins is key to prevent or trigger apoptosis (66, 67). PCSK9 for 1 year in mice, reduced LDL content by more than Lipid loaded endothelial cells show increased Bax protein 50% (81). It also reduced NLRP3 inflammasome expression in levels and decreased Bcl-2 levels that lead to caspase 3 macrophages (81), a powerful inducer for PCSK9 expression and caspase 9 activation inducing cell apoptosis (68). PCSK9 and secretion in macrophages needed for the formation and silencing by siRNA, inhibits apoptosis as silenced PCSK9 cannot progression of atherosclerotic plaques (51). phosphorylate p38 and JNK (both members of the MAPK In humans, PCSK9 inhibitors therapy added to statin therapy signaling pathway) allowing the activation of the apoptosis is capable of increasing fibrous cap thickening in acute coronary inhibitor Bcl-2 (69–72). Interestingly, p38 and JNK are also syndrome patients, reducing plaque vulnerability (82). However, responsible of Bax and Bad phosphorylation that activate the LDL-cholesterol lowering capacities of both PCSK9 inhibitors programmed cell death (73, 74). Hence, PCSK9 may be and statin treatment cannot solely explain the increased fibrous promoting MAPK signaling cascade activation and endothelial cap thickness suggesting that an unknown pleiotropic effect such cell apoptosis [Figure 1], a mechanism that has already been as an anti-inflammatory effect independent of lowering LDL- described in cancer cells (75). cholesterol may be involved (82). PCSK9 inhibitor treatment in an atherogenic mouse model increased the number of circulating endothelial progenitor cells and circulating angiogenic cells, PCSK9 PARTICIPATES IN PLAQUE markers of endothelial and vascular health associated with FORMATION positive outcomes as reduced occurrence of cardiovascular events and death associated to cardiovascular causes (83). Plaque formation is a complex process that includes lipoprotein retention, inflammatory cells recruitment, VSMC proliferation, matrix synthesis, apoptosis, and necrosis (76). Several lines of evidence sustain that PCSK9 promotes plaque formation in mice ROLE OF PCSK9 IN INFLAMMATION IN and human (29, 67, 77). Indeed, PCSK9 increases LDL uptake THE ADAPTIVE IMMUNE SYSTEM by macrophages scavenger receptors contributing to cell foam formation (50); it favors inflammation at the atherosclerotic The role of PCSK9 during atherosclerosis progression in the vascular wall by inducing the expression of adhesion molecules, adaptive immune system has been studied. Dendritic cells (DCs) chemoattractants and inflammatory cytokines (78) and it induces and T lymphocytes are localized in the atherosclerotic plaque, ECs apoptosis reducing vessel stability (69). Furthermore, usually at sites prone to rupture (84). DCs mainly work as increased PCSK9 expression levels are associated to low shear antigen presenting cells to T lymphocytes. They phagocyte stress (60, 61). Therefore, PCSK9 is an efficient target for the antigens and present them to T lymphocytes in a process development therapies toward the prevention and treatment of that involves MHC and TCR complexes (in DCs and T atherosclerotic plaque formation. lymphocytes, respectively). In atheroma plaques, DCs present Anti-PCSK9 therapy in mice reduced by half the plaque oxLDL fragments to T-lymphocytes that are then activated area in the aortic root, and the infiltration of pro-inflammatory (85). The importance of T cell activation in atherosclerosis macrophages in the atherosclerotic plaque was decreased was demonstrated because ApoE knockout and immunodeficient (79). Serum levels of CXCL1, CXCL3, and CXCL10 (known (severe combined immunodeficiency mice without functional B chemoattractants for leukocytes), mainly produced by ECs and and T lymphocytes) mice had less atherosclerotic lesions than VSMCs, were reduced (79). Also, Pcsk9 knockout mice show ApoE knockout mice alone. Furthermore, CD4 T lymphocytes reduced expression of vascular cell adhesion molecule 1 (VCAM- from ApoE knockout mice transferred to immunodeficient ApoE 1), a protein needed for immune cell adhesion to the vascular knockout mice induce the generation of atherosclerotic lesions wall (57). (86). PCSK9 is induced by oxLDL in DCs and enhances the Anti-PCSK9 vaccination is an alternative to monoclonal expression of proteins involved in T cell activation including antibody therapy. Vaccination involves the conjugation of a CD80, CD83, CD86, and HLA-DR and the production of Frontiers in Cardiovascular Medicine | www.frontiersin.org 6 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation pro-inflammatory cytokines including TNFα, IL-1β, and IL- PCSK9 expression (94). Taken together, these results suggest 6. The expression of all these proteins was reduced when that circulating monocytes are pre-conditioned in FH patients PCSK9 was silenced, and TGFβ and IL-10 expression levels due to PCSK9 activity, which enhances their infiltrating were increased (87). T cells activated by oxLDL-stimulated DCs capacity, lipid accumulation and pro-inflammatory activity. produced mainly IFNγ and IL-17, indicating a polarization Indeed, a prospective study with heterozygous FH patients toward an anti-inflammatory Th1/Th17 phenotype. These anti- under standard statin therapy revealed a positive correlation inflammatory T regulatory cells inhibit foam cell formation between circulating levels of PCSK9 and adverse cardiovascular and reverse the pro-inflammatory phenotype of macrophages events (95). reducing atherosclerosis progression (88). PCSK9 is a key molecule in Th17 response as atherosclerotic Pcsk9/Ldlr/Apobec (apolipoprotein B mRNA-editing catalytic polypeptide-1) triple PCSK9 AND PLATELET THROMBOSIS knockout mice had significant lower Th17 production in comparison with atherosclerotic Ldlr/Apobec double knockout Several risk factors associated with cardiovascular disease, mice. This was associated with changes in the different cellular including hyperlipidaemia, induce endothelial dysfunction and sources of Th17 (Th17 lymphocytes or γδTCR+ T cells). Indeed, lead to arterial or venous thrombosis (96). In arteries with mice lacking PCSK9 had a reduced number of Th17 lymphocytes ongoing atherosclerosis progression, atherosclerotic plaque as well as a reduced expression of RORγT, the transcription factor rupture is the main cause for thrombosis (97). needed for Th17 lymphocyte differentiation (89). Pcsk9 knockout mice show reduced carotid artery thrombosis A very recent work shows that PCSK9 downregulates the induced by FeCl3 (a technique to rapidly and accurately induce expression of MHC class I proteins in tumor cells by promoting thrombi formation) in different sized arteries and veins (98). its internalization and degradation in lysosomes (in a similar Upon FeCl3 stimulation, 70% of Pcsk9 knockout mice developed manner to that of PCSK9 with LDLR). Therefore, PCSK9 non-occlusive non-stable thrombi after 30 min while 57% of decreases the cytotoxic T lymphocyte response against the Wt mice showed total artery occlusion before 15 min after tumor (90). In an atherosclerotic context it seems plausible FeCl3 administration suggesting a role for PCSK9 in platelet that modified LDLs could stimulate antigen presenting cells reactivity (98). Furthermore, platelets from Pcsk9 knockout mice such as DCs or B cells to produce a variety of cytokines that show a significant reduction in glycoprotein IIB/IIIA expression would guide T lymphocytes differentiation toward a particular levels, P-selectin expression levels and in circulating platelet- inflammatory subtype. leukocyte aggregates in comparison with Wt mice indicating lower platelet activation in Pcsk9 knockout mice (98). Similarly, Pcsk9 knockout mice also show reduced thrombi formation PCSK9’S ROLE IN FAMILIAL after inferior vena cava ligation in comparison to Wt mice HYPERCHOLESTEROLEMIA (99). Thrombi generated by inferior vena cava ligation in Pcsk9 knockout mice have less leukocyte attachment as leukocyte In 2003, after the discovery of PCSK9 gain-of-function mutations recruitment is dependent on P-selectin and CXCL1, which in FH patients the first monoclonal antibodies against PCSK9 are downregulated in Pcsk9 knockout mice (100). However, were tested in preclinical and clinical studies (including it is unknown whether this inflammatory cell recruitment is ODYSSEY LONG TERM, ODYSSEY OUTCOMES, and downregulated because of PCSK9’s role in lipid uptake or because FOURIER) demonstrating efficacy in reducing LDL cholesterol PCSK9 has lipid-independent functions on platelet’s steady-state plasma levels in patients (91). In 2020 a recent sub analysis (Figure 2 illustrates some of the mechanisms by which PCSK9 of the FOURIER and ODYSSEY OUTCOMES trials revealed induces thrombosis). that PCSK9 inhibition in patients with stable atherosclerosis NETosis is the process by which neutrophils release their and hyperlipidemia on statin therapy significantly reduces nuclei content composed of DNA and antimicrobial proteins the risk of venous thromboembolism supporting a protective including neutrophil elastases and histones, creating networks role for antiPCSK9 antibodies in human cardiovascular of extracellular fibers that trap and facilitate the killing of diseases (92). Monoclonal antibodies against PCSK9 are pathogens (101). NETosis is linked to thrombosis because it also capable of reversing the pro-inflammatory phenotype causes platelet activation, aggregation and adhesion (102) and of atherogenic macrophages in patients with FH. PCSK9 promotes the initiation of the coagulation cascade (100). In Pcsk9 inhibitors reduced CCR2, CX3CR1, and integrins CD11b and knockout mice NETosis is significantly reduced, despite that the CD18 expression in circulating monocytes suggesting a lower total number of blood neutrophils and leukocytes are increased infiltrating and chemoattractant capacity. PCSK9 antibody suggesting that PCSK9 can induce thrombosis by stimulating treatment reduced the production of TNFα by monocytes NETosis [Figure 2; (99)]. while the production of anti-inflammatory cytokine IL-10 The PCSK9-REACT study is an observational, prospective was enhanced (93). In fact, circulating monocytes from FH study where patients with recent acute coronary syndromes patients were enriched with lipid droplets despite non-detectable underwent coronary intervention and received P2Y12 inhibitors LDLR expression but increased expression of CD36 and SRA (103). P2Y12 is a chemoreceptor for adenosine diphosphate [Figure 1; (93)]. Also, ABCA1 protein, a protein responsible (ADP) involved in platelet aggregation (104) and a target for for cholesterol efflux in macrophages, was inhibited upon thromboembolism treatments using antagonists as ticagrelor or Frontiers in Cardiovascular Medicine | www.frontiersin.org 7 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation FIGURE 2 | PCSK9 aggravates thrombosis. Schematic showing the involvement of PCSK9 in different thrombotic processes. prasugrel (105). The study revealed a strong correlation between Cholesterol incorporation into platelet membranes induces PCSK9 blood levels and platelet reactivity (103). It also showed platelet reactivity while cholesterol depletion from membranes is that elevated PCSK9 plasma levels are associated with future associated with platelet stability (109). Thus, PCSK9 inhibition coronary events as 22% of patients with the highest PCSK9 would decrease plasma LDL levels reducing platelet reactivity. plasma levels suffered coronary events while only 2% of the As a matter of fact, statins treatment in hypercholesterolemic patients in the lower tertile experienced coronary events. In patients, is able to reduce platelet membrane cholesterol (110). line with these results, human recombinant PCSK9 added to It remains a matter of discussion whether PCSK9 exerts a direct healthy human plasma was capable of significantly increasing effect on platelets or the effects depend on the dyslipidaemia platelet aggregation and reducing aggregation lag time when generated by PCSK9 binding to LDLR. Dyslipidaemia induces the platelets were stimulated with epinephrine (98). The platelet generation of oxLDL and agLDL, which in turn, facilitate platelet enhancing capacity is because addition of PCSK9 increased the activation by binding scavenger receptors on platelet’s surface total number of platelets that express the activation marker including LOX-1 and CD36 (111–113). Once activated, platelets glycoprotein IIB/IIIA by 36% [Figure 2; (98)]. are capable of oxidizing LDLs, generating a positive feedback of A relationship between PCSK9 plasma levels and total platelet activation (114). PCSK9 inhibition also downregulates number of circulating platelets has also been shown in patients lipoprotein (a) [Lp(a)] serum levels in patients with inherited with stable coronary artery disease (106). Similarly, atrial dyslipidemias (115). Since Lp(a) enhances platelet activation fibrillation patients show a strong correlation between and thrombosis (116–119), PCSK9 may prevent thrombosis by PCSK9 plasma levels and platelet reactivity as elevated lowering Lp(a) levels. A direct effect of PCSK9 on platelets PCSK9 levels positively correlate with elevated risk for this independent of its effects on dyslipidaemia has been recently cardiovascular event (107). These patients also have higher shown as PCSK9 inhibitors can enhance oxidative stress (as a rate of platelet aggregation and recruitment coincidentally result of the activation of the Nox2 and cPLA2 signaling cascades) with higher expression levels of thromboxane B2 (TxB2 , and block platelet activation in Wt human platelets (108). a platelet activation marker), higher release of P-selectin PCSK9 inhibitors are being tested to modulate and enhanced ROS formation (108). Correlation between platelet activation in humans. Patients with primary elevated PCSK9 plasma levels and elevated urine excretion hypercholesterolemia with previous statin treatment were of TxB2 was also found (108). Taken together, these treated for 12 months with alirocumab or evolocumab and results show that not only platelet number but also platelet after only 2 months treatment a significant decrease in the reactivity is enhanced when PCSK9 plasma levels are elevated platelet activation marker CD62P was found (120). Soluble (Figure 2). CD40, soluble P-selectin and platelet factor 4 plasma levels were Frontiers in Cardiovascular Medicine | www.frontiersin.org 8 March 2021 | Volume 8 | Article 639727 Luquero et al. PCSK9 and Cholesterol Regulation also reduced after 12 months of statin and PCSK9 inhibitor inflammation, plaque formation and thrombosis. Hence, the treatment. The study also shows that hypercholesterolemic benefits observed from PCSK9 inhibitory therapies may not patients with additional acetylsalicylic acid administration to only be induced by its plasma lipid-lowering capacities but also statins and PCSK9 inhibitors have decreased platelet aggregation by reducing the impact of several other mechanisms in which (120). A trend in the reduction of platelet aggregation in patients PCSK9 is involved that are actively promoting atherosclerosis. without acetylsalicylic acid administration was observed but Unfortunately, the information on PCSK9 interactome is still there were no significant differences because of the low number limited and further investigations on the role of PCSK9’s activity of patients that followed this treatment (120). on different signaling pathways are still needed to generate a clear PCSK9 is also involved in blood clotting. Clotting formation vision of PCSK9 full potential during atherosclerosis progression. is a complex chemical process were circulating blood clotting Despite PCSK9 has been studied mostly in cardiovascular factors will sequentially induce protein cleavages to generate diseases, it also participates in general mechanisms shared by thrombin and fibrin (121). A correlation between elevated blood many other diseases, and hence it is conceivable that PCSK9 is clotting Factor VIII (FVIII) plasma levels and arterial thrombosis involved in the initiation and progression of other pathologies has been shown in both animal and human studies (122–124). with powerful inflammatory or thrombotic components. FVIII synthesis and clearance (and therefore FVIII plasma levels) are regulated by the liver. Indeed, LDLR and LRP1 expressed in hepatocytes promote FVIII endocytosis and degradation (125– AUTHOR CONTRIBUTIONS 128). Although not demonstrated yet, a connection between AL: research performance, draft manuscript preparation, PCSK9 and FVIII seems plausible. Indeed, downregulation of writing and review. MB-P: conceptualization, funding LDLR expression in hepatocytes cell surface regulated by PCSK9 acquisition, draft manuscript preparation, writing, review, induces an increase in FVIII plasma levels and therefore an and editing. LB: conceptualization and funding acquisition. increased risk of thrombosis and cardiovascular events (127). All authors contributed to the article and approved the PCSK9 can also reduce LRP1 cell surface expression further submitted version. increasing FVIII plasma levels (53, 129). Finally, in patients that produce anti-phospholipidic antibodies, polymorphisms in PCSK9 and LDLR genes are associated with thrombosis FUNDING progression supporting a role in clotting formation for the PCSK9-LDLR axis [Figure 2; (130)]. This work was supported by the Spanish Ministry of Science and Innovation and FEDER funds (PID2019-107160RB-I00 to LB); the Instituto de Salud Carlos III (CIBERCV CB16/11/00411 CONCLUDING REMARKS to LB, TERCEL RD16/0011/018 to LB and FIS2020-01282 to MB-P); the Generalitat of Catalunya-Secretaria d’Universitats Since PCSK9 was first described as the inducer of some i Recerca del Departament d’Economia i Coneixement de FH pathologies, a lot of interest has been placed in the la Generalitat (2017SGR1480 to LB); the Spanish Society achievement of an effective inhibitory treatment. PCSK9 of Cardiology (FEC 2019 to MB-P); and the Fundación inhibitors administered to patients revealed a key role of PCSK9 Investigación Cardiovascular-Fundación Jesus Serra for their in atherosclerotic disease as its inhibition reduced plasma LDL- continuous support. cholesterol levels with improved clinical cardiovascular outcomes demonstrating a multifactorial and pathophysiological role for PCSK9 in atherosclerosis progression. Interestingly, PCSK9 ACKNOWLEDGMENTS functions are far from only regulating LDL-cholesterol plasma levels by reducing hepatic LDLR expression. Indeed, recent We thank S. Huertas and M. A. Velasco for their continuous findings demonstrate that PCSK9 is also actively modulating support to the group. REFERENCES cholesterol. J Biol Chem. (2004) 279:48865–75. doi: 10.1074/jbc.M4096 99200 1. Poirier S, Mayer G. The biology of PCSK9 from the endoplasmic 4. 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Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to Copyright © 2021 Luquero, Badimon and Borrell-Pages. This is an open-access thrombosis and thrombus stability in mice. Blood. (2011) 118:3960–8. article distributed under the terms of the Creative Commons Attribution License (CC doi: 10.1182/blood-2011-06-362814 BY). The use, distribution or reproduction in other forums is permitted, provided 125. Saenko EL, Yakhyaev AV, Mikhailenko I, Strickland DK, Sarafanov the original author(s) and the copyright owner(s) are credited and that the original AG. Role of the low density lipoprotein-related protein receptor in publication in this journal is cited, in accordance with accepted academic practice. mediation of factor VIII catabolism. J Biol Chem. (1999) 274:37685–92. No use, distribution or reproduction is permitted which does not comply with these doi: 10.1074/jbc.274.53.37685 terms. 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