Modified Lipoproteins Induce Arterial Wall Inflammation During Atherogenesis

Circulating apolipoprotein B-containing lipoproteins, notably the low-density lipoproteins, enter the inner layer of the arterial wall, the intima, where a fraction of them is retained and modified by proteases, lipases, and oxidizing agents and enzymes. The modified lipoproteins and various modification products, such as fatty acids, ceramides, lysophospholipids, and oxidized lipids induce inflammatory reactions in the macrophages and the covering endothelial cells, initiating an increased leukocyte diapedesis. Lipolysis of the lipoproteins also induces the formation of cholesterol crystals with strong proinflammatory properties. Modified and aggregated lipoproteins, cholesterol crystals, and lipoproteins isolated from human atherosclerotic lesions, all can activate macrophages and thereby induce the secretion of proinflammatory cytokines, chemokines, and enzymes. The extent of lipoprotein retention, modification, and aggregation have been shown to depend largely on differences in the composition of the circulating lipoprotein particles. These properties can be modified by pharmacological means, and thereby provide opportunities for clinical interventions regarding the prevention and treatment of atherosclerotic vascular diseases.Peer reviewe

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding

Background and aims: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. Methods: Mature human MCs were differentiated from human peripheral blood-derived CD34(+) progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. Results: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. Conclusions: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin-lipoprotein interactions

Atherosclerosis is initiated when lipoproteins bind to proteoglycans (PGs) in arterial walls. The binding is mediated by apolipoprotein apoB-100 and/or apoE, both of which have binding affinity toward heparin. We developed covalently bound heparin coatings for APTES-modified silica capillaries and SiO 2 chips and carried out capillary electrochromatography (CEC) and quartz crystal microbalance (QCM) studies on the interactions of heparin with selected peptide fragments of apoB-100 and apoE and, for CEC, also with low- and high-density lipoproteins (LDL and HDL), the latter with and without apoE. The peptides are known to mediate interactions of HDL and LDL with arterial PGs. Interactions and affinities were expressed in CEC as retention factors and reduced mobilities and in continuous flow QCM techniques as affinity constants. Both techniques showed heparin interactions to be stronger with apoB-100 peptide than with apoE peptide fragment, and they confirmed that the sulfate groups in heparin play an especially important role in interactions with apoB-100 peptide fragments. In addition, CEC confirmed the importance of sulfate groups of heparin in interactions between heparin and LDL and between heparin and apoE-containing HDL. CEC and QCM acted as excellent platforms to mimic these biologically important interactions, with small sample and reagent consumption.Fil: Katriina Lipponen. University of Helsinki, Helsinki, Finland;; ArgentinaFil: Yi Liu. University of Helsinki, Helsinki, Finland;; ArgentinaFil: Stege, Patricia Wanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Química de San Luis; ArgentinaFil: Katariina Ö örni. University of Helsinki, Helsinki, Finland;; ArgentinaFil: Petri T. Kovanen. University of Helsinki, Helsinki, Finland;; ArgentinaFil: Marja-Liisa Riekkola. University of Helsinki, Helsinki, Finland;; Argentin

PCSK9 inhibition alters the lipidome of plasma and lipoprotein fractions

Background and aims: While inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is known to result in dramatic lowering of LDL-cholesterol (LDL-C), it is poorly understood how it affects other lipid species and their metabolism. The aim of this study was to characterize the alterations in the lipidome of plasma and lipoprotein particles after administration of PCSK9 inhibiting antibody to patients with established coronary heart disease. Methods: Plasma samples were obtained from patients undergoing a randomized placebo-controlled phase II trial (EQUATOR) for the safe and effective use of RG7652, a fully human monoclonal antibody inhibiting PCSK9 function. Lipoprotein fractions were isolated by sequential density ultracentrifugation, and both plasma and major lipoprotein classes (VLDL-IDL, LDL, HDL) were subjected to mass spectrometric lipidomic profiling. Results: PCSK9 inhibition significantly decreased plasma levels of several lipid classes, including sphingolipids (dihydroceramides, glucosylceramides, sphingomyelins, ceramides), cholesteryl esters and free cholesterol. Previously established ceramide ratios predicting cardiovascular mortality, or inflammation related eicosanoid lipids, were not altered. RG7652 treatment also affected the overall and relative distribution of lipids in lipoprotein classes. An overall decrease of total lipid species was observed in LDL and VLDL thorn IDL particles, while HDL-associated phospholipids increased. Following the treatment, LDL displayed reduced lipid cargo, whereas relative lipid proportions of the VLDL thorn IDL particles were mostly unchanged, and there were relatively more lipids carried in the HDL particles. Conclusions: Administration of PCSK9 antibody significantly alters the lipid composition of plasma and lipoprotein particles. These changes further shed light on the link between anti-PCSK9 therapies and cardiovascular risk. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Keramidit ja sepelvaltimotauti

English summaryPeer reviewe

Three-Dimensional cryoEM Reconstruction of Native LDL Particles to 16 angstrom Resolution at Physiological Body Temperature

Background Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37°C). Methodology/Principal Findings To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6°C and 37°C resulted in reconstructions at ~16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6°C than at 37°C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6°C, but not at 37°C. At 37°C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6°C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles. Conclusions/Significance Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.Peer reviewe

Low-density lipoprotein aggregation predicts adverse cardiovascular events in peripheral artery disease

Background and aims: Peripheral artery disease (PAD) is a systemic manifestation of atherosclerosis that is associated with a high risk of major adverse cardiovascular events (MACE). LDL aggregation contributes to atherosclerotic plaque progression and may contribute to plaque instability. We aimed to determine if LDL aggregation is associated with MACE in patients with PAD undergoing lower extremity revascularization (LER). Methods: Two hundred thirty-nine patients with PAD undergoing LER had blood collected at baseline and were followed prospectively for MACE (myocardial infarction, stroke, cardiovascular death) for one year. Nineteen age, sex and LDL-C-matched control subjects without cardiovascular disease also had blood drawn. Subject LDL was exposed to sphingomyelinase and LDL aggregate size measured via dynamic light scattering. Results: Mean age was 72.3 10.9 years, 32.6% were female, and LDL-cholesterol was 68 +/- 25 mg/dL. LDL aggregation was inversely associated with triglycerides, but not associated with demographics, LDL-cholesterol or other risk factors. Maximal LDL aggregation occurred significantly earlier in subjects with PAD than in control subjects. 15.9% of subjects experienced MACE over one year. The 1st tertile (shortest time to maximal aggregation) exhibited significantly higher MACE (25% vs. 12.5% in tertile 2 and 10.1% in tertile 3, p = 0.012). After multivariable adjustment for demographics and CVD risk factors, the hazard ratio for MACE in the 1st tertile was 4.57 (95% CI 1.60-13.01; p = 0.004) compared to tertile 3. Inclusion of LDL aggregation in the Framingham Heart Study risk calculator for recurrent coronary heart disease events improved the c-index from 0.57 to 0.63 (p = 0.01). Conclusions: We show that in the setting of very well controlled LDL-cholesterol, patients with PAD with the most rapid LDL aggregation had a significantly elevated MACE risk following LER even after multivariable adjustment. This measure further improved the classification specificity of an established risk prediction tool. Our findings support broader investigation of this assay for risk stratification in patients with atherosclerotic CVD.Peer reviewe

Plant Stanol Esters Reduce LDL (Low-Density Lipoprotein) Aggregation by Altering LDL Surface Lipids The BLOOD FLOW Randomized Intervention Study

OBJECTIVE: Plant stanol ester supplementation (2-3 g plant stanols/d) reduces plasma LDL (low-density lipoprotein) cholesterol concentration by 9% to 12% and is, therefore, recommended as part of prevention and treatment of atherosclerotic cardiovascular disease. In addition to plasma LDL-cholesterol concentration, also qualitative properties of LDL particles can influence atherogenesis. However, the effect of plant stanol ester consumption on the proatherogenic properties of LDL has not been studied. APPROACH AND RESULTS: Study subjects (n=90) were randomized to consume either a plant stanol ester-enriched spread (3.0 g plant stanols/d) or the same spread without added plant stanol esters for 6 months. Blood samples were taken at baseline and after the intervention. The aggregation susceptibility of LDL particles was analyzed by inducing aggregation of isolated LDL and following aggregate formation. LDL lipidome was determined by mass spectrometry. Binding of serum lipoproteins to proteoglycans was measured using a microtiter well-based assay. LDL aggregation susceptibility was decreased in the plant stanol ester group, and the median aggregate size after incubation for 2 hours decreased from 1490 to 620 nm,P=0.001. Plant stanol ester-induced decrease in LDL aggregation was more extensive in participants having body mass index CONCLUSIONS: Consumption of plant stanol esters decreases the aggregation susceptibility of LDL particles by modifying LDL lipidome. The resulting improvement of LDL quality may be beneficial for cardiovascular health. REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01315964. GRAPHIC ABSTRACT: A graphic abstract is available for this article.Peer reviewe

The human liver lipidome is significantly related to the lipid composition and aggregation susceptibility of low-density lipoprotein (LDL) particles

Publisher Copyright: © 2022 The AuthorsBackground and aims: The susceptibility of low-density lipoprotein (LDL) to aggregation predicts atherosclerotic cardiovascular disease. However, causes of interindividual variation in LDL lipid composition and aggregation susceptibility remain unclear. We examined whether the lipid composition and aggregation susceptibility of LDL reflect the lipid composition of the human liver. Methods: Liver biopsies and blood samples for isolation of LDL particles were obtained from 40 obese subjects (BMI 45.9 ± 6.1 kg/m2, age 43 ± 8 years). LDL was isolated using sequential ultracentrifugation and lipidomic analyses of liver and LDL samples were determined using ultra-high performance liquid chromatography–mass spectrometry. LDL aggregation susceptibility ex vivo was analyzed by inducing aggregation by human recombinant secretory sphingomyelinase and following aggregate formation. Results: The composition (acyl carbon number and double bond count) of hepatic triglycerides, phosphatidylcholines, and sphingomyelins (SMs) was closely associated with that of LDL particles. Hepatic dihydroceramides and ceramides were positively correlated with concentrations of the corresponding SM species in LDL as well with LDL aggregation. These relationships remained statistically significant after adjustment for age, sex, and body mass index. Conclusions: Lipid composition of LDL reflects that of the human liver in obese patients. Changes in hepatic sphingolipid metabolism may contribute to interindividual variation of LDL lipid composition and susceptibility to aggregation.Peer reviewe