Effects of PCSK9 Targeting: Alleviating Oxidation, Inflammation, and Atherosclerosis

Authors, Journal, Affiliations, Type, DOI

• Authors: Emily Punch, Justus Klein, Patrick Diaba-Nuhoho, Henning Morawietz, Mahdi Garelnabi
• Journal: Journal of the American Heart Association, 2022;11:e023328
• Affiliations: University of Massachusetts Lowell (Punch, Garelnabi); Technische Universität Dresden / University Hospital Carl Gustav Carus (Klein, Diaba-Nuhoho, Morawietz)
• Type: Contemporary review
• DOI: https://doi.org/10.1161/JAHA.121.023328

Overview

This contemporary review consolidates preclinical evidence for PCSK9's roles in atherosclerosis beyond LDL-C regulation — specifically its involvement in oxidative stress and inflammatory signalling within the arterial plaque. Preclinical data (cell culture and murine models) show PCSK9 directly stimulates macrophage proinflammatory cytokine production via TLR4/NF-κB, independent of LDL-R. A structural discovery provides biological plausibility: PCSK9's unique C-terminal cysteine-rich domain (CRD) is structurally homologous to resistin's CRD, which is known to activate TLR4 and CAP1. However, clinical trials (FOURIER) showed no reduction in CRP with evolocumab, and the hypothesis of LDL-R-independent anti-inflammatory PCSK9 inhibitor effects remains unproven in humans.

Keywords

atherosclerosis, PCSK9, inflammation, TLR4, NF-κB, oxidized LDL, foam cells, resistin, CRD, oxidative stress

Key Takeaways

Atherosclerosis Pathophysiology — Oxidation and Inflammation

• Atherosclerosis begins with endothelial damage (triggered by excess LDL-C, cigarette smoke, hypertension) → deposition of circulating LDL-C in the intimal space → spontaneous oxidation → oxidized LDL (Ox-LDL)
• Ox-LDL acquires damage-associated molecular patterns recognized by pattern recognition receptors (CD36, TLR4, LOX-1, CRP) → proinflammatory cytokine production and macrophage internalization of oxidised species
• Ox-LDL-activated macrophages become foam cells → proinflammatory cytokine release (IL-1β, IL-6, TNF-α, MCP-1) → monocyte recruitment → positive feedback loop of plaque progression
• VSMCs migrate and transform into macrophage-like cells when fatty deposits accumulate → contribute to foam cell pool and further plaque deposition
• Minimally oxidized LDL: stimulates adhesion molecules, chemokines, cytokines → extravasation of immune cells into arterial wall; does not efficiently trigger scavenger receptor-mediated foam cell formation
• Extensively oxidized LDL: stimulates VSMC proliferation; recognized by macrophage scavenger receptors → foam cell formation and fatty plaque accumulation
• Oxidizing enzymes: lipoxygenase (enzymatic LDL oxidation in macrophages + free radical byproducts) and myeloperoxidase (expressed in neutrophils/monocytes/macrophages; hypochlorous acid + radical oxidants) are primary contributors
• HDL normally mediates antioxidant effects and reverse cholesterol transport; oxidative modification by peroxyl/hydroxyl radicals and myeloperoxidase-derived species (malondialdehyde, 4-hydroxynonenal, acrolein) impairs HDL's cardioprotective function

PCSK9 Discovery and Cholesterol Regulation (Summary)

• 2003: PCSK9 gain-of-function mutation identified as cause of autosomal dominant FH (Abifadel et al); primarily expressed in hepatocytes
• PCSK9 expression regulated by SREBP-2 (sterol regulatory element-binding protein-2) — feedback link to intracellular cholesterol levels
• Mechanism: PCSK9 binds EGF-A domain of LDL-R → receptor-ligand complex internalized → PCSK9 prevents conformational change enabling LDL-R recycling → LDL-R degraded in lysosome
• CAP1 as a new coreceptor: adenylyl cyclase-associated protein 1 (CAP1) was identified as a new binding partner of PCSK9, mediating caveolae-dependent endocytosis and lysosomal degradation of LDL-R; PCSK9 binds CAP1 via the SH3 binding domain on the CRD — the same domain through which resistin binds CAP1
• PCSK9 also binds other LDL-R family members: VLDL-R, CD36, ApoER2, LRP1 — suggesting broader roles in lipoprotein metabolism
• PCSK9 is expressed in VSMCs (in addition to hepatocytes), with direct effects on LDL-R expression in macrophages within the arterial plaque

PCSK9 and Inflammation — Evidence for LDL-R-Independent Roles

• Systemic inflammation increases PCSK9 expression: lipopolysaccharide, zymosan, and turpentine each upregulate PCSK9 mRNA in hepatic tissue of C57BL/6 mice (Feingold et al)
• PCSK9 levels correlate with inflammatory biomarkers in clinical studies (Table 1):
  • Positively correlated with CRP in healthy subjects and in patients with CAD and SLE
  • Positively correlated with white blood cell count in patients with CAD
  • Higher PCSK9 in HIV-infected subjects correlates with systemic monocyte markers
  • Serum PCSK9 correlates with necrotic core tissue in coronary artery plaques, independent of LDL-C (Cheng et al)
• In vitro (THP-1 macrophages and human primary macrophages; Ricci et al): incubation with recombinant PCSK9 → significant ↑ IL-1β, IL-6, TNF-α, CXCL2, MCP-1 mRNA; effect is not exclusively LDL-R-dependent (also seen in LDL-R+/+ bone marrow macrophages with 31-fold TNF-α increase)
• In vitro (Tang et al 2012; Ox-LDL model): PCSK9 siRNA → suppressed proinflammatory cytokine expression (IL-1α, IL-6, TNF-α) and attenuated NF-κB nuclear translocation in Ox-LDL-stimulated THP-1 macrophages
• In vivo (Tang et al 2017; ApoE-/- mice): PCSK9 shRNA lentiviral silencing → significantly decreased TLR4 and NF-κB expression in atherosclerotic aortas; decreased atherosclerotic plaque area, macrophage content, and vascular proinflammatory proteins (TNF-α, IL-1β, MCP-1); LV-PCSK9 (overexpression) had reverse effects
• Anti-PCSK9 vaccine (AT04A; APOE*3Leiden.CETP mice; Landlinger et al): AT04A vaccine → significant ↓ plasma proinflammatory markers (SAA, MIP-1β/CCL4, MDC/CCL22, SCF, VEGF-A); ↓ atherosclerotic lesion area; ↓ total aortic lesions; ↓ aortic inflammation
• Alirocumab (APOE*3Leiden.CETP mice; Kühnast et al): alirocumab → reduced monocyte recruitment to plaques, total macrophage content, and necrotic core content

PCSK9 and TLR4/NF-κB Pathway

• TLR4 is a membrane-spanning pattern recognition receptor on innate immune cells (macrophages); normally activated by LPS from gram-negative bacteria; triggers MyD88 recruitment → IκB phosphorylation → IκB degradation → NF-κB nuclear translocation → proinflammatory cytokines (IL-1, IL-6, IL-12, TNF-α, MCP-1)
• TLR4 expression is upregulated in atherosclerotic plaques; TLR4 deletion in ApoE-/- mice → significant decrease in aortic atherosclerosis, lipid accumulation, and macrophage infiltration (Michelsen et al 2004) — suggesting endogenous ligands drive plaque TLR4 activation
• PCSK9 overexpression → TLR4 upregulation and NF-κB activation and translocation (Tang et al); PCSK9 siRNA → reversal
• The CD36 scavenger receptor on macrophages amplifies TLR4/NF-κB signalling in response to Ox-LDL — a potential synergistic pathway with PCSK9

PCSK9 Structure and Resistin Structural Homology — The Key Mechanistic Hypothesis

• PCSK9 is unique among proprotein convertases: its catalytic active site is sterically blocked by the prodomain (non-covalent interaction) — it does not use its protease activity, yet exerts multiple effects via different binding domains
• PCSK9's C-terminal domain contains a highly distinctive cysteine-rich domain (CRD) with three submodules forming a jelly-roll structure — this 3-jelly-roll structure has been identified in only ONE other protein: resistin
• Resistin (FIZZ family) causes proinflammatory effects via its CRD:
  • Binds TLR4 directly → NF-κB nuclear translocation → proinflammatory cytokines (IL-6, IL-12, TNF-α)
  • Binds CAP1 at the SH3 binding domain → ↑ cAMP, PKA activity, NF-κB-related inflammatory transcription
  • Promotes LDL-R degradation (independent of PCSK9; PCSK9 siRNA → ↑ LDL-R; addition of resistin ablates this increase)
  • Expressed in atherosclerotic lesion macrophages → VSMC migration; foam cell formation via CD36 upregulation
• PCSK9-CAP1 binding confirmed: PCSK9 binds CAP1 via the SH3 binding domain on its CRD (Jang et al; HEK293 coimmunoprecipitation) — to facilitate endocytosis and lysosomal degradation of LDL-R (confirmed in liver and kidney cells)
• Hypothesis (not yet confirmed): PCSK9's CRD may bind TLR4 directly (as resistin's CRD does), triggering NF-κB → proinflammatory cytokines in atherosclerotic plaques; this would constitute a true LDL-R-independent inflammatory mechanism

PCSK9 Redox State

• PCSK9's CRD contains disulfide bonds formed during ER post-translational modification; the atherosclerotic plaque is highly oxidative → may promote disulfide bond retention and enhance PCSK9 CRD function
• Resistin function is redox-sensitive (requires oxidative state for biological activity; hQSOX1b sulfhydryl oxidase is required for proper function of murine FIZZ1); by analogy, PCSK9 CRD function may similarly depend on its oxidative state
• Evolocumab significantly prevents cytotoxicity induced by hydrogen peroxide and reduces hydroperoxides and malondialdehyde levels in human umbilical vein endothelial cells — suggesting an antioxidant effect of PCSK9 inhibition (in vitro)

Limitations of the Document

• Preclinical-clinical discordance: the central hypothesis (LDL-R-independent pro-inflammatory role of PCSK9) is supported by cell culture and murine experiments, but clinical trials have NOT confirmed this: evolocumab (FOURIER) had no effect on CRP in humans; no RCT has demonstrated inflammation reduction with PCSK9 inhibition independent of LDL-C lowering
• Conflicting clinical data: (1) Plasma PCSK9 correlated with carotid atherosclerosis progression in one study but not with carotid intima-media thickness in another; (2) One study found PCSK9 correlates with plaque necrotic core independently of LDL-C, but another showed evolocumab did NOT affect plaque composition
• PROSPER study finding (Table 1): genetic variations in PCSK9 that decreased LDL-C did NOT decrease CHD risk in this elderly cohort — contradicts simple cholesterol-only mechanism but also complicates the inflammation hypothesis
• Murine model limitations: studies use ApoE-/- mice and APOE*3Leiden.CETP mice; inflammatory phenotype may not translate directly to humans
• In vitro limitations: siRNA/shRNA and overexpression models do not replicate pharmacological PCSK9 inhibition; LDL-R was often present in these models, making LDL-R-independent conclusions difficult to isolate
• PCSK9-TLR4 direct binding unconfirmed: the core structural hypothesis (CRD binds TLR4 in immune cells as resistin does) has not been tested; only indirect evidence and structural analogies
• Review article — no original data: narrative synthesis; no systematic search methodology; potential selection bias toward positive findings supporting the hypothesis
• Author disclosures: Morawietz has German Research Foundation grants; Garelnabi and Punch from UMass Lowell; no pharmaceutical conflicts disclosed

Key Concepts Mentioned

• concepts/PCSK9-Inhibitors — primary subject; LDL-R-independent roles added as contradiction/open question
• concepts/Atherosclerosis-Pathophysiology — comprehensive overview of plaque initiation, oxidation, foam cell formation, and PCSK9's role in the inflammatory cycle

Key Entities Mentioned

• entities/Evolocumab — antioxidant effect in vitro (H2O2 model); but no CRP reduction in FOURIER

Wiki Pages Updated

• Created: wiki/sources/pcsk9-jaha-2022.md
• Created: wiki/concepts/Atherosclerosis-Pathophysiology.md
• Updated: wiki/concepts/PCSK9-Inhibitors.md
• Updated: wiki/entities/Evolocumab.md
• Updated: wiki/sourceindex.md
• Updated: wiki/wikiindex.md
• Updated: log.md