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ObjectivesMetabolic inflammation is a hallmark of obesity and related disorders, afflicting substantial morbidity and mortality to individuals worldwide. White visceral and subcutaneous adipose tissue not only serves as energy storage but also controls metabolism. Adipose tissue inflammation, commonly observed in human obesity, is considered a critical driver of metabolic perturbation while molecular hubs are poorly explored. Metabolic stress evoked by e.g. long-chain fatty acids leads to oxidative perturbation of adipocytes and production of inflammatory cytokines, fuelling macrophage infiltration and systemic low-grade inflammation. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation, accumulation of oxygen-specific epitopes and cell death, collectively referred to as ferroptosis. Here, we explore the function of adipocyte GPX4 in mammalian metabolism.MethodsWe studied the regulation and function of GPX4 in differentiated mouse adipocytes derived from 3T3-L1 fibroblasts. We generated two conditional adipocyte-specific Gpx4 knockout mice by crossing Gpx4fl/fl mice with Adipoq-Cre+ (Gpx4−/−AT) or Fabp4-Cre+ (Gpx4+/−Fabp4) mice. Both models were metabolically characterized by a glucose tolerance test and insulin resistance test, and adipose tissue lipid peroxidation, inflammation and cell death were assessed by quantifying oxygen-specific epitopes, transcriptional analysis of chemokines, quantification of F4/80+ macrophages and TUNEL labelling.ResultsGPX4 expression was induced during and required for adipocyte differentiation. In mature adipocytes, impaired GPX4 activity spontaneously evoked lipid peroxidation and expression of inflammatory cytokines such as TNF-α, interleukin 1β (IL-1β), IL-6 and the IL-8 homologue CXCL1. Gpx4−/−AT mice spontaneously displayed adipocyte hypertrophy on a chow diet, which was paralleled by the accumulation of oxygen-specific epitopes and macrophage infiltration in adipose tissue. Furthermore, Gpx4−/−AT mice spontaneously developed glucose intolerance, hepatic insulin resistance and systemic low-grade inflammation, when compared to wildtype littermates, which was similarly recapitulated in Gpx4+/−Fabp4 mice. Gpx4−/−AT mice exhibited no signs of adipocyte death.ConclusionAdipocyte GPX4 protects against spontaneous metabolic dysregulation and systemic low-grade inflammation independent from ferroptosis, which could be therapeutically exploited in the future.

Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders.

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.

The inverse relationship between HDL cholesterol and cardiovascular mortality is weakened in coronary artery disease (CAD). We aimed to investigate the associations of HDL particle concentrations with cardiovascular mortality and the impact of CAD on these associations. We also sought to comparatively evaluate HDL cholesterol and HDL particle concentrations in predicting cardiovascular mortality. Total and subclass HDL particle concentrations were measured by nuclear magnetic resonance spectroscopy in 2290 participants of the LUdwigshafen RIsk and Cardiovascular Health study referred for coronary angiography. The participants were prospectively followed over a median (interquartile range) duration of 10.0 (6.1-10.6) years. The mean (SD) age of the participants (1575 males, 715 females) was 62.9 (10.4) years; body mass index, 27.6 (4.1) kg/m ; HDL cholesterol, 39 (11) mg/dL [1 (0.29) mmol/L]; and total HDL particle concentration, 24.1 (5.8) μmol/L. Of the participants, 434 died from cardiovascular diseases. In multivariate analyses, tertiles of total HDL particle concentrations were inversely related to cardiovascular mortality (hazard ratio for third vs first tertile = 0.55, < 0.001). This association was primarily mediated by small HDL particles ( < 0.001). Adding total or small HDL particle concentrations rather than HDL cholesterol to multivariate prediction models improved performance metrics for cardiovascular mortality. The presence of CAD had no impact on the associations between HDL particle concentrations and cardiovascular mortality. High HDL particle concentration is consistently and independently of CAD associated with decreased cardiovascular mortality. Whether the inverse relationship between HDL particle concentration and cardiovascular mortality may be translated into novel therapies is under investigation.

Intervertebral disc (IVD) degeneration that accelerates the loss of disc structural and functional integrities is recognized as one of the major factors of chronic back pain. Cardiovascular risk factors, such as deficits of apolipoproteins that elevate the levels of cholesterol and triglycerides, are considered critical for the progress of atherosclerosis; notably in the abdominal aorta and its lumbar branching arteries that supply lumbar vertebrae and IVDs. Obstruction of the lumbar arteries by atherosclerosis is presumed to promote lumbar disc degeneration and low back pain. APOE-knockout rabbits have recently been shown to generate hyperlipidemia with increased levels of cholesterol and triglycerides that mimic the symptoms of atherosclerosis in humans. Here, we analysed IVD degeneration in the lumbar spines of ten homozygous APOE-knockout and four wild-type New Zealand White rabbits of matching age to prove accelerated IVD degeneration in APOE-knockout rabbits, since APOE-knockout rabbits could be a beneficial model for therapeutic approaches of degenerative IVD disorders. Experiments were performed using T1/T2-weighted magnetic resonance imaging, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, glucose-oxidase assay, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot. APOE-knockout lumbar spines showed more advanced IVD degeneration, obstructed lumbar arteries and lower enhancement of contrast agent in IVDs. Moreover, lower concentration of glucose, lower number of viable cells and lower concentrations of aggrecan, collagen II and higher concentration of collagen I were detected in APOE-knockout IVDs (p < 0.0001). APOE-knockout in rabbits could induce structurally deteriorating premature IVD degeneration that mimics the symptoms of accelerated IVD degeneration in humans. APOE-knockout rabbits could be used as beneficial model, as they can bypass the standard surgical interventions that are commonly applied in research animals for the induction of enhanced IVD degeneration. Their parallel use in therapeutic approaches of IVD disorders and atherosclerosis could reduce the number of research animals to be used and contribute to the principles of 3Rs (Replacement, Reduction and Refinement).

Introduction: Cardiovascular disease (CVD) is a systemic multifocal illness called atherosclerosis that causes artery constriction and blockage. By causing cholesterol to build up in the artery wall, hypercholesterolemia is a major factor in the pathophysiology of atherosclerotic plaque development. Reverse cholesterol transport is the process of transporting cholesterol from the periphery back to the liver through cholesterol efflux mediated by high-density lipoprotein (HDL). It was suggested that the cholesterol efflux capacity (CEC), which is inversely linked with cardiovascular risk, can serve as a stand-in measure for reverse cholesterol transport. In this work, we sought to investigate a potential link between the peripheral plaque volume (PV) and CEC. Methods: Since lipid-lowering therapy interferes with CEC, we performed a cross-sectional study of 176 patients (48.9% females) with one cardiovascular risk factor or known CVD that did not currently take lipid-lowering medication. CEC was determined using cAMP-treated 3H-cholesterol-labeled J774 cells. Cholesterol ester transfer protein (CETP)-mediated cholesterol ester transfer was measured by quantifying the transfer of cholesterol ester from radiolabeled exogenous HDL cholesterol to Apolipoprotein B-containing lipoproteins. PV in the carotid and the femoral artery, defined as the total PV, was measured using a 3D ultrasound system equipped with semi-automatic software. Results: In our patients, we discovered an inverse relationship between high total PV and CEC (p = 0.027). However, there was no connection between total PV and low-density lipoprotein cholesterol, lipoprotein (a), or CETP-mediated cholesterol ester transfer. Conclusion: In patients not receiving lipid-lowering treatment, CEC inversely correlates with peripheral atherosclerosis, supporting its role in the pathophysiology of atherosclerosis.

(1) Background and Aims: Efforts to reduce coronary artery disease (CAD) by raising high-density lipoprotein (HDL) cholesterol (HDL-C) have not been uniformly successful. A more important factor than HDL-C may be cellular cholesterol efflux mediated by HDL, which has been shown to be associated with CAD. In this report, we analyzed the influence of cardiovascular biomarkers and risk factors on cholesterol efflux in a prospective observational study of patients referred to coronary angiography. (2) Methods: HDL-mediated efflux capacity was determined for 2468 participants of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study who were referred to coronary angiography at baseline between 1997 and 2000. Median follow-up time was 9.9 years. Primary and secondary endpoints were cardiovascular and all-cause mortality, respectively. (3) Results: Cholesterol efflux strongly correlated with HDL-related markers including HDL cholesterol, HDL phospholipids, and apolipoproteins AI and AII, as well as HDL particle concentration, which was not seen for low density lipoprotein (LDL) markers including LDL cholesterol and apoB. Cholesterol efflux was associated negatively with C-reactive protein (CRP), fibrinogen, interleukin-6 (IL-6), and serum amyloid A. Cardiovascular mortality was higher in patients in the lowest cholesterol efflux quartile. This association was weakened, but not fully abolished, after adjustment for HDL cholesterol. (4) Conclusions: We demonstrate that cholesterol efflux was associated with HDL-composition as well as inflammatory burden in patients referred for coronary angiography, and that this inversely predicts cardiovascular mortality independently of HDL cholesterol.

Background: Impaired renal function is associated with cardiovascular and all-cause mortality. In the general population, HDL-cholesterol is associated with cardiovascular events, which is not true in patients with chronic kidney disease (CKD). This has been attributed to abnormal HDL function in CKD. Methods: In this study, we analyzed the association of genetic markers for kidney function with cholesterol efflux capacity as one of the major HDL functions, as well as with cardiovascular mortality, in 2469 patients of the Ludwigshafen Risk and Cardiovascular Health Study who all underwent coronary angiography. Results: A genetic score of 53 SNPs associated with GRF and the uromodulin SNP rs12917707 were inversely correlated with cholesterol efflux capacity. This was in line with the observed association between cholesterol efflux capacity and kidney function in these patients. Adjustment for eGFR and uromodulin as markers of kidney function did not affect the relationship between cholesterol efflux and cardiovascular mortality. Conclusions: Our data propose the view that cholesterol efflux and kidney function are exerting their effects on cardiovascular mortality via different and independent pathways. Decreased cholesterol efflux may therefore not mediate the effects of impaired kidney function on cardiovascular mortality.

Background: Cholesterol efflux is an important mechanism by which high-density lipoproteins (HDLs) protect against cardiovascular disease. As peripheral artery disease (PAD) is associated with high mortality rates, mainly due to cardiovascular causes, we investigated whether cholesterol efflux capacity (CEC) of apolipoprotein B (apoB)-depleted plasma, a widely used surrogate of HDL function, may serve as a predictive marker for mortality in this patient population. Methods: In this prospective single-center study (median follow-up time: 9.3 years), apoB-containing lipoproteins were precipitated from plasma of 95 patients with PAD and incubated with J744-macrophages, which were loaded with radiolabeled cholesterol. CEC was defined as the fractional radiolabel released during 4 h of incubation. Results: Baseline CEC was lower in PAD patients that currently smoked (p = 0.015) and had a history of myocardial infarction (p = 0.011). Moreover, CEC showed a significant correlation with HDL-cholesterol (p = 0.003) and apolipoprotein A-I levels (p = 0.001) as well as the ankle-brachial index (ABI, p = 0.018). However, CEC did not differ between survivors and non-survivors. Neither revealed Kaplan–Meier and Cox regression analyses any significant association of CEC with all-cause mortality rates. Conclusion: Taken together, CEC is associated with ABI but does not predict all-cause mortality in patients with PAD.

Bacterial sepsis results in high mortality rates, and new therapeutics to control infection are urgently needed. Here, we investigate the therapeutic potential of fibrates in the treatment of bacterial sepsis and examine their effects on innate immunity. Fibrates significantly improved the survival from sepsis in mice infected with Salmonella typhimurium , which was paralleled by markedly increased neutrophil influx to the site of infection resulting in rapid clearance of invading bacteria. As a consequence of fibrate‐mediated early control of infection, the systemic inflammatory response was repressed in fibrate‐treated mice. Mechanistically, we found that fibrates preserve chemotaxis of murine neutrophils by blocking LPS‐induced phosphorylation of ERK. This results in a decrease of G protein‐coupled receptor kinase‐2 expression, thereby inhibiting the LPS‐mediated downregulation of CXCR2, a chemokine receptor critical for neutrophil recruitment. Accordingly, application of a synthetic CXCR2 inhibitor completely abrogated the protective effects of fibrates in septicemia in vivo . Our results unravel a novel function of fibrates in innate immunity and host response to infection and suggest fibrates as a promising adjunct therapy in bacterial sepsis. Synopsis In this study, fibrates are shown to protect against bacteria‐induced sepsis by promoting neutrophil influx to the primary site of infection, aiding in an efficient elimination of bacteria, and contributing to improved clinical outcomes from sepsis. Survival of septic mice infected with Salmonella typhimurium is improved through treatment with fibrates. Increased migratory efficiency of neutrophils results in early clearance of bacteria. Expression of chemokine receptor CXCR2 on neutrophils in sepsis is preserved. CXCR2 expression is maintained through a ERK/GRK2‐dependent mechanism. Graphical Abstract In this study, fibrates are shown to protect against bacteria‐induced sepsis by promoting neutrophil influx to the primary site of infection, aiding in an efficient elimination of bacteria, and contributing to improved clinical outcomes from sepsis.