Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome.

BACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.

Evaluation of soluble suppression of tumorigenicity 2 (sST2) as serum marker for liver fibrosis.

BACKGROUND & AIMS: With the increase in patients at risk of advanced liver disease due to the obesity epidemic, there will be a need for simple screening tools for advanced liver fibrosis. Soluble suppression of tumorigenicity 2 (sST2) is a serum biomarker for fibrotic processes. The aim of this study was to evaluate sST2 as marker for liver fibrosis in patients successfully treated for chronic hepatitis C. METHODS: 424 patients from the Swiss Hepatitis C Cohort Study were screened for inclusion in this post-hoc cohort study. Inclusion criteria were sustained virological response (SVR), available elastography (VCTE) and serum samples for biomarker analysis before and after treatment. For the validation of sST2, values were compared to VCTE, FIB-4 and APRI using Spearman's correlation and AUROC analyses. RESULTS: Data of 164 subjects were finally analyzed. Median sST2 values slightly increased with VCTE-derived fibrosis stages and remained stable after reaching SVR within the respective fibrosis stage, suggesting that sST2 is not influenced by liver inflammation. However, correlation of sST2 pre- and post-treatment with VCTE was fair (Spearman's rho = 0.39 and rho = 0.36). The area under the curve (AUROC) for sST2 in detecting VCTE-defined F4 fibrosis (vs. F0-F3) before therapy was 0.74 (95%CI 0.65-0.83), and 0.67(95%CI 0.56-0.78) for the discrimination of F3/F4 fibrosis vs. F0-F2. Adding sST2 to either APRI or FIB-4, respectively, increased diagnostic performance of both tests. CONCLUSIONS: sST2 can potentially identify patients with advanced fibrosis as a single serum marker and in combination with APRI and FIB-4.

Comparison of a two-step Tempus600 hub solution single-tube vs. container-based, one-step pneumatic transport system.

OBJECTIVES: Efficient and timely transportation of clinical samples is pivotal to ensure accurate diagnoses and effective patient care. During the transportation process, preservation of sample integrity is crucial to avoid pre-analytical aberrations on laboratory results. Here, we present a comparative analysis between a two-step Tempus600 hub solution single-tube and a one-step, container-based pneumatic transport system (PTS) from Airco, for the in-house transportation of blood samples. METHODS: Ten blood samples from healthy volunteers were split in 10 mL collection tubes filled at full or half capacity for transportation with the two PTS (about 250 m). To compare the impact of transportation, markers of hemolysis such as lactate dehydrogenase (LDH), potassium (K+), and the hemolysis index (HI), were determined. Additionally, differences in HI in routine samples and repeated transportation was investigated. To assess and compare the mechanistic impact profiles, we recorded the acceleration profiles of the two PTS using a shock data logger. RESULTS: Transportation using the Tempus600 hub solution resulted in 49 and 46 % higher HI with samples filled to total or half capacity, respectively. Routine samples transported with the Tempus600 hub solution showed a higher median HI by 23 and 33 %. Additionally, shock logger analysis showed an elevated amount of shocks (6.5 fold) and shock intensities (1.8 fold). CONCLUSIONS: The Tempus600 hub solution caused an increased number of unreportable LDH or K+ results based on the hemolysis index. However, it was only statistically significant for LDH (p<0.01 and p<0.08) - while the comparisons for K+ were not statistically significant (p<0.28 and p<0.56).

High-density lipoprotein revisited: biological functions and clinical relevance.

Previous interest in high-density lipoproteins (HDLs) focused on their possible protective role in atherosclerotic cardiovascular disease (ASCVD). Evidence from genetic studies and randomized trials, however, questioned that the inverse association of HDL-cholesterol (HDL-C) is causal. This review aims to provide an update on the role of HDL in health and disease, also beyond ASCVD. Through evolution from invertebrates, HDLs are the principal lipoproteins, while apolipoprotein B-containing lipoproteins first developed in vertebrates. HDLs transport cholesterol and other lipids between different cells like a reusable ferry, but serve many other functions including communication with cells and the inactivation of biohazards like bacterial lipopolysaccharides. These functions are exerted by entire HDL particles or distinct proteins or lipids carried by HDL rather than by its cholesterol cargo measured as HDL-C. Neither does HDL-C measurement reflect the efficiency of reverse cholesterol transport. Recent studies indicate that functional measures of HDL, notably cholesterol efflux capacity, numbers of HDL particles, or distinct HDL proteins are better predictors of ASCVD events than HDL-C. Low HDL-C levels are related observationally, but also genetically, to increased risks of infectious diseases, death during sepsis, diabetes mellitus, and chronic kidney disease. Additional, but only observational, data indicate associations of low HDL-C with various autoimmune diseases, and cancers, as well as all-cause mortality. Conversely, extremely high HDL-C levels are associated with an increased risk of age-related macular degeneration (also genetically), infectious disease, and all-cause mortality. HDL encompasses dynamic multimolecular and multifunctional lipoproteins that likely emerged during evolution to serve several physiological roles and prevent or heal pathologies beyond ASCVD. For any clinical exploitation of HDL, the indirect marker HDL-C must be replaced by direct biomarkers reflecting the causal role of HDL in the respective disease.

Dipeptidyl peptidase 3 plasma levels predict cardiogenic shock and mortality in acute coronary syndromes.

BACKGROUND AND AIMS: Dipeptidyl peptidase 3 (DPP3) is a protease involved in the degradation of angiotensin II which disturbs peripheral blood pressure regulation and compromises left ventricular function. This study examined the relationship of circulating DPP3 (cDPP3) with cardiogenic shock (CS) and mortality in patients presenting with acute coronary syndromes (ACS). METHODS: Plasma cDPP3 levels were assessed at baseline and 12-24 h after presentation in patients with ACS prospectively enrolled into the multi-centre SPUM-ACS study (n = 4787). RESULTS: Circulating DPP3 levels were associated with in-hospital CS when accounting for established risk factors including the ORBI risk score [per log-2 increase, hazard ratio (HR) 1.38, 95% confidence interval (CI) 1.05-1.82, P = .021]. High cDPP3 was an independent predictor of mortality at 30 days (HR 1.87, 95% CI 1.36-2.58, P < .001) and at one year (HR 1.61, 95% CI 1.28-2.02, P < .001) after adjustment for established risk factors and the GRACE 2.0 score. Compared to values within the normal range, persistently elevated cDPP3 levels at 12-24 h were associated with 13.4-fold increased 30-day mortality risk (HR 13.42, 95% CI 4.86-37.09, P < .001) and 5.8-fold increased 1-year mortality risk (HR 5.79, 95% CI 2.70-12.42, P < .001). Results were consistent across various patient subgroups. CONCLUSIONS: This study identifies cDPP3 as a novel marker of CS and increased mortality in patients with ACS. Circulating DPP3 offers prognostic information beyond established risk factors and improves early risk assessment.

Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.

International Validation of the Canadian Syncope Risk Score : A Cohort Study.

BACKGROUND: The Canadian Syncope Risk Score (CSRS) was developed to predict 30-day serious outcomes not evident during emergency department (ED) evaluation. OBJECTIVE: To externally validate the CSRS and compare it with another validated score, the Osservatorio Epidemiologico della Sincope nel Lazio (OESIL) score. DESIGN: Prospective cohort study. SETTING: Large, international, multicenter study recruiting patients in EDs in 8 countries on 3 continents. PARTICIPANTS: Patients with syncope aged 40 years or older presenting to the ED within 12 hours of syncope. MEASUREMENTS: Composite outcome of serious clinical plus procedural events (primary outcome) and the primary composite outcome excluding procedural interventions (secondary outcome). RESULTS: Among 2283 patients with a mean age of 68 years, the primary composite outcome occurred in 7.2%, and the composite outcome excluding procedural interventions occurred in 3.1% at 30 days. Prognostic performance of the CSRS was good for both 30-day composite outcomes and better compared with the OESIL score (area under the receiver-operating characteristic curve [AUC], 0.85 [95% CI, 0.83 to 0.88] vs. 0.74 [CI, 0.71 to 0.78] and 0.80 [CI, 0.75 to 0.84] vs. 0.69 [CI, 0.64 to 0.75], respectively). Safety of triage, as measured by the frequency of the primary composite outcome in the low-risk group, was higher using the CSRS (19 of 1388 [0.6%]) versus the OESIL score (17 of 1104 [1.5%]). A simplified model including only the clinician classification of syncope (cardiac syncope, vasovagal syncope, or other) variable at ED discharge-a component of the CSRS-achieved similar discrimination as the CSRS (AUC, 0.83 [CI, 0.80 to 0.87] for the primary composite outcome). LIMITATION: Unable to disentangle the influence of other CSRS components on clinician classification of syncope at ED discharge. CONCLUSION: This international external validation of the CSRS showed good performance in identifying patients at low risk for serious outcomes outside of Canada and superior performance compared with the OESIL score. However, clinician classification of syncope at ED discharge seems to explain much of the performance of the CSRS in this study. The clinical utility of the CSRS remains uncertain. PRIMARY FUNDING SOURCE: Swiss National Science Foundation & Swiss Heart Foundation.

Soluble lectin-like oxidized low-density lipoprotein receptor-1 predicts premature death in acute coronary syndromes.

AIMS: The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and its shedding product [soluble LOX-1 (sLOX-1)] are implicated in atherosclerotic cardiovascular disease (ASCVD) pathogenesis. Herein, we examined the relationship of sLOX-1 with both fatal events and plaque progression in patients with acute coronary syndromes (ACS). METHODS AND RESULTS: Plasma sLOX-1 was assessed at baseline in ACS and chronic coronary syndrome (CCS) patients prospectively recruited in the multicentre SPUM-ACS study, with sex- and age-matched healthy subjects serving as additional controls (n = 2924). Compared with both CCS and controls, ACS patients showed markedly elevated sLOX-1 levels (median, 2.00 and 2.00 vs. 35.08 pg/mL; P < 0.0001) which were independently associated with increased mortality risk over 30-day [tertile (T)3: adjusted hazard ratio (HR), 3.11; 95% confidence interval (CI), 1.44-10.61; P = 0.0055] and 1-year intervals (T3: adjusted HR, 2.04; 95% CI, 1.19-3.92; P = 0.0098). Results remained consistent after adjustment for GRACE 2.0 (T3: adjusted HR, 1.86; 95% CI, 1.04-3.74; P = 0.0391) and were primarily driven by the pronounced relationship of sLOX-1 with cardiovascular mortality at 30 days (T3: adjusted HR, 3.81; 95% CI, 1.62-19.62; P = 0.0036) and at 1 year (T3: adjusted HR, 2.29; 95% CI, 1.19-5.34; P = 0.0148). In ACS patients undergoing serial intracoronary imaging and statin therapy, sLOX-1 dropped significantly in those with coronary plaque regression at 1 year (ΔsLOX-1: -4.64 ± 1.80; P = 0.0057), and showed a good discrimination for predicting plaque progression (area under the curve = 0.74; 95% CI, 0.59-0.86; P = 0.0031). CONCLUSION: Plasma sLOX-1 levels are increased during ACS and predict fatal events beyond traditional and emerging risk factors. Persistently high sLOX-1 associates with coronary plaque progression in patients with established ASCVD. CLINICAL TRIAL REGISTRATION: NCT01000701.

Lipoprotein(a) in atherosclerotic cardiovascular disease and aortic stenosis: a European Atherosclerosis Society consensus statement.

This 2022 European Atherosclerosis Society lipoprotein(a) [Lp(a)] consensus statement updates evidence for the role of Lp(a) in atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis, provides clinical guidance for testing and treating elevated Lp(a) levels, and considers its inclusion in global risk estimation. Epidemiologic and genetic studies involving hundreds of thousands of individuals strongly support a causal and continuous association between Lp(a) concentration and cardiovascular outcomes in different ethnicities; elevated Lp(a) is a risk factor even at very low levels of low-density lipoprotein cholesterol. High Lp(a) is associated with both microcalcification and macrocalcification of the aortic valve. Current findings do not support Lp(a) as a risk factor for venous thrombotic events and impaired fibrinolysis. Very low Lp(a) levels may associate with increased risk of diabetes mellitus meriting further study. Lp(a) has pro-inflammatory and pro-atherosclerotic properties, which may partly relate to the oxidized phospholipids carried by Lp(a). This panel recommends testing Lp(a) concentration at least once in adults; cascade testing has potential value in familial hypercholesterolaemia, or with family or personal history of (very) high Lp(a) or premature ASCVD. Without specific Lp(a)-lowering therapies, early intensive risk factor management is recommended, targeted according to global cardiovascular risk and Lp(a) level. Lipoprotein apheresis is an option for very high Lp(a) with progressive cardiovascular disease despite optimal management of risk factors. In conclusion, this statement reinforces evidence for Lp(a) as a causal risk factor for cardiovascular outcomes. Trials of specific Lp(a)-lowering treatments are critical to confirm clinical benefit for cardiovascular disease and aortic valve stenosis.

HDL in the 21st Century: A Multifunctional Roadmap for Future HDL Research.

Low high-density lipoprotein cholesterol (HDL-C) characterizes an atherogenic dyslipidemia that reflects adverse lifestyle choices, impaired metabolism, and increased cardiovascular risk. Low HDL-C is also associated with increased risk of inflammatory disorders, malignancy, diabetes, and other diseases. This epidemiologic evidence has not translated to raising HDL-C as a viable therapeutic target, partly because HDL-C does not reflect high-density lipoprotein (HDL) function. Mendelian randomization analyses that have found no evidence of a causal relationship between HDL-C levels and cardiovascular risk have decreased interest in increasing HDL-C levels as a therapeutic target. HDLs comprise distinct subpopulations of particles of varying size, charge, and composition that have several dynamic and context-dependent functions, especially with respect to acute and chronic inflammatory states. These functions include reverse cholesterol transport, inhibition of inflammation and oxidation, and antidiabetic properties. HDLs can be anti-inflammatory (which may protect against atherosclerosis and diabetes) and proinflammatory (which may help clear pathogens in sepsis). The molecular regulation of HDLs is complex, as evidenced by their association with multiple proteins, as well as bioactive lipids and noncoding RNAs. Clinical investigations of HDL biomarkers (HDL-C, HDL particle number, and apolipoprotein A through I) have revealed nonlinear relationships with cardiovascular outcomes, differential relationships by sex and ethnicity, and differential patterns with coronary versus noncoronary events. Novel HDL markers may also have relevance for heart failure, cancer, and diabetes. HDL function markers (namely, cholesterol efflux capacity) are associated with coronary disease, but they remain research tools. Therapeutics that manipulate aspects of HDL metabolism remain the holy grail. None has proven to be successful, but most have targeted HDL-C, not metrics of HDL function. Future therapeutic strategies should focus on optimizing HDL function in the right patients at the optimal time in their disease course. We provide a framework to help the research and clinical communities, as well as funding agencies and stakeholders, obtain insights into current thinking on these topics, and what we predict will be an exciting future for research and development on HDLs.

Novel plasma biomarkers predicting biventricular involvement in arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease characterized by fibrofatty replacement of the myocardium and ventricular arrhythmias. Biventricular involvement in ARVC may lead to heart failure. This study aimed to investigate the role of plasma biomarkers soluble (s)ST2, Galectin-3 (Gal-3) and GDF-15 in predicting biventricular involvement and adverse outcomes in ARVC. METHODS AND RESULTS: ARVC patients from 2 independent cohorts, were studied. The Bejing (Chinese) cohort (n = 108) was the discovery cohort, whereas the Zurich (Swiss) cohort (n = 47) served as validation. All patients had a definite ARVC diagnosis at time of blood withdrawal. Biomarkers were independently correlated with NT-proBNP and left ventricular (LV)-function. ARVC patients with LV involvement had higher levels of sST2 and GDF-15 as compared to controls and patients with isolated right ventricle (RV) involvement. sST2 and GDF-15 were significantly correlated with late gadolinium enhancement in CMR and with adverse heart failure outcomes. Gal-3 was elevated in ARVC patients with and without LV involvement. The combined use of the three biomarkers (sST2, GDF-15 and NT-proBNP) showed the best performance in predicting LV involvement in both cohorts. Plasma drawn from the coronary arteries and coronary sinus indicated a transmyocardial elevation of sST2, but no transmyocardial gradient of GDF-15. After heart transplantation, both sST2 and GDF-15 returned to near-normal levels. CONCLUSION: Our study showed that sST2 and GDF-15 may predict biventricular involvement in ARVC. The combined use of sST2, GDF-15 and NT-proBNP showed the best prediction of biventricular involvement in ARVC.

Controlled-Level EVERolimus in Acute Coronary Syndrome (CLEVER-ACS) - A phase II, randomized, double-blind, multi-center, placebo-controlled trial.

BACKGROUND: Activation of inflammatory pathways during acute myocardial infarction contributes to infarct size and left ventricular (LV) remodeling. The present prospective randomized clinical trial was designed to test the efficacy and safety of broad-spectrum anti-inflammatory therapy with a mammalian target of rapamycin (mTOR) inhibitor to reduce infarct size. DESIGN: Controlled-Level EVERolimus in Acute Coronary Syndrome (CLEVER-ACS, clinicaltrials.gov NCT01529554) is a phase II randomized, double-blind, multi-center, placebo-controlled trial on the effects of a 5-day course of oral everolimus on infarct size, LV remodeling, and inflammation in patients with acute ST-elevation myocardial infarction (STEMI). Within 5 days of successful primary percutaneous coronary intervention (pPCI), patients are randomly assigned to everolimus (first 3 days: 7.5 mg every day; days 4 and 5: 5.0 mg every day) or placebo, respectively. The primary efficacy outcome is the change from baseline (defined as 12 hours to 5 days after pPCI) to 30-day follow-up in myocardial infarct size as measured by cardiac magnetic resonance imaging (CMRI). Secondary endpoints comprise corresponding changes in cardiac and inflammatory biomarkers as well as microvascular obstruction and LV volumes assessed by CMRI. Clinical events, laboratory parameters, and blood cell counts are reported as safety endpoints at 30 days. CONCLUSION: The CLEVER-ACS trial tests the hypothesis whether mTOR inhibition using everolimus at the time of an acute STEMI affects LV infarct size following successful pPCI.

Abnormal high-density lipoprotein induces endothelial dysfunction via activation of Toll-like receptor-2.

Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.

Apolipoprotein M and Sphingosine-1-Phosphate Receptor 1 Promote the Transendothelial Transport of High-Density Lipoprotein.

Objective: ApoM enriches S1P (sphingosine-1-phosphate) within HDL (high-density lipoproteins) and facilitates the activation of the S1P1 (S1P receptor type 1) by S1P, thereby preserving endothelial barrier function. Many protective functions exerted by HDL in extravascular tissues raise the question of how S1P regulates transendothelial HDL transport. Approach and Results: HDL were isolated from plasma of wild-type mice, Apom knockout mice, human apoM transgenic mice or humans and radioiodinated to trace its binding, association, and transport by bovine or human aortic endothelial cells. We also compared the transport of fluorescently-labeled HDL or Evans Blue, which labels albumin, from the tail vein into the peritoneal cavity of apoE-haploinsufficient mice with (apoE-haploinsufficient mice with endothelium-specific knockin of S1P1) or without (control mice, ie, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1) endothelium-specific knockin of S1P1. The binding, association, and transport of HDL from Apom knockout mice and human apoM-depleted HDL by bovine aortic endothelial cells was significantly lower than that of HDL from wild-type mice and human apoM-containing HDL, respectively. The binding, uptake, and transport of 125I-HDL by human aortic endothelial cells was increased by an S1P1 agonist but decreased by an S1P1 inhibitor. Silencing of SR-BI (scavenger receptor BI) abrogated the stimulation of 125I-HDL transport by the S1P1 agonist. Compared with control mice, that is, apoE-haploinsufficient mice without endothelium-specific knockin of S1P1, apoE-haploinsufficient mice with endothelium-specific knockin of S1P1 showed decreased transport of Evans Blue but increased transport of HDL from blood into the peritoneal cavity and SR-BI expression in the aortal endothelium. Conclusions: ApoM and S1P1 promote transendothelial HDL transport. Their opposite effect on transendothelial transport of albumin and HDL indicates that HDL passes endothelial barriers by specific mechanisms rather than passive filtration.

High Density Lipoproteins: Is There a Comeback as a Therapeutic Target?

Low plasma levels of High Density Lipoprotein (HDL) cholesterol (HDL-C) are associated with increased risks of atherosclerotic cardiovascular disease (ASCVD). In cell culture and animal models, HDL particles exert multiple potentially anti-atherogenic effects. However, drugs increasing HDL-C have failed to prevent cardiovascular endpoints. Mendelian Randomization studies neither found any genetic causality for the associations of HDL-C levels with differences in cardiovascular risk. Therefore, the causal role and, hence, utility as a therapeutic target of HDL has been questioned. However, the biomarker "HDL-C" as well as the interpretation of previous data has several important limitations: First, the inverse relationship of HDL-C with risk of ASCVD is neither linear nor continuous. Hence, neither the-higher-the-better strategies of previous drug developments nor previous linear cause-effect relationships assuming Mendelian randomization approaches appear appropriate. Second, most of the drugs previously tested do not target HDL metabolism specifically so that the futile trials question the clinical utility of the investigated drugs rather than the causal role of HDL in ASCVD. Third, the cholesterol of HDL measured as HDL-C neither exerts nor reports any HDL function. Comprehensive knowledge of structure-function-disease relationships of HDL particles and associated molecules will be a pre-requisite, to test them for their physiological and pathogenic relevance and exploit them for the diagnostic and therapeutic management of individuals at HDL-associated risk of ASCVD but also other diseases, for example diabetes, chronic kidney disease, infections, autoimmune and neurodegenerative diseases.

Lipoproteins in chronic kidney disease: from bench to bedside.

Chronic kidney disease (CKD) is associated with high cardiovascular risk. CKD patients exhibit a specific lipoprotein pattern termed 'uraemic dyslipidaemia', which is characterized by rather normal low-density lipoprotein cholesterol, low high-density lipoprotein cholesterol, and high triglyceride plasma levels. All three lipoprotein classes are involved in the pathogenesis of CKD-associated cardiovascular diseases (CVDs). Uraemia leads to several modifications of the structure of lipoproteins such as changes of the proteome and the lipidome, post-translational protein modifications (e.g. carbamylation) and accumulation of small-molecular substances within the lipoprotein moieties, which affect their functionality. Lipoproteins from CKD patients interfere with lipid transport and promote inflammation, oxidative stress, endothelial dysfunction as well as other features of atherogenesis, thus contributing to the development of CKD-associated CVD. While, lipid-modifying therapies play an important role in the management of CKD patients, their efficacy is modulated by kidney function. Novel therapeutic agents to prevent the adverse remodelling of lipoproteins in CKD and to improve their functional properties are highly desirable and partially under development.

Lipoprotein(a) is associated with large artery atherosclerosis stroke aetiology and stroke recurrence among patients below the age of 60 years: results from the BIOSIGNAL study.

AIMS: Lipoprotein(a) [Lp(a)] is a recognized causal risk factor for atherosclerotic cardiovascular disease but its role for acute ischaemic stroke (AIS) is controversial. In this study, we evaluated the association of Lp(a) with large artery atherosclerosis (LAA) stroke and risk of recurrent cerebrovascular events in AIS patients. METHODS AND RESULTS: For this analysis of the prospective, observational, multicentre BIOSIGNAL cohort study we measured Lp(a) levels in plasma samples of 1733 primarily Caucasian (98.6%) AIS patients, collected within 24 h after symptom onset. Primary outcomes were LAA stroke aetiology and recurrent cerebrovascular events (ischaemic stroke or transient ischaemic attack) within 1 year. We showed that Lp(a) levels are independently associated with LAA stroke aetiology [adjusted odds ratio 1.48, 95% confidence interval (CI) 1.14-1.90, per unit log10Lp(a) increase] and identified age as a potent effect modifier (Pinteraction =0.031) of this association. The adjusted odds ratio for LAA stroke in patients aged <60 years was 3.64 (95% CI 1.76-7.52) per unit log10Lp(a) increase and 4.04 (95% CI 1.73-9.43) using the established cut-off ≥100 nmol/l. For 152 recurrent cerebrovascular events, we did not find a significant association in the whole cohort. However, Lp(a) levels ≥100 nmol/l were associated with an increased risk for recurrent events among patients who were either <60 years [adjusted hazard ratio (HR) 2.40, 95% CI 1.05-5.47], had evident LAA stroke aetiology (adjusted HR 2.18, 95% CI 1.08-4.40), or had no known atrial fibrillation (adjusted HR 1.60, 95% CI 1.03-2.48). CONCLUSION: Elevated Lp(a) was independently associated with LAA stroke aetiology and risk of recurrent cerebrovascular events among primarily Caucasian individuals aged <60 years or with evident arteriosclerotic disease.

Decoding Functional High-Density Lipoprotein Particle Surfaceome Interactions.

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

Metabolism of HSAN1- and T2DM-associated 1-deoxy-sphingolipids inhibits the migration of fibroblasts.

Hereditary sensory neuropathy type 1 (HSAN1) is a rare axonopathy, characterized by a progressive loss of sensation (pain, temperature, and vibration), neuropathic pain, and wound healing defects. HSAN1 is caused by several missense mutations in the serine palmitoyltransferase long-chain base subunit 1 and serine palmitoyltransferase long-chain base subunit 2 of the enzyme serine palmitoyltransferase-the key enzyme for the synthesis of sphingolipids. The mutations change the substrate specificity of serine palmitoyltransferase, which then forms an atypical class of 1-deoxy-sphinglipids (1-deoxySLs). Similarly, patients with type 2 diabetes mellitus also present with elevated 1-deoxySLs and a comparable clinical phenotype. The effect of 1-deoxySLs on neuronal cells was investigated in detail, but their impact on other cell types remains elusive. Here, we investigated the consequences of externally added 1-deoxySLs on the migration of fibroblasts in a scratch assay as a simplified cellular wound-healing model. We showed that 1-deoxy-sphinganine (1-deoxySA) inhibits the migration of NIH-3T3 fibroblasts in a dose- and time-dependent manner. This was not seen for a non-native, L-threo stereoisomer. Supplemented 1-deoxySA was metabolized to 1-deoxy-(dihydro)ceramide and downstream to 1-deoxy-sphingosine. Inhibiting downstream metabolism by blocking N-acylation rescued the migration phenotype. In contrast, adding 1-deoxy-sphingosine had a lesser effect on cell migration but caused the massive formation of intracellular vacuoles. Further experiments showed that the effect on cell migration was primarily mediated by 1-deoxy-dihydroceramides rather than by the free base or 1-deoxyceramides. Based on these findings, we suggest that limiting the N-acylation of 1-deoxySA could be a therapeutic approach to improve cell migration and wound healing in patients with HSAN1 and type 2 diabetes mellitus.

Reproducible Determination of High-Density Lipoprotein Proteotypes.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.