The role of PCSK9 in heart failure and other cardiovascular diseases-mechanisms of action beyond its effect on LDL cholesterol

Abstract

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a protein that regulates low-density lipoprotein (LDL) cholesterol metabolism by binding to the hepatic LDL receptor (LDLR), ultimately leading to its lysosomal degradation and an increase in LDL cholesterol (LDLc) levels. Treatment strategies have been developed based on blocking PCSK9 with specific antibodies (alirocumab, evolocumab) and on blocking its production with small regulatory RNA (siRNA) (inclisiran). Clinical trials evaluating these drugs have confirmed their high efficacy in reducing serum LDLc levels and improving the prognosis in patients with atherosclerotic cardiovascular diseases. Most studies have focused on the action of PCSK9 on LDLRs and the subsequent increase in LDLc concentrations. Increasing evidence suggests that the adverse cardiovascular effects of PCSK9, particularly its atherosclerotic effects on the vascular wall, may also result from mechanisms independent of its effects on lipid metabolism. PCSK9 induces the expression of pro-inflammatory cytokines contributing to inflammation within the vascular wall and promotes apoptosis, pyroptosis, and ferroptosis of cardiomyocytes and is thus involved in the development and progression of heart failure. The elimination of PCSK9 may, therefore, not only be a treatment for hypercholesterolaemia but also for atherosclerosis and other cardiovascular diseases. The mechanisms of action of PCSK9 in the cardiovascular system are not yet fully understood. This article reviews the current understanding of the mechanisms of PCSK9 action in the cardiovascular system and its contribution to cardiovascular diseases. Knowledge of these mechanisms may contribute to the wider use of PCSK9 inhibitors in the treatment of cardiovascular diseases.

Keywords: Atherosclerosis; Heart failure; Inflammation; PCSK9; PCSK9 inhibitors.

Fig. 1

Mechanism of action of PCSK9 in a hepatocyte. PCSK9 is involved in regulating LDLc levels by binding to hepatic LDLR, which ultimately leads to its lysosomal degradation. When the LDLR-LDLc complex is formed, it is internalised and enters endosomes. There, in the acidic environment (pH 5.4), LDLR undergoes some conformational changes that lead to the release of LDLc from the complex. LDLc is then transported to lysosomes and undergoes degradation, while LDLR returns to the cell surface. When PCSK9 attaches to LDLR, this LDLR-PCSK9 complex also undergoes internalisation and enters endosomes, where the aforementioned acidic environment makes the binding between LDLR and PCSK9 much stronger. As a result, LDLR is unable to dissociate from PCSK9 and the entire LDLR-PCSK9 complex enters lysosomes and is degraded. As a result, LDLR is unable to return to the cell surface and bind to LDLc. Explanation of abbreviations: PCSK9, proprotein convertase subtilisin/keksin type-9; PCSK9 mRNA, mRNA for proprotein convertase subtilisin/keksin type-9; LDLc, low-density lipoprotein cholesterol; LDLR, LDL receptor

Fig. 2

The role of PCSK9 in the development and progression of heart failure through its involvement in such biological processes as apoptosis, pyroptosis, and ferroptosis of cardiomyocytes. Particularly high levels of PCSK9 expression have been found in cardiomyocytes exposed to such harmful factors as hypoxia, ischaemia/reperfusion, and hypoxia/reoxygenation. An increased release of PCSK9 by cardiomyocytes promotes cardiomyocyte apoptosis by activating the NF-kB pathway and, by the stimulation of macrophages, releasing pro-inflammatory cytokines. PCSK9 increases mitochondrial ROS release and contributes to increased expression of the pro-apoptotic caspase 9, caspase 3, and Bax and decreased expression of the anti-apoptotic protein Bcl-2. PCSK9 may also be involved in the development and progression of HF by initiating mitochondrial DNA damage and releasing ROS, leading to NLRP3 activation and pyroptosis. Activation of pyroptosis in cardiomyocytes begins with accumulation of the NLRP3 inflammasome with the involvement of DAMPs, leading to activation of caspase-1. Activated caspase-1 causes the conversion of GSDMD to N-GSDMD, which leads to the formation of holes in the cell membrane. A damaged cell membrane allows the contents of the cell to escape, leading to a strong inflammatory response. The second effect of caspase-1 activation is the conversion of inactive forms of IL-1 β and IL-18 into their active forms. Together, this leads to cell death. PCSK9 also affects another process associated with the development and progression of HF-ferroptosis. This is a type of programmed cell death in which iron overload induces the Fenton reaction, ROS production, and lipid peroxidation, ultimately leading to cell death. The mechanisms regulating ferroptosis in cardiomyocytes are complex, involving several metabolic pathways and ROS production. The major source of ROS in the cell is the mitochondria. PCSK9 induces mitochondrial dysfunction and increases ROS production in cardiomyocytes. It is thought that PCSK9 may regulate ferroptosis in cardiomyocytes by regulating mitochondrial function and ROS production. PCSK9 may also regulate ferroptosis in cardiomyocytes via TLR4 which inhibits GPX4, thereby enhancing ferroptosis. Explanation of abbreviations: PCSK9, proprotein convertase subtilisin/keksin type-9; NF-kB, nuclear factor kappa B; ROS, reactive oxygen species; NLRP3, NOD-like receptor family, pyrin domain containing 3; DAMPs, damage-associated molecular patterns; GSDMD, Gasdermin D; N-GSDMD, N-terminal Gasdermin D; IL-1β, interleukin-1beta; IL-18, interleukin-18; STEAP3, the six-transmembrane epithelial antigen of prostate family member 3; TLR4, toll-like receptor 4; GPX4, glutathione peroxidase 4; NADP⁺, nicotinamide adenine dinucleotide (oxidised); NADPH, nicotinamide adenine dinucleotide (reduced); Glu, glutamate; Gly, glycine; GR, glutathione reductase; GSSG, glutathione disulfide; GSH, glutathione; FABP, fatty-acid-binding protein; FAT, ester of fatty acid; CD36, fatty acid translocase, PUFA, polyunsaturated fatty acid; PUFA-Pl, polyunsaturated fatty acid—containing phospholipids; PLOOH, phospholipid hydroperoxide; PLOH, phospholipid alcohol

Fig. 3

Mechanisms of the atherosclerotic and pro-inflammatory effects of PCSK9 on the arterial wall. VSMCs are the major source of PCSK9 in the vascular wall, while PCSK9 expression has also been confirmed in endothelial cells and macrophages. In the early stages of atherosclerosis, a key process is the formation of foam cells in the vascular wall. These foam cells are mainly created by macrophages engulfing oxLDL. OxLDL enters not only macrophages but also monocytes, VSMCs and fibroblasts via SRs such as LOX-1, SR-A, CD36, and TLR4. LOX-1 is the most important of these SRs. PCSK9 increases the expression of all SRs, but especially LOX-1, in macrophages and VSMCs. In these cells, PCSK9 and LOX-1 increase each other’s expression, resulting in increased uptake of oxLDL by these cells. This interaction between PCSK9 and LOX-1 has important implications for the development of atherosclerotic lesions. LOX-1 is the major oxLDL receptor in ECs, but is also present at high levels in VSMCs. LOX-1 activation enhances oxLDL uptake, increases adhesion molecule expression, mitochondrial ROS production, and inflammation. Increased inflammation, in turn, increases LOX-1 expression, creating a self-perpetuating atherosclerotic ‘vicious cycle’. PCSK9, interacting with LOX-1, is directly involved in this process. PCSK9 also promotes foam cell formation by another mechanism. PCSK9 inhibits cholesterol efflux in macrophages by inhibiting the expression of ABCA1, one of the membrane transporters through which most cholesterol efflux in macrophages occurs. PCSK9 also inhibits, to a lesser extent, the expression of SR-BI, another membrane transporter through which cholesterol efflux occurs. This promotes the formation of foam cells. PCSK9 increases the secretion of pro-inflammatory cytokines by macrophages. At the cellular level, this stimulating effect of PCSK9 on pro-inflammatory cytokine production is mediated by NF-kB. PCSK9 induces NF-kB translocation to the nucleus in macrophages, resulting in increased mRNA levels for pro-inflammatory cytokines and TLR4. The resulting inflammation is also associated with increased production of ROS in the mitochondria. ROS induce endothelial dysfunction, induce and sustain inflammation, increase inflammatory cell infiltration and activation, and enhance apoptosis of ECs and VSMCs. Increased levels of ROS also increase the expression of PCSK9 and LOX-1. Pro-inflammatory cytokines, ROS, and also shear stress induce the differentiation of contractile VSMCs into synthetic VSMCs, which synthesise extracellular matrix, proteases, and cytokines, and have a greater capacity for proliferation and migration. Synthetic VSMCs also have enhanced lipid synthesis and increased expression of SRs, which contribute to foam cell formation. PCSK9, which is significantly upregulated in VSMCs under shear stress, induces the differentiation of VSMCs from a contractile phenotype to synthetic VSMCs and enhances VSMC proliferation and migration. Explanation of abbreviations: PCSK9, proprotein convertase subtilisin/keksin type-9; LDLc, low-density lipoprotein cholesterol; oxLDL, oxidised LDL; SRs, scavenger receptors; SR-A, class A scavenger receptor; SR-BI, class B scavenger receptor type I; LOX-1, lectin-like oxLDL receptor-1; ECs, endothelial cells; VSMCs, vascular smooth muscle cells; ROS, reactive oxygen species; ABCA1, ATP-binding cassette A1; ABCG1, ATP-binding cassette sub-family G member 1; NF-kB, nuclear factor kappa B; TLR4, toll-like receptor 4; ICAM-1, intercellular adhesion molecule 1; VCAM-1, vascular cell adhesion molecule 1; TNFa, tumor necrosis factor alpha; IL-1β, interleukin-1beta; IL-6, interleukin-6

Fig. 4

PCSK9 inhibition strategies. a The first strategy involves the use of anti-PCSK9 antibodies (evolocumab and alirocumab), which inhibit the interaction between circulating PCSK9 and LDLR on the surface of hepatocytes. As a consequence of these antibodies binding to PCSK9, they prevent PCSK9 from binding to LDLR on the surface of hepatocytes. This results in LDLR binding to LDLc and the formation of an LDLR-LDLc complex without PCSK9. This complex is then internalised and enters endosomes. There, in an acidic environment (pH 5.4), LDLR undergoes some conformational changes that lead to the release of LDLc from this complex. LDLc is then transported to lysosomes and undergoes degradation while LDLR, unhindered, returns to the cell surface. b The second strategy involves the administration of siRNA in the form of lipid nanoparticles (inclisiran). Inclisiran siRNA consists of complementary 21 sense and 23 antisense oligonucleotide sequences. To facilitate the uptake of siRNA by hepatocytes, Ga1NAc is fused to a sense strand and, because of this, it binds to ASGR1 which is highly expressed on hepatocytes. This allows specific delivery of siRNA to the liver. When endosomal uptake of siRNA occurs, small portions of siRNA are released into the cytoplasm where the dissociated antisense strand binds to PCSK9 mRNA and, with the participation of several proteins, forms RISC. This leads to degradation of PCSK9 mRNA resulting in the discontinuation of PCSK9 production. Explanation of abbreviations: PCSK9, proprotein convertase subtilisin/keksin type-9; LDLc, low-density lipoprotein cholesterol; LDLR, LDL receptor; PCSK9-mRNA, mRNA for proprotein convertase subtilisin/keksin type-9; siRNA, small interfering RNA; ASGR1, asialoglycoprotein receptor 1; Ga1NAc, N-acetylgalactosamine; RISC, RNA-induced silencing complex