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

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages

IntroductionLipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined.Methods and resultsTo explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1β and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1β and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1β release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation.DiscussionOur data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory

Etyyliesteri-eikosapentaeenihapporavintolisän vaikutus ihmisen LDL-hiukkasten aggregaatioherkkyyteen ja plasman lipidikonsentraatioihin

Tiivistelmä Johdanto: Sydän- ja verisuonitaudit ovat suurin yksittäinen rasite terveydenhuoltojärjestelmällemme ja ne aiheuttavat eniten ennenaikaisia kuolemia maailmassa. Sydäntautiriskiä voi alentaa terveellisillä ruokailu- ja liikuntatottumuksilla sekä useilla lääkkeillä ja ravintolisillä, kuten etyyli-eikosapentaeenihapolla (E-EPA). Tässä tutkimuksessa tutkimme E-EPA-ravintolisän vaikutusta tunnettuihin sydän- ja verisuonitautien riskitekijöihin, kuten veren lipoproteiinikonsentraatioihin sekä uuteen riskitekijään, LDLhiukkasten aggregaatioherkkyyteen. Tutkimusasetelma: Tutkimusryhmä, johon kuului yhteensä 39 miestä ja naista, osallistui 4- viikon pituiseen tutkimukseen, jossa tutkimushenkilöt lisäsivät ruokavalioonsa 3.9 g E-EPA:a päivässä. E-EPA-kapseleissa oli lisäksi mukana 75 µg päiväannos D-vitamiinia. Tutkimushenkilöiltä kerättiin verinäytteet tutkimuksen aloitusta edeltävänä päivänä, sekä yksi viikko E-EPA:n syönnin aloittamisen jälkeen, E-EPA:n syönnin loppuessa 4-viikon kohdalla sekä 1 viikko E-EPA:n syönnin lopettamisen jälkeen. Tutkimus oli avoin tutkimus, jossa tutkimushenkilöissä tapahtuvia muutoksia verrattiin tutkimushenkilöiden omiin lähtöarvoihin. Tulokset: Plasman kokonaiskolesterolikonsentraation keskiarvo laski 3,8 mmol/l:sta 3,6 mmol/l:aan (p=0.0038 one-way ANOVA) yhden viikon E-EPA ravintolisän jälkeen, missä se pysyi tutkimuksen loppuun asti. Myös plasman LDL-kolesterolipitoisuus oli laskenut neljän viikon kohdalla (p=0.0028 one-way ANOVA) ja plasman triglyseridipitoisuus jo ensimmäisen viikon kohdalla (p=0.0004 one-way ANOVA). D-vitamiinitasot nousivat keskimäärin 18%, mikä on vähemmän kuin mitä vastaavissa tutkimuksissa on havaittu aiemmin. Ero johtuu luultavasti tutkimushenkilöidemme korkeista veren D-vitamiinipitoisuuden lähtöarvoista ja voi selittyä negatiivisen palautejärjestelmän roolilla D-vitamiinin synteesissä. LDL-kolesterolin aggregoitumisherkkyys ei merkittävästi muuttunut tutkimusryhmässä. Havaitsimme kuitenkin, että E-EPA hidastaa LDL-hiukkasten aggregaatiota henkilöillä, joiden LDL aggregoituu lähtötilanteessa herkästi: LDL-hiukkasten aggregoitumisnopeuden muutos korreloi käänteisesti lähtötilanteen aggregoitumisherkkyyden kanssa (r = -0.451, p = 0.0039). On mahdollista, että henkilöt, joiden LDL-hiukkaset ovat herkkiä aggregoitumaan, voisivat hyötyä E-EPA ravintolisästä.Abstract Introduction: Atherosclerotic cardiovascular diseases (ASCVD) cause the biggest burden on our healthcare system and cause most premature deaths. Risk for ASCVD can be lowered by lifestyle choices and medication, as well as several therapeutics such as ethyl eicosapentaenoic acid (E-EPA) supplementation. Here we aimed to investigate the effect of EEPA intervention on known ASCVD risk factors including circulating lipoprotein levels as well as low-density lipoprotein (LDL) aggregation susceptibility, a new independent risk factor for ASCVD. Study design: A study group of 39 healthy men and women participated in a 4-week long dietary supplement trial with 3.9 g/day of E-EPA. A dose of 75 µg/day of vitamin D was included in the E-EPA capsules. Blood samples were drawn before the trial, at weeks 1 and 4 of the intervention and 1 week after the intervention. The study was an open design where participants’ own baseline measurements were used to measure changes. Outcomes: The mean plasma cholesterol concentration was reduced from 3.8 mmol/l to 3.6 mmol/l (p=0.0038 one-way ANOVA) after one week of E-EPA supplementation and remained the same until the end of study period. This change was followed by a change in plasma LDL (p=0.0028 one-way ANOVA) and triglyceride (p=0.0004 one-way ANOVA) concentrations after four week and one week of E-EPA supplementation, respectively. Vitamin D levels increased on average by 18%, showcasing a lower relative response than seen in other vitamin D trials, which can be attributed to high effective baseline concentrations of vitamin D in our study group and the related negative feedback system. LDL aggregation susceptibility did not significantly change in the entire group. However, we discovered that the change in LDL aggregation susceptibility correlated negatively ( = -0.451, p = 0.0039) with the baseline LDL aggregation susceptibility. Thus, LDL aggregation decreased in participants having aggregation-prone LDL at baseline. This finding highlights a possibility that participants with higher LDL aggregation susceptibility may benefit from addition of E-EPA to their diet

Lipoprotein(a) induces caspase-1 activation and IL-1 signaling in human macrophages

IntroductionLipoprotein(a) (Lp(a)) is an LDL-like particle with an additional apolipoprotein (apo)(a) covalently attached. Elevated levels of circulating Lp(a) are a risk factor for atherosclerosis. A proinflammatory role for Lp(a) has been proposed, but its molecular details are incompletely defined. Methods and resultsTo explore the effect of Lp(a) on human macrophages we performed RNA sequencing on THP-1 macrophages treated with Lp(a) or recombinant apo(a), which showed that especially Lp(a) induces potent inflammatory responses. Thus, we stimulated THP-1 macrophages with serum containing various Lp(a) levels to investigate their correlations with cytokines highlighted by the RNAseq, showing significant correlations with caspase-1 activity and secretion of IL-1 beta and IL-18. We further isolated both Lp(a) and LDL particles from three donors and then compared their atheroinflammatory potentials together with recombinant apo(a) in primary and THP-1 derived macrophages. Compared with LDL, Lp(a) induced a robust and dose-dependent caspase-1 activation and release of IL-1 beta and IL-18 in both macrophage types. Recombinant apo(a) strongly induced caspase-1 activation and IL-1 beta release in THP-1 macrophages but yielded weak responses in primary macrophages. Structural analysis of these particles revealed that the Lp(a) proteome was enriched in proteins associated with complement activation and coagulation, and its lipidome was relatively deficient in polyunsaturated fatty acids and had a high n-6/n-3 ratio promoting inflammation. DiscussionOur data show that Lp(a) particles induce the expression of inflammatory genes, and Lp(a) and to a lesser extent apo(a) induce caspase-1 activation and IL-1 signaling. Major differences in the molecular profiles between Lp(a) and LDL contribute to Lp(a) being more atheroinflammatory.Peer reviewe