Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 µm2 compared with 339 ± 15 × 103 µm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

LXR Agonist T0901317's Hepatic Impact Overrules Its Atheroprotective Action in Macrophages, Driving Early Atherogenesis in Chow-Diet-Fed Male Apolipoprotein E Knockout Mice.

Preclinical studies regarding the potential of liver X receptor (LXR) agonists to inhibit macrophage foam cell formation and the development of atherosclerotic lesions are generally executed in mice fed with Western-type diets enriched in cholesterol and fat. Here, we investigated whether LXR agonism remains anti-atherogenic under dietary conditions with a low basal hepatic lipogenesis rate. Hereto, atherosclerosis-susceptible male apolipoprotein E knockout mice were fed a low-fat diet with or without 10 mg/kg/day LXR agonist T0901317 supplementation for 8 weeks. Importantly, T0901317 significantly stimulated atherosclerosis susceptibility, despite an associated increase in the macrophage gene expression levels of cholesterol efflux transporters ABCA1 and ABCG1. The pro-atherogenic effect of T0901317 coincided with exacerbated hypercholesterolemia, hypertriglyceridemia, and a significant rise in hepatic triglyceride stores and macrophage numbers. Furthermore, T0901317-treated mice exhibited elevated plasma MCP-1 levels and monocytosis. In conclusion, these findings highlight that the pro-atherogenic hepatic effects of LXR agonism are dominant over the anti-atherogenic effects in macrophages in determining the overall atherosclerosis outcome under low-fat diet feeding conditions. A low-fat diet experimental setting, as compared to the commonly used high-fat-diet-based preclinical setup, thus appears more sensitive in uncovering the potential relevance of the off-target liver effects of novel anti-atherogenic therapeutic approaches that target macrophage LXR.

Gene Editing for the Treatment of Hypercholesterolemia.

PURPOSE OF REVIEW: Here, we summarize the key findings from preclinical studies that tested the concept that editing of hepatic genes can lower plasma low-density lipoprotein (LDL)-cholesterol levels to subsequently reduce atherosclerotic cardiovascular disease risk. RECENT FINDINGS: Selective delivery of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing tools targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) to hepatocytes, i.e., through encapsulation into N-acetylgalactosamine-coupled lipid nanoparticles, is able to induce a stable ~ 90% decrease in plasma PCSK9 levels and a concomitant 60% reduction in LDL-cholesterol levels in mice and non-humane primates. Studies in mice have shown that this state-of-the-art technology can be extended to include additional targets related to dyslipidemia such as angiopoietin-like 3 and several apolipoproteins. The use of gene editors holds great promise to lower plasma LDL-cholesterol levels also in the human setting. However, gene editing safety has to be guaranteed before this approach can become a clinical success.

Scavenger Receptor BI Deficiency in Mice Is Associated With Plasma Ceramide and Sphingomyelin Accumulation and a Reduced Cholesteryl Ester Fatty Acid Length and Unsaturation Degree.

Objective: Scavenger receptor class B type I (SR-BI) is primarily known for its role in the selective uptake of cholesteryl esters (CEs) from high-density lipoproteins (HDLs). Here we investigated whether SR-BI deficiency is associated with other potentially relevant changes in the plasma lipidome than the established effect of HDL-cholesterol elevation. Methods: Targeted ultra-high-performance liquid chromatography-tandem mass spectrometry was utilized to measure lipid species in plasma from female wild-type and SR-BI knockout mice. Results: SR-BI deficiency was associated with a reduction in the average CE fatty acid length (-2%; p<0.001) and degree of CE fatty acid unsaturation (-18%; p<0.001) due to a relative shift from longer, polyunsaturated CE species CE (20:4), CE (20:5), and CE (22:6) towards the mono-unsaturated CE (18:1) species. Sphingomyelin (SM) levels were 64% higher (p<0.001) in SR-BI knockout mice without a parallel change in (lyso)phosphatidylcholine (LPC) concentrations, resulting in an increase in the SM/LPC ratio from 0.102±0.005 to 0.163±0.003 (p<0.001). In addition, lower LPC lengths (-5%; p<0.05) and fatty acid unsaturation degrees (-20%; p<0.01) were detected in SR-BI knockout mice. Furthermore, SR-BI deficiency was associated with a 4.7-fold increase (p<0.001) in total plasma ceramide (Cer) levels, with a marked >9-fold rise (p<0.001) in Cer (d18:1/24:1) concentrations. Conclusion: We have shown that SR-BI deficiency in mice not only impacts the CE concentrations, length, and saturation index within the plasma compartment, but is also associated with plasma accumulation of several Cer and SM species that may contribute to the development of specific hematological and metabolic (disease) phenotypes previously detected in SR-BI knockout mice.

ANGPTL3 (Angiopoietin-Like 3) Preferentially Resides on High-Density Lipoprotein in the Human Circulation, Affecting Its Activity.

Background ANGPTL3 (angiopoietin-like protein 3) is an acknowledged crucial regulator of lipid metabolism by virtue of its inhibitory effect on lipoprotein lipase and endothelial lipase. It is currently unknown whether and to which lipoproteins ANGPTL3 is bound and whether the ability of ANGPTL3 to inhibit lipase activity is affected by binding to lipoproteins. Methods and Results Incubation of ultracentrifugation-isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL) fractions from healthy volunteers with recombinant ANGPTL3 revealed that ANGPTL3 associates with both HDL and LDL particles ex vivo. Plasma from healthy volunteers and a patient deficient in HDL was fractionated by fast protein liquid chromatography, and ANGPTL3 distribution among lipoprotein fractions was measured. In healthy volunteers, ≈75% of lipoprotein-associated ANGPTL3 resides in HDL fractions, whereas ANGPTL3 was largely bound to LDL in the patient deficient in HDL. ANGPTL3 activity was studied by measuring lipolysis and uptake of 3H-trioleate by brown adipocyte T37i cells. Unbound ANGPTL3 did not suppress lipase activity, but when given with HDL or LDL, ANGPTL3 suppressed lipase activity by 21.4±16.4% (P=0.03) and 25.4±8.2% (P=0.006), respectively. Finally, in a subset of the EPIC (European Prospective Investigation into Cancer) Norfolk study, plasma HDL cholesterol and amount of large HDL particles were both positively associated with plasma ANGPTL3 concentrations. Moreover, plasma ANGPTL3 concentrations showed a positive association with incident coronary artery disease (odds ratio, 1.25 [95% CI, 1.01-1.55], P=0.04). Conclusions Although ANGPTL3 preferentially resides on HDL, its activity was highest once bound to LDL particles.

High-density lipoproteins and non-alcoholic fatty liver disease.

Background and aims: Non-alcoholic fatty liver disease (NAFLD), a high incidence liver pathology, is associated with a ∼1.5-fold higher cardiovascular disease risk. This phenomenon is generally attributed to the NAFLD-associated increase in circulating levels of pro-atherogenic apolipoprotein B100-containing small dense low-density lipoprotein and plasma hypertriglyceridemia. However, also a significant reduction in cholesterol transported by anti-atherogenic high-density lipoproteins (HDL) is frequently observed in subjects suffering from NAFLD as compared to unaffected people. In this review, we summarize data regarding the relationship between NAFLD and plasma HDL-cholesterol levels, with a special focus on highlighting potential causality between the NAFLD pathology and changes in HDL metabolism. Methods and results: Publications in PUBMED describing the relationship between HDL levels and NAFLD susceptibility and/or disease severity, either in human clinical settings or genetically-modified mouse models, were critically reviewed for subsequent inclusion in this manuscript. Furthermore, relevant literature describing effects on lipid loading in cultured hepatocytes of models with genetic alterations related to HDL metabolism have been summarized. Conclusions: Although in vitro observations suggest causality between HDL formation by hepatocytes and protection against NAFLD-like lipid accumulation, current literature remains inconclusive on whether relative HDL deficiency is actually driving the development of fatty liver disease in humans. In light of the current obesity pandemic and the associated marked rise in NAFLD incidence, it is of clear scientific and societal interest to gain further insight into the relationship between HDL-cholesterol levels and fatty liver development to potentially uncover the therapeutic potential of pharmacological HDL level and/or function modulation.

Metyrapone Treatment Protects Low-Density Lipoprotein Receptor Knockout Mice against Hypercorticosteronemia Development without Changing Atherosclerosis Susceptibility.

The steroid 11beta-hydroxylase inhibitor metyrapone is able to effectively reverse the hypercortisolemia detected in human Cushing's Syndrome patients. In this current preclinical study, we investigated whether metyrapone monotherapy can also reverse the hypercortisolemia-associated increase in atherosclerotic cardiovascular disease risk. In this instance, female low-density lipoprotein receptor knockout mice fed a cholic acid-containing high cholesterol/high fat diet to induce the development of hypercorticosteronemia and atherosclerotic lesions were treated twice daily with 100 mg/kg metyrapone for 4 weeks. Metyrapone effectively protected against hypercorticosteronemia development with endpoint plasma corticosterone levels remaining 43% lower than in controls (p < 0.01). Gene expression analysis in livers and adrenals validated that glucocorticoid receptor signaling was also reduced. Importantly, metyrapone treatment did not impact plasma cholesterol levels or alter atherosclerotic plaque areas or lesional collagen contents. However, metyrapone induced significant systemic lymphocytopenia as evident from marked decreases in splenic white pulp contents and thymus weights (-48% and -41%, respectively; p < 0.001). In conclusion, we have shown that treatment with metyrapone diminishes hypercorticosteronemia without affecting atherosclerosis susceptibility in cholic acid-containing high cholesterol/high fat diet-fed low-density lipoprotein receptor knockout mice. These preclinical findings highlight that restoring plasma glucocorticoid levels to normal is not necessarily sufficient to overcome the cardiovascular co-morbidities associated with human Cushing's disease.

The impact of CYP2C19 genotype on phenoconversion by concomitant medication.

Introduction: Pharmacogenetics-informed drug prescribing is increasingly applied in clinical practice. Typically, drug metabolizing phenotypes are determined based on genetic test results, whereupon dosage or drugs are adjusted. Drug-drug-interactions (DDIs) caused by concomitant medication can however cause mismatches between predicted and observed phenotypes (phenoconversion). Here we investigated the impact of CYP2C19 genotype on the outcome of CYP2C19-dependent DDIs in human liver microsomes. Methods: Liver samples from 40 patients were included, and genotyped for CYP2C19*2, *3 and *17 variants. S-mephenytoin metabolism in microsomal fractions was used as proxy for CYP2C19 activity, and concordance between genotype-predicted and observed CYP2C19 phenotype was examined. Individual microsomes were subsequently co-exposed to fluvoxamine, voriconazole, omeprazole or pantoprazole to simulate DDIs. Results: Maximal CYP2C19 activity (Vmax) in genotype-predicted intermediate metabolizers (IMs; *1/*2 or *2/*17), rapid metabolizers (RMs; *1/*17) and ultrarapid metabolizers (UMs; *17/*17) was not different from Vmax of predicted normal metabolizers (NMs; *1/*1). Conversely, CYP2C19*2/*2 genotyped-donors exhibited Vmax rates ∼9% of NMs, confirming the genotype-predicted poor metabolizer (PM) phenotype. Categorizing CYP2C19 activity, we found a 40% concordance between genetically-predicted CYP2C19 phenotypes and measured phenotypes, indicating substantial phenoconversion. Eight patients (20%) exhibited CYP2C19 IM/PM phenotypes that were not predicted by their CYP2C19 genotype, of which six could be linked to the presence of diabetes or liver disease. In subsequent DDI experiments, CYP2C19 activity was inhibited by omeprazole (-37% ± 8%), voriconazole (-59% ± 4%) and fluvoxamine (-85% ± 2%), but not by pantoprazole (-2 ± 4%). The strength of CYP2C19 inhibitors remained unaffected by CYP2C19 genotype, as similar percental declines in CYP2C19 activity and comparable metabolism-dependent inhibitory constants (Kinact/KI) of omeprazole were observed between CYP2C19 genotypes. However, the consequences of CYP2C19 inhibitor-mediated phenoconversion were different between CYP2C19 genotypes. In example, voriconazole converted 50% of *1/*1 donors to a IM/PM phenotype, but only 14% of *1/*17 donors. Fluvoxamine converted all donors to phenotypic IMs/PMs, but *1/*17 (14%) were less likely to become PMs than *1/*1 (50%) or *1/*2 and *2/*17 (57%). Conclusion: This study suggests that the differential outcome of CYP2C19-mediated DDIs between genotypes are primarily dictated by basal CYP2C19 activity, that may in part be predicted by CYP2C19 genotype but likely also depends on disease-related factors.

Perspective: Hepatocyte-Directed Base Editing as Novel Treatment for Human Dyslipidemia-Current Status and Remaining Challenges.

Hyperlipidemia is a major risk factor for the development of atherosclerotic cardiovascular disease. Lipid-lowering drug therapies therefore still form the heart of the ongoing battle against the occurrence of cardiovascular events. However, in light of the important improvements in gene interference and editing that have been made during the last 2 decades, gene therapy-the genetic modification of cells to produce a permanent therapeutic effect-is currently employed to relief hypercholesterolemic subjects from their potential (chronic) cardiovascular disease burden. In this perspective, we review the current status regarding hepatocyte-directed base editing to treat human dyslipidemia and provide suggestions for further technological improvement.

PRMT5 inhibition induces pro-inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice.

Protein arginine methyltransferase 5 (PRMT5) controls inflammation and metabolism through modulation of histone methylation and gene transcription. Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1 ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5 mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p < 0.05) and significantly increased hepatic triglyceride levels (+50%; p < 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.

Zebrafish as outgroup model to study evolution of scavenger receptor class B type I functions.

BACKGROUND AND AIMS: Scavenger receptor class B1 (SCARB1) - also known as the high-density lipoprotein (HDL) receptor - is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 - which is important for cholesterol handling - is highly conserved across multiple vertebrates, except in zebrafish. METHODS: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1 -1 nt) and scarb1 715_716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology. RESULTS: We demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmentation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (-50 %, p < 0.001). Importantly, these alterations were independent of changes in free (wild-type 2.4 ± 0.2 µg/µl versus scarb1-/- 2.0 ± 0.1 µg/µl) as well as total (wild-type 4.2 ± 0.4 µg/µl versus scarb1-/- 4.0 ± 0.3 µg/µl) plasma cholesterol levels. Uptake of HDL in the liver of scarb1-/- zebrafish larvae was reduced (-86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient canaries, which also lack carotenoids in their plumage, similarly as scarb1-/- zebrafish, failed to show an increase in plasma free- and total cholesterol levels. CONCLUSION: Our findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution.

Time to run: Late rather than early exercise training in mice remodels the gut microbiome and reduces atherosclerosis development.

The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients.

2-Hydroxypropyl-beta-cyclodextrin Treatment Does Not Induce Atherosclerotic Lesion Regression in Western-Type Diet-Fed Apolipoprotein E Knockout Mice.

2-Hydroxypropyl-beta-cyclodextrin (2HPßCD) is able to bind and solubilize unesterified cholesterol and may therefore be able to reverse the deposition of cholesterol in macrophages within the aortic vessel wall, a hallmark of atherosclerotic cardiovascular disease. However, conflicting results regarding the potential of 2HPßCD to induce regression of established atherosclerotic lesions have been described. In the current study, we therefore also investigated the ability of 2HPßCD to stimulate cholesterol removal from macrophage foam cells in vitro and induce the regression of established atherosclerotic lesions in apolipoprotein E knockout (APOE KO) mice. In vitro studies using murine thioglycollate-elicited peritoneal macrophages verified that 2HPßCD is able to induce cholesterol efflux from macrophages in an ATP-binding cassette transporter-independent manner. Switching Western-type-diet-fed APOE KO mice with established atherosclerotic lesions back to a chow diet was associated with a reduction in the hypercholesterolemia extent and an increase in the absolute lesion size and plaque collagen-to-macrophage ratio. Importantly, parallel subcutaneous administration of 2HPßCD was not able to prevent the diet-switch-associated lesion growth or induce atherosclerosis regression. Although in our hands, 2HPßCD does effectively stimulate cellular cholesterol efflux from macrophages, we do not consider it worthwhile to further pursue 2HPßCD as therapeutic moiety in the atherosclerosis regression context.

Hypocholesterolemic phospholipid transfer protein knockout mice exhibit a normal glucocorticoid response to food deprivation.

OBJECTIVES: Glucocorticoids, adrenal-derived steroid hormones, facilitate the physiological response to stress. High-density lipoproteins (HDL) are considered the primary source of cholesterol used for glucocorticoid synthesis in mice. Phospholipid transfer protein (PLTP) is a key player in HDL formation. In the current study we tested the hypothesis that HDL deficiency associated with genetic lack of PLTP negatively impacts the adrenal steroid function. METHODS: We determined the glucocorticoid response to overnight food deprivation stress and the adrenal lipid and genetic phenotype of wild-type and PLTP knockout mice. RESULTS: Basal plasma corticosterone levels, adrenal weights, and adrenocortical neutral lipid stores were not different between wild-type and PLTP knockout mice. Strikingly, plasma corticosterone levels were also equally high in the two groups of mice under fasting conditions (two-way ANOVA genotype effect: P>0.05). However, compensatory mechanisms were active to overcome adrenal lipid depletion, since gene expression levels of cholesterol synthesis, acquisition and mobilization proteins were ~2-fold higher in PLTP knockout adrenals versus wild-type adrenals. In support of an overall similar glucocorticoid stress response, hepatic relative mRNA expression levels of the glucocorticoid receptor target/glucocorticoid-sensitive genes PEPCK, ANGPTL4, FGF21, TDO2 and HMGCS2 were also not different. CONCLUSIONS: We have shown that hypocholesterolemic PLTP knockout mice exhibit a normal glucocorticoid response to food deprivation. These novel data (1) highlight that the effect of HDL deficiency on adrenal glucocorticoid output in mice is model dependent and (2) imply that other (lipoprotein) cholesterol sources than HDL can also generate the pool utilized by adrenocortical cells to synthesize glucocorticoids.

Epidermal 1-O-acylceramides appear with the establishment of the water permeability barrier in mice and are produced by maturating keratinocytes.

1-O-Acylceramides (1-OACs) have a fatty acid esterified to the 1-hydroxyl of the sphingosine head group of the ceramide, and recently we identified these lipids as natural components of human and mouse epidermis. Here we show epidermal 1-OACs arise shortly before birth during the establishment of the water permeability barrier in mice. Fractionation of human epidermis indicates 1-OACs concentrate in the stratum corneum. During in vitro maturation into reconstructed human epidermis, human keratinocytes dramatically increase 1-OAC levels indicating they are one source of epidermal 1-OACs. In search of potential enzymes responsible for 1-OAC synthesis in vivo, we analyzed mutant mice with deficiencies of ceramide synthases (Cers2, Cers3, or Cers4), diacylglycerol acyltransferases (Dgat1 or Dgat2), elongase of very long fatty acids 3 (Elovl3), lecithin cholesterol acyltransferase (Lcat), stearoyl-CoA desaturase 1 (Scd1), or acidic ceramidase (Asah1). Overall levels of 1-OACs did not decrease in any mouse model. In Cers3 and Dgat2-deficient epidermis they even increased in correlation with deficient skin barrier function. Dagt2 deficiency reshapes 1-OAC synthesis with an increase in 1-OACs with N-linked non-hydroxylated fatty acids and a 60% decrease compared to control in levels of 1-OACs with N-linked hydroxylated palmitate. As none of the single enzyme deficiencies we examined resulted in a lack of 1-OACs, we conclude that either there is functional redundancy in forming 1-OAC and more than one enzyme is involved, and/or an unknown acyltransferase of the epidermis performs the final step of 1-OAC synthesis, the implications of which are discussed.

Stabilin 1 and 2 are important regulators for cellular uptake of apolipoprotein B-containing lipoproteins in zebrafish.

BACKGROUND AND AIMS: Scavenger receptors form a superfamily of membrane-bound receptors that bind and internalize different types of ligands, including pro-atherogenic oxidized low-density lipoproteins (oxLDLs). In vitro studies have indicated a role for the liver sinusoidal endothelial cell receptors stabilin 1 (stab1) and 2 (stab2) in oxLDL clearance. In this study, we evaluated the potential role of stab1 and stab2 in lipoprotein uptake in zebrafish, an upcoming model for studying cholesterol metabolism and atherosclerosis. METHODS: Lipoproteins were injected in the duct of Cuvier of wild-type (ABTL) or stab1 and stab2 mutant (stab1-/-stab2-/-) zebrafish larvae at 3 days post-fertilization. To examine the effect of stabilin deficiency on lipoprotein and cholesterol metabolism, zebrafish larvae were challenged with a high cholesterol diet (HCD; 4% w/w) for 10 days. RESULTS: Lipoprotein injections showed impaired uptake of both LDL and oxLDL into the vessel wall of caudal veins of stab1-/-stab2-/- zebrafish, which was paralleled by redistribution to tissue macrophages. Total body cholesterol levels did not differ between HCD-fed stab1-/-stab2-/- and ABTL zebrafish. However, stab1-/-stab2-/- larvae exhibited 1.4-fold higher mRNA expression levels of ldlra involved in (modified) LDL uptake, whereas the expression levels of scavenger receptors scarb1 and cd36 were significantly decreased. CONCLUSIONS: We have shown that stabilins 1 and 2 have an important scavenging function for apolipoprotein B-containing lipoproteins in zebrafish and that combined deficiency of these two proteins strongly upregulates the clearance of lipoproteins by macrophages within the caudal vein. Our current study highlights the use of zebrafish as model to study lipoprotein metabolism and liver sinusoidal endothelial cell function.

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice.

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.

Apolipoprotein A1 deficiency in mice primes bone marrow stem cells for T cell lymphopoiesis.

The bone marrow has emerged as a potentially important target in cardiovascular disease as it generates all leukocytes involved in atherogenesis. In the current study, we evaluated whether a change in bone marrow functionality underlies the increased atherosclerosis susceptibility associated with high-density lipoprotein (HDL) deficiency. We found that HDL deficiency in mice due to the genetic lack of hepatocyte-derived apolipoprotein A1 (APOA1) was associated with an increase in the Lin-Sca-1+Kit+ (LSK) bone marrow stem cell population and lymphoid-primed multipotent progenitor numbers, which translated into a higher production and systemic flux of T cell subsets. In accordance with APOA1 deficiency-associated priming of stem cells to increase T lymphocyte production, atherogenic diet-fed low-density lipoprotein receptor knockout mice transplanted with bone marrow from APOA1-knockout mice displayed marked lymphocytosis as compared to wild-type bone marrow recipients. However, atherosclerotic lesion sizes and collagen contents were similar in the two groups of bone marrow recipients. In conclusion, systemic lack of APOA1 primes bone marrow stem cells for T cell lymphopoiesis. Our data provide novel evidence for a regulatory role of HDL in bone marrow functioning in normolipidemic mice.

PRMT4 inhibitor TP-064 impacts both inflammatory and metabolic processes without changing the susceptibility for early atherosclerotic lesions in male apolipoprotein E knockout mice.

BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease is a metabolic and inflammatory disorder. In vitro studies have suggested that protein arginine methyltransferase 4 (PRMT4) may act as a transcriptional coactivator to modulate inflammatory and metabolic processes. Here we investigated the potential anti-atherogenic effect of PRMT4 inhibitor TP-064 in vivo. METHODS: Male apolipoprotein E knockout mice fed a high cholesterol/high fat Western-type diet were intraperitoneally injected three times a week with 2.5 mg/kg (low dose) or 10 mg/kg (high dose) TP-064 or with DMSO control. RESULTS: TP-064 induced a dose-dependent decrease in lipopolysaccharide-induced ex vivo blood monocyte Tnfα secretion (p < 0.05 for trend) in the context of unchanged blood monocyte concentrations and neutrophilia induction (p < 0.01 for trend). A dose-dependent decrease in gonadal white adipose tissue expression levels of PPARγ target genes was detected, which translated into a reduced body weight gain after high dose TP-064 treatment (p < 0.05). TP-064 treatment also dose-dependently downregulated gene expression of the glycogen metabolism related protein G6pc in the liver (p < 0.001 for trend). In addition, a trend towards lower plasma insulin and higher blood glucose levels was observed, which was paralleled by a reduction in hepatic mRNA expression levels of the insulin-responsive genes Fasn (-55%; p < 0.001) and Gck (-47%; p < 0.001) in high dose-treated mice. Plasma triglyceride levels were reduced by high dose TP-064 treatment (-30%; p < 0.05). However, no change was observed in the size or composition of aortic root atherosclerotic lesions. CONCLUSIONS: The PRMT4 inhibitor TP-064 impacts both inflammatory and metabolic processes without changing atherosclerosis susceptibility of male apolipoprotein E knockout mice.