LDL aggregation susceptibility as a novel risk factor for atherosclerosis

Atherosclerotic cardiovascular disease (ASCVD) is the leading worldwide cause of mortality and morbidity, being both an economic burden for healthcare systems and a source of great individual suffering. The low density lipoprotein cholesterol (LDL-C) concentration in plasma is a causal and modifiable risk factor for ASCVD. Atherogenesis is initiated and then driven by retention, modification, and aggregation of low density lipoprotein (LDL) particles in the arterial intima. Experimental work by our group and others has demonstrated that aggregated LDL particles can induce lipid accumulation in macrophages and this can also activate intimal cells and thereby induce inflammation in the arterial wall. Here, it was hypothesized that it is not only the plasma concentration of LDL particles that influences atherogenesis, but also their characteristics. This thesis aims at testing whether subjects with aggregation-prone LDL have an increased risk for ASCVD and/or ASCVD death. For this purpose, a method to measure LDL aggregation susceptibility was developed, and it was further studied if LDL aggregation susceptibility is modifiable. In addition, it is studied here if LDL aggregates cause a heightened inflammatory response in cells present in the artery wall. The first publication was a hypothesis-generating study, where it was discovered that LDL particles from ASCVD patients are more prone to aggregate in comparison to those from healthy individuals, and importantly, that aggregation-prone LDL predicted future ASCVD death in a group of patients with established ASCVD. It was found that the aggregation-prone LDL particles are rich in sphingolipids but have less phosphatidylcholines than their aggregation-resistant LDL counterparts. Three interventions in animal models aimed at altering the LDL composition, were observed not only to lower the susceptibility LDL particles to aggregate but also to slow the development of atherosclerosis. Similar compositional changes induced in humans by proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition or adoption of a healthy Nordic diet also lowered the LDL aggregation susceptibility. In the second publication it was demonstrated that genetic background, here ethnicity, influenced LDL aggregation susceptibility. LDL particles from South Asians were more prone to aggregate compared to those from white Caucasians, a finding that may partly explain why South Asians are at higher risk for ASCVD. The third study was a dietary intervention to investigate if different macronutrients could alter LDL particles aggregation susceptibility. Saturated fats were found to increase LDL aggregation, while unsaturated fats or simple sugars had no effect. In addition, the consumption of plant stanol esters, that are known to reduce the LDL-C concentration, was found to decrease the LDL aggregation susceptibility. This thesis propose that the aggregation susceptibility of LDL particles is a novel modifiable risk factor of ASCVD; its assessment may add predictive power to the conventional ASCVD risk estimation. The evaluation of this biomarker may facilitate the identification of those patients who would benefit most from aggressive LDL-C-lowering therapies.Ateroskleroosi eli valtimonkovettumatauti on maailman yleisin kuolinsyy ja se kehittyy vuosien kuluessa lapsuudesta lähtien. Plasman LDL-hiukkasten kolesterolipitoisuus on merkittävä riskitekijä taudin kehittymiselle, mikä on näytetty geneettisissä, epidemiologisissa ja kliinisissä tutkimuksissa. LDL-hiukkasten pitoisuus valtimon seinämän sisäkerroksessa, intimassa, on sama kuin plasmassa. LDL-hiukkasten kertyminen valtimon seinämään alkaa plasman LDL kolesteroli pitoisuuden ollessa suurempi kuin fysiologinen LDL kolesteroli pitoisuus (1-1,5 mmol/l). Intimassa LDL-hiukkaset takertuvat solunulkoiseen tukiverkkoon ja ovat alttiita entsymaattisille ja hapettaville muutoksille. Muuntuneet LDL-hiukkaset ovat alttiita aggregoitumaan eli takertumaan toisiinsa, ja aggregoituneet LDL-hiukkaset puolestaan tarttuvat entistä tiukemmin soluväliaineeseen. LDL-hiukkasten kerääntyminen intimaan houkuttelee paikalle makrofageja, jotka fagosytosoivat erityisesti aggregoituneita hiukkasia, mikä johtaa vaahtosolujen muodostumiseen ja paikalliseen tulehdukseen. Valtimoplakkien tiedetään sisältävän aggregoituneita LDL-hiukkasia ja eläinkokeissa on näytetty, että plasman LDL aggregoituu valtimon seinämässä vain kahden tunnin kuluessa siitä, kun eläimeen injektoidaan LDL-hiukkasia ja plasman LDL-kolesterolipitoisuus kohoaa. Tässä väitöskirjatyössä kehitin menetelmän mitata LDL-hiukkasten aggregoitumisherkkyyttä verinäytteistä. Osoitin, että LDL-hiukkasten herkkyys aggregoitua vaihtelee ihmisten välillä ja johtuu eroista LDL-hiukkasten pinnan rasvojen suhteista, fosfatidyylikoliinien ja sfingomyeliinien suhteellisesta osuudesta. Sfingomyeliiniä paljon sisältävät LDL-hiukkaset aggregoituvat herkemmin, ja fosfatidyylikoliinia paljon sisältävät LDL-hiukkaset hitaammin. Osoitin, että LDL-hiukkaset aggregoituvat herkemmin sepelvaltimotautipotilailla kuin terveillä verrokeilla ja mikä tärkeintä, LDL- hiukkasten aggregoitumisherkkyys ennusti tulevaa sydänkuolemaa sepelvaltimotautia sairastavilla potilailla. LDL-hiukkasten aggregoitumisherkkyys osoittautui itsenäiseksi riskitekijäksi, eikä korreloinut esimerkiksi plasman LDL-kolesterolipitoisuuden tai iän kanssa, eikä eronnut miesten ja naisten välillä. Lisäksi osoitin, että LDL-hiukkasten herkkyys aggregoitua riippuu mahdollisesti ainakin osittain geneettisistä tekijöistä, sillä eteläaasialaisten LDL aggregoitui herkemmin kuin länsimaalaisten. Kliinisesti on tärkeää tietää voiko LDL-hiukkasten aggregoitumisherkkyyteen vaikuttaa elintavoilla tai lääkityksellä. Näytin tässä työssä, että ravinnon rasvoilla on merkitystä, sillä tyydyttyneet rasvat huononsivat LDL-hiukkasten laatua ja lisäsivät LDL-hiukkasten aggregoitumisherkkyyttä. Pohjoismainen terveellinen ruokavalio, erityisesti siihen kuuluvat kasviöljyt paransivat LDL- hiukkasten laatua ja vähensivät aggregoitumista. PCSK9 (proprotein convertase subtilisin/kexin 9) estäjä, plasman kolesterolipitoisuutta laskeva monoklonaalinen vasta- ainelääke vähensi LDL-hiukkasten aggregoitumista parantamalla LDL-hiukkasten laatua. Lisäksi havaittiin, että kasviöljypohjaisen kasvistanolia sisältävän levitteen käyttö puolestaan paransi LDL-hiukkasten laatua ja vähensi LDL-hiukkasten aggregoitumisherkkyyttä. Tämä väitöskirjatyö esittelee LDL-hiukkasten aggregoitumisherkkyyden uutena muokattavissa olevana riskitekijänä ateroskleroottisille valtimosairauksille. LDL- hiukkasten aggregoitumisherkkyys voi tulevaisuudessa auttaa tunnistamaan potilaat, jotka hyötyisivät eniten aggressivisesta LDL kolesterolia alentavasta lääkityksestä

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

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

LDL aggregation susceptibility is higher in healthy South Asian compared with white Caucasian men

BACKGROUND: South Asians are more prone to develop atherosclerotic cardiovascular disease (ASCVD) compared with white Caucasians, which is not fully explained by classical risk factors. We recently reported that the presence of aggregation-prone low-density lipoprotein (LDL) in the circulation is associated with increased ASCVD mortality. OBJECTIVE: We hypothesized that LDL of South Asians is more prone to aggregate, which may be explained by differences in their LDL lipid composition. METHODS: In this cross-sectional hypothesis-generating study, LDL was isolated from plasma of healthy South Asians (n = 12) and age- and BMI-matched white Caucasians (n = 12), and its aggregation susceptibility and lipid composition were analyzed. RESULTS: LDL from South Asians was markedly more prone to aggregate compared with white Caucasians. Among all measured lipids, sphingomyelin 24:0 and triacylglycerol 56:8 showed the highest positive correlation with LDL aggregation. In addition, LDL from South Asians was enriched in arachidonic acid containing phosphatidylcholine 38:4 and had less phosphatidylcholines and cholesteryl esters containing monounsaturated fatty acids. Interestingly, body fat percentage, which was higher in South Asians (+26%), positively correlated with LDL aggregation and highly positively correlated with triacylglycerol 56:8, sphingomyelin 24:0, and total sphingomyelin. CONCLUSIONS: LDL aggregation susceptibility is higher in healthy young South Asians compared with white Caucasians. This may be partly explained by the higher body fat percentage of South Asians, leading to sphingomyelin enrichment of LDL. We anticipate that the presence of sphingomyelin-rich, aggregation -prone LDL particles in young South Asians may increase LDL accumulation in the arterial wall and thereby contribute to their increased risk of developing ASCVD later in life. (C) 2019 National Lipid Association. Published by Elsevier Inc.Peer reviewe

The effect of intakes of fish and Camelina sativa oil on atherogenic and anti-atherogenic functions of LDL and HDL particles : A randomized controlled trial

Background and aims: Omega-3 fatty acids are known to have several cardioprotective effects. Our aim was to investigate the effects of intakes of fish and Camelina sativa oil (CSO), rich in alpha-linolenic acid, on the atherogenic and anti-atherogenic functions of LDL and HDL particles. Methods: Altogether, 88 volunteers with impaired glucose metabolism were randomly assigned to CSO (10 g of alpha-linolenic acid/day), fatty fish (4 fish meals/week), lean fish (4 fish meals/week) or control group for 12 weeks. 79 subjects completed the study. The binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determined in vitro. Results: Intake of CSO decreased the binding of lipoproteins to aortic proteoglycans in a non-normalized model (p = 0.006). After normalizing with serum concentrations of non-HDL cholesterol, apolipoprotein B (apoB) or LDL cholesterol, which decreased in the CSO group, the change was no longer statistically significant. In the fish groups, there were no changes in the binding of lipoproteins to proteoglycans. Regarding other lipoprotein functions, there were no changes in any of the groups. Conclusions: Intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum LDL cholesterol concentration, which suggests that the level of apoB-containing lipoproteins in the circulation is the main driver of lipoprotein retention within the arterial wall. Intake of fish or CSO has no effects on other lipoprotein functions.Peer reviewe

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

Background 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.Keywords: Atherosclerosis; Cardiovascular disease; Ceramides; Cholesterol; Lipidomics; Phosphatidylcholines; Triglycerides.</p

Uudistuva postitoiminta – Valtiosihteerityöryhmän raportti: Arvioita posti- ja jakelumarkkinan nykytilanteesta, tulevaisuuden näkymistä ja sääntelykehyksen muutostarpeista

Valtioneuvoston kanslia asetti 14.1.2020 ministeriöiden välisen työryhmän arvioimaan posti- ja jakelumarkkinan nykytilannetta ja tulevaisuuden näkymiä ja muutostarpeita mm. postilainsäädäntöön. Työryhmä koostui eräiden ministeriöiden valtiosihteereistä, puheenjohtajanaan valtiosihteeri Olli Koski, VNK. Työryhmän tavoitteena oli tuottaa taustaa ja ohjeistusta lain valmistelun ja yleispalvelun järjestämiseksi sekä valmistella postilain tarkastelun yhteydessä kannanotto muuttuvaan markkinaan sopeutumisessa. Työryhmän tehtävänä oli hallitusohjelman mukaisesti selvittää kokonaisvaltaisesti vaihtoehtoja postin ja sanomalehtien jakelun turvaamiseksi koko maassa moniäänisen tiedonvälityksen varmistamiseksi sekä turvaamaan postinjakelun harvaan asutuilla alueilla sekä saaristossa saaristolain mukaisesti. Työryhmän tuli kiinnittää erityisesti huomiota yleispalveluvelvoitteen hoitamisen kustannustehokkuuteen ja tämän mahdollistaviin toimintamalleihin, kartoittaa yhteistyömahdollisuuksia muiden yksityisten, valtion toimijoiden ja ministeriöiden kanssa, sekä pohtia postimarkkinan muutoksesta johtuvia tarpeita postilainsäädännön uudistukseen. Työryhmä käsitteli palveluiden organisointiin liittyviä yhteistyömalleja ja -rakenteita, mutta niiden mahdolliset vaikutukset Posti Group Oyj:n omistusrakenteisiin eivät kuuluneet kuitenkaan työryhmän tehtäviin. Raportti koostuu kahdesta osasta: osa A Valtiosihteeriryhmän yhteiskuntapoliittiset johtopäätökset ja osa B taustatietoja

Children with familial hypercholesterolemia display changes in LDL and HDL function : A cross-sectional study

Publisher Copyright: © 2021 The Association for the Publication of the Journal of Internal Medicine.Background: The functional status of lipoprotein particles contributes to atherogenesis. The tendency of plasma low-density lipoprotein (LDL) particles to aggregate and the ability of igh-density lipoprotein (HDL) particles to induce and mediate reverse cholesterol transport associate with high and low risk for cardiovascular disease in adult patients, respectively. However, it is unknown whether children with familial hypercholesterolemia (FH) display lipoprotein function alterations. Hypothesis: We hypothesized that FH children had disrupted lipoprotein functions. Methods: We analyzed LDL aggregation susceptibility and HDL-apoA-I exchange (HAE), and activity of four proteins that regulate lipoprotein metabolism (cholesteryl ester transfer protein, lecithin–cholesterol acyltransferase, phospholipid transfer protein, and paraoxonase-1) in plasma samples derived from children with FH (n = 47) and from normocholesterolemic children (n = 56). Variation in lipoprotein functions was further explored using an nuclear magnetic resonance-based metabolomics profiling approach. Results: LDL aggregation was higher, and HAE was lower in FH children than in normocholesterolemic children. LDL aggregation associated positively with LDL cholesterol (LDL-C) and negatively with triglycerides, and HAE/apoA-I associated negatively with LDL-C. Generally, the metabolomic profile for LDL aggregation was opposite of that of HAE/apoA-I. Conclusions: FH children displayed increased atherogenicity of LDL and disrupted HDL function. These newly observed functional alterations in LDL and HDL add further understanding of the risk for atherosclerotic cardiovascular disease in FH children.Peer reviewe