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.

Prevalence of Subclinical Cardiovascular Disease in Patients with Non-Alcoholic-Fatty Liver Disease: Analysis of the Paracelsus 10.000 Cohort Study.

BACKGROUND: In patients with non-alcoholic fatty liver disease (NAFLD) cardiovascular diseases are more often the cause of death than the liver disease itself. However, the prevalence of atherosclerotic manifestations in individuals with NAFLD is still uncertain. This study aimed to explore the association between NAFLD and coronary artery calcification (CAC) in a Central European population. METHODS: A total of 1,743 participants from the Paracelsus 10,000 study were included. The participants underwent CAC scoring and were assessed for fatty liver index (FLI), fibrosing non-alcoholic steatohepatitis Index (FNI) and fibrosis-4 index (FIB-4 score), which are indicators for steatosis and fibrosis. Multivariable logistic regression models were calculated. RESULTS: Results revealed an association between liver steatosis/fibrosis and CAC. A FLI > 60 was associated with higher odds of NAFLD (OR 3.38, 95% CI: 2.61-4.39, p < 0.01) and increased prevalence of CAC-Score >300 compared to FLI <30 (9% vs. 3%, p < 0.01), even after adjusting for traditional cardiometabolic risk factors. While the crude odds ratios of the FIB-4 scores ≥ 1.3 and FNI score were significantly associated with increased odds of CAC, they became non-significant after adjusting for age, sex, and MetS. CONCLUSION: This study reveals a significant association between NAFLD and CAC. The findings suggest that assessing liver fat and fibrosis could enhance assessment of cardiovascular risk, but further research is needed to determine whether hepatic fat plays an independent role in the development of atherosclerosis and whether targeting liver steatosis can mitigate vascular risk.

Ethyl glucuronide in hair detects a high rate of harmful alcohol consumption in presumed non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) and alcohol-related liver disease (ALD) cannot reliably be distinguished by routine diagnostics, and the role of alcohol consumption in metabolic dysfunction-associated fatty liver disease (MAFLD) remains unclear. We investigated alcohol consumption in patients with presumed NAFLD and ALD using novel objective alcohol markers. METHODS: In total, 184 consecutive patients were included in this prospective observational study. Alcohol intake was assessed by ethylglucuronide in hair (hEtG) and urine (uEtG); the utility of these measures for alcohol detection was compared to Alcohol Use Disorders Identification Test-Consumption (AUDIT-C), carbohydrate deficient transferrin (CDT), mean corpuscular volume (MCV), gamma-glutamyltransferase (GGT), and ALD/NAFLD index (ANI). Clinical characteristics of patients with NAFLD and ALD were re-assessed after reclassification based on repeated moderate (≥10 g <60 g EtOH/day) and excessive (≥60 g EtOH/day) alcohol consumption, and patients were retrospectively reclassified based on MAFLD criteria. RESULTS: Repeated moderate to excessive alcohol consumption was detected in 28.6%, 28.5%, and 25.0% of patients with presumed NAFLD, ALD or MAFLD, respectively. ANI score, AUDIT-C, uEtG, and hEtG showed AUCs of 0.628, 0.733, 0.754, and 0.927 for the detection of repeated moderate to excessive alcohol consumption, respectively. The indirect markers CDT, MCV and GGT were not reliable. Patients with repeated moderate or excessive alcohol consumption were significantly more often male, had a significantly lower BMI, and suffered significantly less often from type 2 diabetes or impaired glucose tolerance. CONCLUSIONS: In total, 28.6% of patients with presumed NAFLD, and 25.0% with MAFLD are at risk of alcohol-related liver damage. AUDIT-C, uEtG and hEtG should be used to screen for alcohol consumption in patients with fatty liver disease. LAY SUMMARY: Fatty liver disease can be caused by metabolic factors and/or alcohol consumption. The diagnosis of non-alcoholic fatty liver disease (NAFLD) is based on the exclusion of harmful alcohol consumption, while metabolic dysfunction-associated fatty liver disease (MAFLD), which has been proposed as a new name for NAFLD, is based on the presence of metabolic comorbidities and allows for alcohol consumption. Herein, we show that up to 29% of patients diagnosed with NAFLD and 25% with MAFLD are at risk of alcohol-related liver damage. We show that ethyl glucuronide (a metabolite of alcohol) in the hair and urine can accurately detect potentially harmful alcohol consumption in these patients - as such, these tests should be integrated into routine diagnostic work-up for patients with fatty liver disease.

Afamin predicts the prevalence and incidence of nonalcoholic fatty liver disease.

OBJECTIVES: In the general population, increased afamin concentrations are associated with the prevalence and incidence of metabolic syndrome as well as type 2 diabetes. Although metabolic syndrome is commonly associated with nonalcoholic fatty liver disease (NAFLD), there exist no information on afamin and NAFLD. METHODS: Afamin concentrations were cross-sectionally measured in 146 Austrian patients with NAFLD, in 45 patients without NAFLD, and in 292 age- and sex-matched healthy controls. Furthermore, the feasibility of afamin to predict incident NAFLD was evaluated in 1,434 adult participants in the population-based Cardiovascular Risk in Young Finns Study during a 10-year follow-up. RESULTS: Median afamin concentrations were significantly higher in NAFLD patients (83.6 mg/L) than in patients without NAFLD (61.6 mg/L, p<0.0001) or in healthy controls (63.9 mg/L, p<0.0001). In age- and sex-adjusted logistic regression analyses a 10 mg/L increase of afamin was associated with a 1.5-fold increase of having NAFLD as compared with patients without NAFLD and the risk was even two-fold when compared with healthy controls. In the population-based cohort, afamin concentrations at baseline were significantly lower in participants without NAFLD (n=1,195) than in 239 participants who developed NAFLD (56.5 vs. 66.9 mg/L, p<0.0001) during the 10-year follow up, with highest afamin values observed in individuals developing severe forms of NAFLD. After adjustment for several potentially confounding parameters, afamin remained an independent predictor for the development of NAFLD (OR=1.37 [95% CI 1.23-1.54] per 10 mg/L increase, p<0.0001). CONCLUSIONS: Afamin concentrations are increased in patients with NAFLD and independently predict the development of NAFLD in a population-based cohort.

The haemochromatosis gene Hfe and Kupffer cells control LDL cholesterol homeostasis and impact on atherosclerosis development.

AIMS: Imbalances of iron metabolism have been linked to the development of atherosclerosis. However, subjects with hereditary haemochromatosis have a lower prevalence of cardiovascular disease. The aim of our study was to understand the underlying mechanisms by combining data from genome-wide association study analyses in humans, CRISPR/Cas9 genome editing, and loss-of-function studies in mice. METHODS AND RESULTS: Our analysis of the Global Lipids Genetics Consortium (GLGC) dataset revealed that single nucleotide polymorphisms (SNPs) in the haemochromatosis gene HFE associate with reduced low-density lipoprotein cholesterol (LDL-C) in human plasma. The LDL-C lowering effect could be phenocopied in dyslipidaemic ApoE-/- mice lacking Hfe, which translated into reduced atherosclerosis burden. Mechanistically, we identified HFE as a negative regulator of LDL receptor expression in hepatocytes. Moreover, we uncovered liver-resident Kupffer cells (KCs) as central players in cholesterol homeostasis as they were found to acquire and transfer LDL-derived cholesterol to hepatocytes in an Abca1-dependent fashion, which is controlled by iron availability. CONCLUSION: Our results disentangle novel regulatory interactions between iron metabolism, KC biology and cholesterol homeostasis which are promising targets for treating dyslipidaemia but also provide a mechanistic explanation for reduced cardiovascular morbidity in subjects with haemochromatosis.

Mesenchymal iron deposition is associated with adverse long-term outcome in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Approximately one-third of patients with non-alcoholic fatty liver disease (NAFLD) show signs of mild-to-moderate iron overload. The impact of histological iron deposition on the clinical course of patients with NAFLD has not been established. METHODS & RESULTS: For this retrospective study, 299 consecutive patients with biopsy-proven NAFLD and a mean follow-up of 8.4 (±4.1; range: 0.3-18.0) years were allocated to one of four groups according to presence of hepatic iron in the reticuloendothelial system (RES) and/or hepatocytes (HC): 156 subjects (52%) showed no stainable iron (NONE), 58 (19%) exclusively reticuloendothelial (xRES), 19 (6%) exclusively hepatocellular (xHC) and 66 (22%) showed a mixed (HC/RES) pattern of iron deposition. A long-term analysis for overall survival, hepatic, cardiovascular or extrahepatic-malignant events was conducted. Based on multivariate Cox proportional hazards models any reticuloendothelial iron was associated with fatal and non-fatal hepatic events. Specifically, xRES showed a cause-specific hazard ratio (csHR) of 2.4 (95%-CI, 1.0-5.8; P = .048) for hepatic as well as cardiovascular fatal and non-fatal events combined (csHR 3.2; 95%-CI, 1.2-8.2; P = .015). Furthermore, the mixed HC/RES iron pattern showed a higher rate of combined hepatic fatal and non-fatal events (csHR 3.6; 95%-CI, 1.4-9.5; P = .010), while xHC iron deposition was not associated with any defined events. CONCLUSIONS: The presence of reticuloendothelial-accentuated hepatic iron distribution patterns is associated with detrimental long-term outcomes reflected in a higher rate of both liver-related and cardiovascular fatal and non-fatal events.

Comparison of visual, automatic and semiautomatic methods to determine ventilatory indices in 50 to 60 years old adults.

The aim of this study was to compare different methods of detecting ventilatory indices (VI) and to investigate the impact of cardiorespiratory fitness (CRF) level on VI detection. Fifty females and fifty males completed a graded exercise test until volitional exhaustion with continuous gas-exchange measurement. The first and second ventilatory indices (VI-1, VI-2) were detected through different single automatic methods and through a semiautomatic method which combines visual and automatic detection methods. Additionally, the VIs were detected visually by two experts which served as the study specific gold standard. When comparing the semiautomatic method at VI-1 (intraclass correlation coefficients (ICC) 0.88 [0.81, 0.92], Bland-Altman bias ± limits of agreement (LoA) 55 ± 334 ml O2 · min-1) and VI-2 (ICC 0.97 [0.96, 0.98], LoA 1 ± 268 ml O2 · min-1) to the visually detected VI, high levels of agreements and no significant differences were found. This was not the case for any of the other automatic methods. Additionally, we couldn't find any relevant differences regarding the CRF level.We therefore concluded that the semiautomatic detection method should be used for VI detection, as results are more accurate than in any of the single-automatic methods.Abbreviations: CPET: cardiopulmonary exercise test; CRF: Cardiorespiratory fitness; VO2peak: peak oxygen uptake; VI-1: first ventilatory indices; VI-2: second ventilatory indices; LoA: Bland-Altman bias ± limits of agreement; ICC: intraclass correlation coefficient.

A comprehensive map of single-base polymorphisms in the hypervariable LPA kringle IV type 2 copy number variation region.

Lipoprotein (a) [Lp(a)] concentrations are among the strongest genetic risk factors for cardiovascular disease and present pronounced interethnic and interindividual differences. Approximately 90% of Lp(a) variance is controlled by the LPA gene, which contains a 5.6-kb-large copy number variation [kringle IV type 2 (KIV-2) repeat] that generates >40 protein isoforms. Variants within the KIV-2 region are not called in common sequencing projects, leaving up to 70% of the LPA coding region currently unaddressed. To completely assess the variability in LPA, we developed a sequencing strategy for this region and report here the first map of genetic variation in the KIV-2 region, a comprehensively evaluated ultradeep sequencing protocol, and an easy-to-use variant analysis pipeline. We sequenced 123 Central-European individuals and reanalyzed public data of 2,504 individuals from 26 populations. We found 14 different loss-of-function and splice-site mutations, as well as >100, partially even common, missense variants. Some coding variants were frequent in one population but absent in others. This provides novel candidates to explain the large ethnic and individual differences in Lp(a) concentrations. Importantly, our approach and pipeline are also applicable to other similar copy number variable regions, allowing access to regions that are not captured by common genome sequencing.

Hypoglycemia but Not Hyperglycemia Is Associated with Mortality in Critically Ill Patients with Diabetes.

BACKGROUND: Both severe hyperglycemia (> 200 mg/dL) and hypoglycemia (≤70 mg/dL) are known to be associated with increased mortality in critically ill patients. Therefore, we investigated associations of a single episode of blood glucose deviation (concentration either ≤70 mg/dL and/or > 200 mg/dL) during an intensive care unit (ICU) stay with mortality in these patients. METHODS: A total of 4,986 patients (age 65 ± 15 years; 39% female; 14% type 2 diabetes [T2DM] based on medical records) admitted to a German ICU in a tertiary care hospital were investigated retrospectively. The intra-ICU and long-term mortality of patients between 4 and 7 years after their ICU submission were assessed. RESULTS: A total 62,659 glucose measurements were analyzed. A single glucose deviation was associated with adverse outcomes compared to patients without a glucose deviation, represented by both intra-ICU mortality (22 vs. 10%; OR 2.62; 95% CI 2.23-3.09; p < 0.001) and long-term mortality (HR 2.01; 95% CI 1.81-2.24; p < 0.001). In patients suffering from T2DM hypoglycemia (30 vs. 13%; OR 2.94; 95% CI 2.28-3.80; p < 0.001) but not hyperglycemia (16 vs. 14%; OR 1.05; 95% CI 0.68-1.62; p = 0.84) was associated with mortality. CONCLUSION: In patients with dia-betes, hypo- but not hyperglycemia was associated with increased mortality, whereas in patients without diabetes, both hyper- and hypoglycemia were associated with adverse outcome. Blood glucose concentration might need differential approaches depending on concomitant diseases.

A genome-wide association meta-analysis on apolipoprotein A-IV concentrations.

Apolipoprotein A-IV (apoA-IV) is a major component of HDL and chylomicron particles and is involved in reverse cholesterol transport. It is an early marker of impaired renal function. We aimed to identify genetic loci associated with apoA-IV concentrations and to investigate relationships with known susceptibility loci for kidney function and lipids. A genome-wide association meta-analysis on apoA-IV concentrations was conducted in five population-based cohorts (n = 13,813) followed by two additional replication studies (n = 2,267) including approximately 10 M SNPs. Three independent SNPs from two genomic regions were significantly associated with apoA-IV concentrations: rs1729407 near APOA4 (P = 6.77 × 10 - 44), rs5104 in APOA4 (P = 1.79 × 10-24) and rs4241819 in KLKB1 (P = 5.6 × 10-14). Additionally, a look-up of the replicated SNPs in downloadable GWAS meta-analysis results was performed on kidney function (defined by eGFR), HDL-cholesterol and triglycerides. From these three SNPs mentioned above, only rs1729407 showed an association with HDL-cholesterol (P = 7.1 × 10 - 07). Moreover, weighted SNP-scores were built involving known susceptibility loci for the aforementioned traits (53, 70 and 38 SNPs, respectively) and were associated with apoA-IV concentrations. This analysis revealed a significant and an inverse association for kidney function with apoA-IV concentrations (P = 5.5 × 10-05). Furthermore, an increase of triglyceride-increasing alleles was found to decrease apoA-IV concentrations (P = 0.0078). In summary, we identified two independent SNPs located in or next the APOA4 gene and one SNP in KLKB1 The association of KLKB1 with apoA-IV suggests an involvement of apoA-IV in renal metabolism and/or an interaction within HDL particles. Analyses of SNP-scores indicate potential causal effects of kidney function and by lesser extent triglycerides on apoA-IV concentrations.