Diacylglycerols and Lysophosphatidic Acid, Enriched on Lipoprotein(a), Contribute to Monocyte Inflammation.

BACKGROUND: Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). METHODS: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. RESULTS: Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68+IL-18+TLR4+ (toll-like receptor) TREM2+ (triggering receptor expressed on myeloid cells) resident macrophages and CD68+CASP1+ (caspase) IL-1B+SELL+ (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]). CONCLUSIONS: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.

Major Facilitator Superfamily Domain Containing 5 Inhibition Reduces Lipoprotein(a) Uptake and Calcification in Valvular Heart Disease.

BACKGROUND: High circulating levels of Lp(a) (lipoprotein[a]) increase the risk of atherosclerosis and calcific aortic valve disease, affecting millions of patients worldwide. Although atherosclerosis is commonly treated with low-density lipoprotein-targeting therapies, these do not reduce Lp(a) or risk of calcific aortic valve disease, which has no available drug therapies. Targeting Lp(a) production and catabolism may provide therapeutic benefit, but little is known about Lp(a) cellular uptake. METHODS: Here, unbiased ligand-receptor capture mass spectrometry was used to identify MFSD5 (major facilitator superfamily domain containing 5) as a novel receptor/cofactor involved in Lp(a) uptake. RESULTS: Reducing MFSD5 expression by a computationally identified small molecule or small interfering RNA suppressed Lp(a) uptake and calcification in primary human valvular endothelial and interstitial cells. MFSD5 variants were associated with aortic stenosis (P=0.027 after multiple hypothesis testing) with evidence suggestive of an interaction with plasma Lp(a) levels. CONCLUSIONS: MFSD5 knockdown suppressing human valvular cell Lp(a) uptake and calcification, along with meta-analysis of MFSD5 variants associating with aortic stenosis, supports further preclinical assessment of MFSD5 in cardiovascular diseases, the leading cause of death worldwide.

Oxidized phospholipids in cardiovascular disease.

Prolonged or excessive exposure to oxidized phospholipids (OxPLs) generates chronic inflammation. OxPLs are present in atherosclerotic lesions and can be detected in plasma on apolipoprotein B (apoB)-containing lipoproteins. When initially conceptualized, OxPL-apoB measurement in plasma was expected to reflect the concentration of minimally oxidized LDL, but, surprisingly, it correlated more strongly with plasma lipoprotein(a) (Lp(a)) levels. Indeed, experimental and clinical studies show that Lp(a) particles carry the largest fraction of OxPLs among apoB-containing lipoproteins. Plasma OxPL-apoB levels provide diagnostic information on the presence and extent of atherosclerosis and improve the prognostication of peripheral artery disease and first and recurrent myocardial infarction and stroke. The addition of OxPL-apoB measurements to traditional cardiovascular risk factors improves risk reclassification, particularly in patients in intermediate risk categories, for whom improving decision-making is most impactful. Moreover, plasma OxPL-apoB levels predict cardiovascular events with similar or greater accuracy than plasma Lp(a) levels, probably because this measurement reflects both the genetics of elevated Lp(a) levels and the generalized or localized oxidation that modifies apoB-containing lipoproteins and leads to inflammation. Plasma OxPL-apoB levels are reduced by Lp(a)-lowering therapy with antisense oligonucleotides and by lipoprotein apheresis, niacin therapy and bariatric surgery. In this Review, we discuss the role of role OxPLs in the pathophysiology of atherosclerosis and Lp(a) atherogenicity, and the use of OxPL-apoB measurement for improving prognosis, risk reclassification and therapeutic interventions.

Effect of Eplontersen on Cardiac Structure and Function in Patients With Hereditary Transthyretin Amyloidosis.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTRv) is associated with polyneuropathy, cardiomyopathy, or both. The effects of eplontersen on cardiac structure and function were assessed. METHODS AND RESULTS: NEURO-TTRansform was an open-label trial involving 144 adults with ATTRv polyneuropathy (49 patients [34%] with cardiomyopathy) receiving eplontersen throughout and compared with a historical placebo group (n = 60; 30 patients [50%] with cardiomyopathy) from the NEURO-TTR trial at week 65. Treatment effect (eplontersen vs placebo), presented as mean difference (95% confidence interval) was analyzed after adjusting for age, sex, region, baseline value, ATTRv disease stage, previous ATTRv treatment, and V30M transthyretin variant. There were notable differences at baseline between the eplontersen group and historical placebo. In the cardiomyopathy subgroup, 65 weeks of eplontersen treatment was associated with improvement from baseline relative to placebo in left ventricular ejection fraction of 4.3% (95% confidence interval 1.40-21.01; P = .049) and stroke volume 10.64 mL (95% confidence interval 3.99-17.29; P = .002) while the remainder of echocardiographic parameters remained stable. CONCLUSIONS: Eplontersen was associated with stable or improved measures of cardiac structure and function vs historical placebo in patients with ATTRv polyneuropathy and cardiomyopathy. Further investigation into eplontersen's effect on transthyretin amyloid cardiomyopathy is being conducted in the CARDIO-TTRansform trial.

Lipoprotein(a) and carotid intima-media thickness in children with familial hypercholesterolaemia in the Netherlands: a 20-year follow-up study.

BACKGROUND: Elevated lipoprotein(a) and familial hypercholesterolaemia are both independent risk conditions for cardiovascular disease. Although signs of atherosclerosis can be observed in children with familial hypercholesterolaemia, it is unknown whether elevated lipoprotein(a) is an additional risk factor for atherosclerosis in these young patients. Therefore, we aimed to assess the contribution of lipoprotein(a) concentrations to arterial wall thickening (as measured by carotid intima-media thickness) in children with familial hypercholesterolaemia who were followed up into adulthood. METHODS: We conducted a 20-year follow-up study of 214 children (aged 8-18 years) with heterozygous familial hypercholesterolaemia who were randomly assigned in a statin trial in Amsterdam (Netherlands) between Dec 7, 1997, and Oct 4, 1999. At baseline, and at 2, 10, and 20 years thereafter, blood samples were taken and carotid intima-media thickness was measured. Linear mixed-effects models were used to evaluate the association between lipoprotein(a) and carotid intima-media thickness during follow-up. We adjusted for sex, age, corrected LDL-cholesterol, statin use, and BMI. FINDINGS: Our study population comprised 200 children who had a carotid intima-media thickness measurement and a measured lipoprotein(a) concentration from at least one visit available. Mean age at baseline was 13·0 years (SD 2·9), 106 (53%) children were male, and 94 (47%) were female. At baseline, median lipoprotein(a) concentration was 18·5 nmol/L (IQR 8·7-35·5) and mean carotid intima-media thickness was 0·4465 mm (SD 0·0496). During follow-up, higher lipoprotein(a) concentrations contributed significantly to progression of carotid intima-media thickness (ß adjusted 0·0073 mm per 50 nmol/L increase in lipoprotein(a) [95% CI 0·0013-0·0132]; p=0·017). INTERPRETATION: Our findings suggest that lipoprotein(a) concentrations contribute significantly to arterial wall thickening in children with familial hypercholesterolaemia who were followed-up until adulthood, suggesting that lipoprotein(a) is an independent and additional risk factor for early atherosclerosis in those already at increased risk. Lipoprotein(a) measurement in young patients with familial hypercholesterolaemia is crucial to identify those at potentially highest risk for cardiovascular disease. FUNDING: Silence Therapeutics.

On-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways is not affected by pelacarsen.

BACKGROUND: Pelacarsen decreases plasma levels of lipoprotein(a) [Lp(a)] and oxidized phospholipids (OxPL). It was previously reported that pelacarsen does not affect the platelet count. We now report the effect of pelacarsen on on-treatment platelet reactivity. METHODS: Subjects with established cardiovascular disease and screening Lp(a) levels ≥60 mg per deciliter (~ ≥150 nmol/L) were randomized to receive pelacarsen (20, 40, or 60 mg every 4 weeks; 20 mg every 2 weeks; or 20 mg every week), or placebo for 6-12 months. Aspirin Reaction Units (ARU) and P2Y12 Reaction Units (PRU) were measured at baseline and the primary analysis timepoint (PAT) at 6 months. RESULTS: Of the 286 subjects randomized, 275 had either an ARU or PRU test, 159 (57.8%) were on aspirin alone and 94 (34.2%) subjects were on dual anti-platelet therapy. As expected, the baseline ARU and PRU were suppressed in subjects on aspirin or on dual anti-platelet therapy, respectively. There were no significant differences in baseline ARU in the aspirin groups or in PRU in the dual anti-platelet groups. At the PAT there were no statistically significant differences in ARU in subjects on aspirin or PRU in subjects on dual anti-platelet therapy among any of the pelacarsen groups compared to the pooled placebo group (p > 0.05 for all comparisons). CONCLUSION: Pelacarsen does not modify on-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways.

Toll-Like Receptor 3 Mediates Aortic Stenosis Through a Conserved Mechanism of Calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/ß receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/ß receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Impact of C-reactive protein levels on lipoprotein(a)-associated aortic stenosis incidence and progression.

Aims: Elevated lipoprotein(a) [Lp(a)] levels are associated with the risk of coronary artery disease (CAD) and calcific aortic valve stenosis (CAVS). Observational studies revealed that Lp(a) and C-reactive protein (CRP) levels, a biomarker of systemic inflammation, may jointly predict CAD risk. Whether Lp(a) and CRP levels also jointly predict CAVS incidence and progression is unknown. Methods and results: We investigated the association of Lp(a) with CAVS according to CRP levels in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk study (n = 18 226, 406 incident cases) and the UK Biobank (n = 438 260, 4582 incident cases), as well as in the ASTRONOMER study (n = 220), which assessed the haemodynamic progression rate of pre-existing mild-to-moderate aortic stenosis. In EPIC-Norfolk, in comparison to individuals with low Lp(a) levels (<50 mg/dL) and low CRP levels (<2.0 mg/L), those with elevated Lp(a) (>50 mg/dL) and low CRP levels (<2.0 mg/L) and those with elevated Lp(a) (>50 mg/dL) and elevated CRP levels (>2.0 mg/L) had a higher CAVS risk [hazard ratio (HR) = 1.86 (95% confidence intervals, 1.30-2.67) and 2.08 (1.44-2.99), respectively]. A comparable predictive value of Lp(a) in patients with vs. without elevated CRP levels was also noted in the UK Biobank. In ASTRONOMER, CAVS progression was comparable in patients with elevated Lp(a) levels with or without elevated CRP levels. Conclusion: Lp(a) predicts the incidence and possibly progression of CAVS regardless of plasma CRP levels. Lowering Lp(a) levels may warrant further investigation in the prevention and treatment of CAVS, regardless of systemic inflammation.

The contribution of amyloid deposition in the aortic valve to calcification and aortic stenosis.

Calcific aortic valve disease (CAVD) and stenosis have a complex pathogenesis, and no therapies are available that can halt or slow their progression. Several studies have shown the presence of apolipoprotein-related amyloid deposits in close proximity to calcified areas in diseased aortic valves. In this Perspective, we explore a possible relationship between amyloid deposits, calcification and the development of aortic valve stenosis. These amyloid deposits might contribute to the amplification of the inflammatory cycle in the aortic valve, including extracellular matrix remodelling and myofibroblast and osteoblast-like cell proliferation. Further investigation in this area is needed to characterize the amyloid deposits associated with CAVD, which could allow the use of antisense oligonucleotides and/or isotype gene therapies for the prevention and/or treatment of CAVD.

Lp(a), oxidized phospholipids and oxidation-specific epitopes are increased in subjects with keloid formation.

BACKGROUND: Keloid formation following trauma or surgery is common among darkly pigmented individuals. Since lipoprotein(a) [Lp(a)] has been postulated to have a putative role in wound healing, and also mediates atherosclerotic cardiovascular disease, it was assessed whether Lp(a), its associated oxidized phospholipids and other oxidation-specific biomarkers were associated with keloid formation. METHODS: This case-control study included darkly pigmented individuals of African ancestry, 100 with keloid scarring and 100 non-keloid controls. The lipid panel, hsCRP, Lp(a), oxidized phospholipids on apolipoprotein B-100 (OxPL-apoB), IgG and IgM apoB-immune complexes and IgG and IgM autoantibodies to a malondialdehyde mimotope (MDA-mimotope) were measured. Immunohistochemistry of keloid specimens was performed for both Lp(a) and OxPL staining. RESULTS: Cases and controls were well matched for age, sex and lipid profile. Mean Lp(a) (57.8 vs. 44.2 mg/dL; P = 0.01, OxPL-apoB 17.4 vs. 15.7 nmol/L; P = 0.009) and IgG and IgM apoB-immune complexes and IgG and IgM MDA-mimotope levels were significantly higher in keloid cases. Keloid tissue stained strongly for OxPL. CONCLUSION: Darkly pigmented individuals of African ancestry with keloids have higher plasma levels of Lp(a), OxPL-apoB and oxidation-specific epitopes. The commonality of excessive wound healing in keloids and chronic complications from coronary revascularization suggests avenues of investigation to define a common mechanism driven by Lp(a) and the innate response to oxidized lipids.

Association of lipoprotein(a) with intrinsic and on-clopidogrel platelet reactivity.

Lipoprotein(a) [Lp(a)] is an independent, genetically determined, and causal risk factor for cardiovascular disease. Laboratory data have suggested an interaction of Lp(a) with platelet function, potentially caused by its interaction with platelet receptors. So far, the potential association of Lp(a) with platelet activation and reactivity has not been proven in larger clinical cohorts. This study analyzed intrinsic platelet reactivity before loading with clopidogrel 600 mg and on-treatment platelet reactivity tested 24 h following loading in patients undergoing elective coronary angiography. Platelet reactivity was tested by optical aggregometry following stimulation with collagen or adenosine diphosphate as well as by flow cytometry. Lp(a) levels were directly measured in all patients from fresh samples. The present analysis included 1912 patients. Lp(a) levels ranged between 0 and 332 mg/dl. There was a significant association of rising levels of Lp(a) with a higher prevalence of a history of ischemic heart disease (p < 0.001) and more extensive coronary artery disease (p = 0.001). Results for intrinsic (p = 0.80) and on-clopidogrel platelet reactivity (p = 0.81) did not differ between quartiles of Lp(a) levels. Flow cytometry analyses of expression of different platelet surface proteins (CD41, CD62P or PAC-1) confirmed these findings. Correlation analyses of levels of Lp(a) with any of the tested platelet activation markers did not show any correlation. The present data do not support the hypothesis of an interaction of Lp(a) with platelet reactivity.

Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency.

BACKGROUND: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. METHODS: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr-/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2V617F). RESULTS: Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk-/- platelets promoted increased NETosis when incubated with Lnk-/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk-/- platelets, as well as increased priming and response of Lnk-/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk-/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ controls. CONCLUSIONS: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

PCSK9 Activity Is Potentiated Through HDL Binding.

Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

Longitudinal Assessment of Lipoprotein(a) Levels in Perinatally HIV-Infected Children and Adolescents.

HIV is an independent risk factor of cardiovascular disease (CVD); therefore, perinatally HIV-infected (PHIV) children potentially have a greater CVD risk at older age. Lipoprotein(a) (Lp(a)) is an established risk factor for CVD in the general population. To evaluate a potential increased CVD risk for PHIV children, we determined their lipid profiles including Lp(a). In the first substudy, we assessed the lipid profiles of 36 PHIV children visiting the outpatient clinic in Amsterdam between 2012 and 2020. In the second substudy, we enrolled 21 PHIV adolescents and 23 controls matched for age, sex and ethnic background on two occasions with a mean follow-up time of 4.6 years. We assessed trends of lipid profiles and their determinants, including patient and disease characteristics, using mixed models. In the first substudy, the majority of PHIV children were Black (92%) with a median age of 8.0y (5.7-10.8) at first assessment. Persistent elevated Lp(a) levels were present in 21/36 (58%) children (median: 374 mg/L (209-747); cut off = 300). In the second substudy, the median age of PHIV adolescents was 17.5y (15.5-20.7) and of matched controls 16.4y (15.8-19.5) at the second assessment. We found comparable lipid profiles between groups. In both studies, increases in LDL-cholesterol and total cholesterol were associated with higher Lp(a) levels. A majority of PHIV children and adolescents exhibited elevated Lp(a) levels, probably associated with ethnic background. Nonetheless, these elevated Lp(a) levels may additionally contribute to an increased CVD risk.

The Prolyl-tRNA Synthetase Inhibitor Halofuginone Inhibits SARS-CoV-2 Infection.

We identify the prolyl-tRNA synthetase (PRS) inhibitor halofuginone 1 , a compound in clinical trials for anti-fibrotic and anti-inflammatory applications 2 , as a potent inhibitor of SARS-CoV-2 infection and replication. The interaction of SARS-CoV-2 spike protein with cell surface heparan sulfate (HS) promotes viral entry 3 . We find that halofuginone reduces HS biosynthesis, thereby reducing spike protein binding, SARS-CoV-2 pseudotyped virus, and authentic SARS-CoV-2 infection. Halofuginone also potently suppresses SARS-CoV-2 replication post-entry and is 1,000-fold more potent than Remdesivir 4 . Inhibition of HS biosynthesis and SARS-CoV-2 infection depends on specific inhibition of PRS, possibly due to translational suppression of proline-rich proteins. We find that pp1a and pp1ab polyproteins of SARS-CoV-2, as well as several HS proteoglycans, are proline-rich, which may make them particularly vulnerable to halofuginone's translational suppression. Halofuginone is orally bioavailable, has been evaluated in a phase I clinical trial in humans and distributes to SARS-CoV-2 target organs, including the lung, making it a near-term clinical trial candidate for the treatment of COVID-19.

Design and Rationale of the Global Phase 3 NEURO-TTRansform Study of Antisense Oligonucleotide AKCEA-TTR-LRx (ION-682884-CS3) in Hereditary Transthyretin-Mediated Amyloid Polyneuropathy.

INTRODUCTION: AKCEA-TTR-LRx is a ligand-conjugated antisense (LICA) drug in development for the treatment of hereditary transthyretin amyloidosis (hATTR), a fatal disease caused by mutations in the transthyretin (TTR) gene. AKCEA-TTR-LRx shares the same nucleotide sequence as inotersen, an antisense medicine approved for use in hATTR polyneuropathy (hATTR-PN). Unlike inotersen, AKCEA-TTR-LRx is conjugated to a triantennary N-acetylgalactosamine moiety that supports receptor-mediated uptake by hepatocytes, the primary source of circulating TTR. This advanced design increases drug potency to allow for lower and less frequent dosing. The NEURO-TTRansform study will investigate whether AKCEA-TTR-LRx is safe and efficacious, with the aim of improving neurologic function and quality of life in hATTR-PN patients. METHODS/DESIGN: Approximately 140 adults with stage 1 (independent ambulation) or 2 (requires ambulatory support) hATTR-PN are anticipated to enroll in this multicenter, open-label, randomized, phase 3 study. Patients will be assigned 6:1 to AKCEA-TTR-LRx 45 mg subcutaneously every 4 weeks or inotersen 300 mg once weekly until the prespecified week 35 interim efficacy analysis, after which patients receiving inotersen will receive AKCEA-TTR-LRx 45 mg subcutaneously every 4 weeks. All patients will then receive AKCEA-TTR-LRx through the remainder of the study treatment period. The final efficacy analysis at week 66 will compare the AKCEA-TTR-LRx arm with the historical placebo arm from the phase 3 trial of inotersen (NEURO-TTR). The primary outcome measures are between-group differences in the change from baseline in serum TTR, modified Neuropathy Impairment Score + 7, and Norfolk Quality of Life-Diabetic Neuropathy questionnaire. CONCLUSION: NEURO-TTRansform is designed to determine whether targeted delivery of AKCEA-TTR-LRx to hepatocytes with lower and less frequent doses will translate into clinical and quality-of-life benefits for patients with hATTR-PN. TRIAL REGISTRATION: The study is registered at ClinicalTrials.gov (NCT04136184) and EudraCT (2019-001698-10).

Hereditary transthyretin amyloidosis with peripheral neuropathy (hATTR-PN for short) is a rare inherited condition. In hATTR-PN, a protein called transthyretin (TTR for short) builds up and damages nerves throughout the body. This neuropathy causes symptoms such as weakness, loss of sensation, and pain. Currently available medicines can slow disease progression, but researchers are looking for more effective treatments with fewer side effects. AKCEA-TTR-L_Rx is an investigational treatment for hATTR-PN. AKCEA-TTR-L_Rx prevents the liver from making TTR, reducing the amount that causes disease progression. It is similar to an existing treatment called inotersen, but designed for better delivery to the liver and is more potent. This article describes the NEURO-TTRansform study that will evaluate how effective AKCEA-TTR-L_Rx is for treating hATTR-PN. Around 140 adults with hATTR-PN from the USA, Canada, and Europe will be able to take part in this study. The study treatment period will be 85 weeks long. People will receive injections underneath the skin of either: AKCEA-TTR-L_Rx every 4 weeks, or Inotersen once a week for 35 weeks, followed by a switch to AKCEA-TTR-L_Rx every 4 weeks. People may continue to receive AKCEA-TTR-L_Rx after the study treatment period ends. In this study, researchers will compare results from people who received AKCEA-TTR-L_Rx to results from people who received no active ingredients (called placebo) in a similar study (called NEURO-TTR). Researchers will measure the differences in peoples': Neuropathy symptoms. Quality of life. TTR protein levels in the blood.

Effect of bariatric surgery on plasma levels of oxidised phospholipids, biomarkers of oxidised LDL and lipoprotein(a).

BACKGROUND: Obesity is associated with adverse cardiovascular outcomes and this is improved following bariatric surgery. Oxidised phospholipids (OxPL) are thought to reflect the pro-inflammatory effects of lipoprotein(a) [Lp(a)], and both are independent predictors of cardiovascular disease. OBJECTIVE: Our study sought to determine the impact of bariatric surgery on OxPL, biomarkers of oxidised LDL (OxLDL) and Lp(a). METHODS: This is a prospective, observational study of 59 patients with severe obesity undergoing bariatric surgery. Blood samples were obtained prior to surgery and at 6 and 12 months after. Sixteen patients attending the tertiary medical weight management clinic at the same centre were also recruited for comparison. Lipid and metabolic blood parameters, OxLDL, OxPL on apolipoprotein B-100 (OxPL-apoB), IgG and IgM autoantibodies to MDA-LDL, IgG and IgM apoB-immune complexes and Lp(a) were measured. RESULTS: Reduction in body mass index (BMI) was significant following bariatric surgery, from median 48 kg/m2 at baseline to 37 kg/m2 at 6 months and 33 kg/m2 at 12 months. OxPL-apoB levels decreased significantly at 12 months following surgery [5.0 (3.2-7.4) to 3.8 (3.0-5.5) nM, p = 0.001], while contrastingly, Lp(a) increased significantly [10.2 (3.8-31.9) to 16.9 (4.9-38.6) mg/dl, p = 0.002]. There were significant post-surgical decreases in IgG and IgM biomarkers, particularly at 12 months, while OxLDL remained unchanged. CONCLUSIONS: Bariatric surgery results in a significant increase in Lp(a) but reductions in OxPL-apoB and other biomarkers of oxidised lipoproteins, suggesting increased synthetic capacity and reduced oxidative stress. These biomarkers might be clinically useful to monitor physiological effects of weight loss interventions.

Intracellular AIBP (Apolipoprotein A-I Binding Protein) Regulates Oxidized LDL (Low-Density Lipoprotein)-Induced Mitophagy in Macrophages.

OBJECTIVE: Atherosclerotic lesions are often characterized by accumulation of OxLDL (oxidized low-density lipoprotein), which is associated with vascular inflammation and lesion vulnerability to rupture. Extracellular AIBP (apolipoprotein A-I binding protein; encoded by APOA1BP gene), when secreted, promotes cholesterol efflux and regulates lipid rafts dynamics, but its role as an intracellular protein in mammalian cells remains unknown. The aim of this work was to determine the function of intracellular AIBP in macrophages exposed to OxLDL and in atherosclerotic lesions. Approach and Results: Using a novel monoclonal antibody against human and mouse AIBP, which are highly homologous, we demonstrated robust AIBP expression in human and mouse atherosclerotic lesions. We observed significantly reduced autophagy in bone marrow-derived macrophages, isolated from Apoa1bp-/- compared with wild-type mice, which were exposed to OxLDL. In atherosclerotic lesions from Apoa1bp-/- mice subjected to Ldlr knockdown and fed a Western diet, autophagy was reduced, whereas apoptosis was increased, when compared with that in wild-type mice. AIBP expression was necessary for efficient control of reactive oxygen species and cell death and for mitochondria quality control in macrophages exposed to OxLDL. Mitochondria-localized AIBP, via its N-terminal domain, associated with E3 ubiquitin-protein ligase PARK2 (Parkin), MFN (mitofusin)1, and MFN2, but not BNIP3 (Bcl2/adenovirus E1B 19-kDa-interacting protein-3), and regulated ubiquitination of MFN1 and MFN2, key components of mitophagy. CONCLUSIONS: These data suggest that intracellular AIBP is a new regulator of autophagy in macrophages. Mitochondria-localized AIBP augments mitophagy and participates in mitochondria quality control, protecting macrophages against cell death in the context of atherosclerosis.

Ligand conjugated antisense oligonucleotide for the treatment of transthyretin amyloidosis: preclinical and phase 1 data.

AIMS: Amyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment. METHODS AND RESULTS: AKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P < 0.001). A maximum mean (SD) reduction in TTR levels of -86.3% (6.5) from baseline was achieved after a single dose of 120 mg AKCEA-TTR-LRx . CONCLUSIONS: These findings suggest an improved safety and tolerability profile with the increase in potency achieved by productive receptor-mediated uptake of AKCEA-TTR-LRx by hepatocytes and supports further development of AKCEA-TTR-LRx for the treatment of ATTR polyneuropathy and cardiomyopathy.

The interconnection between lipoprotein(a), lipoprotein(a) cholesterol and true LDL-cholesterol in the diagnosis of familial hypercholesterolemia.

PURPOSE OF REVIEW: Elevated levels of lipoprotein(a) [Lp(a)] are present in 30-50% of patients with familial hypercholesterolemia. The contribution of Lp(a) towards risk stratification of patients with familial hypercholesterolemia has been recently recognized, with studies showing a significantly worse prognosis if Lp(a) is elevated. However, the role of elevated Lp(a) in diagnosis of familial hypercholesterolemia is less well defined or accepted. RECENT FINDINGS: An important confounder in the diagnosis of familial hypercholesterolemia is the significant contribution of the cholesterol content on Lp(a) (Lp(a)-C) in individuals with elevated Lp(a). Because Lp(a)-C is incorporated into all clinical LDL-C measurements, it can contribute significantly to the cholesterol threshold diagnostic criteria for familial hypercholesterolemia used in most clinical algorithms. SUMMARY: In this review, we discuss the interrelationship of Lp(a), Lp(a)-C and correct LDL-C in the diagnosis and prognosis of familial hypercholesterolemia. Future studies of accurately measuring correct LDL-C or in using apoB-100 and Lp(a) criteria may overcome the limitations of using estimated LDL-C in the diagnosis of familial hypercholesterolemia in individuals with concomitant elevation of Lp(a).