Deficiency of B vitamins leads to cholesterol-independent atherogenic transformation of the aorta.

Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors, including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in vivo models of atherosclerosis that resemble the human situation. Here, we have developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development, even at low balloon pressure. Our automated system helped to glean vital details of cholesterol-independent changes in the aortic wall of balloon-injured rabbits. We show that deficiency of B vitamins, which are required for homocysteine degradation, leads to atherogenic transformation of the aorta resulting in accumulation of macrophages and lipids, impairment of its biomechanical properties and disorganization of aortic collagen/elastin in the absence of hypercholesterolemia. A combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity compared to any single condition. Our findings suggest that deficiency of B vitamins leads to atherogenic transformation of the aorta even in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.

Phosphatidylethanolamine N-Methyltransferase Knockout Modulates Metabolic Changes in Aging Mice.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated with longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as the brain and kidneys during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage, and energy consumption, we employed NMR-based metabolomics to study the liver, plasma, intestine (duodenum, jejunum, and ileum), brown/white adipose tissues (BAT and WAT), and skeletal muscle of young (9-10 weeks) and old (91-132 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. A deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to the WT mice, whereas the metabolome of the jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of the metabolome during the aging of the liver, WAT, duodenum, and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously underexplored, critical role in both aging and energy metabolism.

Tissue-Specific Landscape of Metabolic Dysregulation during Ageing.

The dysregulation of cellular metabolism is a hallmark of ageing. To understand the metabolic changes that occur as a consequence of the ageing process and to find biomarkers for age-related diseases, we conducted metabolomic analyses of the brain, heart, kidney, liver, lung and spleen in young (9-10 weeks) and old (96-104 weeks) wild-type mice [mixed genetic background of 129/J and C57BL/6] using NMR spectroscopy. We found differences in the metabolic fingerprints of all tissues and distinguished several metabolites to be altered in most tissues, suggesting that they may be universal biomarkers of ageing. In addition, we found distinct tissue-clustered sets of metabolites throughout the organism. The associated metabolic changes may reveal novel therapeutic targets for the treatment of ageing and age-related diseases. Moreover, the identified metabolite biomarkers could provide a sensitive molecular read-out to determine the age of biologic tissues and organs and to validate the effectiveness and potential off-target effects of senolytic drug candidates on both a systemic and tissue-specific level.

Metabolomic Profiles of Mouse Tissues Reveal an Interplay between Aging and Energy Metabolism.

Energy metabolism, including alterations in energy intake and expenditure, is closely related to aging and longevity. Metabolomics studies have recently unraveled changes in metabolite composition in plasma and tissues during aging and have provided critical information to elucidate the molecular basis of the aging process. However, the metabolic changes in tissues responsible for food intake and lipid storage have remained unexplored. In this study, we aimed to investigate aging-related metabolic alterations in these tissues. To fill this gap, we employed NMR-based metabolomics in several tissues, including different parts of the intestine (duodenum, jejunum, ileum) and brown/white adipose tissues (BAT, WAT), of young (9-10 weeks) and old (96-104 weeks) wild-type (mixed genetic background of 129/J and C57BL/6) mice. We, further, included plasma and skeletal muscle of the same mice to verify previous results. Strikingly, we found that duodenum, jejunum, ileum, and WAT do not metabolically age. In contrast, plasma, skeletal muscle, and BAT show a strong metabolic aging phenotype. Overall, we provide first insights into the metabolic changes of tissues essential for nutrient uptake and lipid storage and have identified biomarkers for metabolites that could be further explored, to study the molecular mechanisms of aging.

Global analysis of protein arginine methylation.

Quantitative information about the levels and dynamics of post-translational modifications (PTMs) is critical for an understanding of cellular functions. Protein arginine methylation (ArgMet) is an important subclass of PTMs and is involved in a plethora of (patho)physiological processes. However, because of the lack of methods for global analysis of ArgMet, the link between ArgMet levels, dynamics, and (patho)physiology remains largely unknown. We utilized the high sensitivity and robustness of nuclear magnetic resonance (NMR) spectroscopy to develop a general method for the quantification of global protein ArgMet. Our NMR-based approach enables the detection of protein ArgMet in purified proteins, cells, organoids, and mouse tissues. We demonstrate that the process of ArgMet is a highly prevalent PTM and can be modulated by small-molecule inhibitors and metabolites and changes in cancer and during aging. Thus, our approach enables us to address a wide range of biological questions related to ArgMet in health and disease.

The anticoagulant effects of ethyl pyruvate in whole blood samples.

BACKGROUND: Ethyl pyruvate (EP), the ethyl ester of pyruvate, has proven antiinflammatory and antioxidative properties. Additionally, anticoagulant properties have been suggested recently. EP, therefore, is a potentially antiatherosclerotic drug. We aimed to investigate whether EP possesses antiplatelet and anticoagulant properties particularly in the physiological environment of whole blood. METHODS: We investigated the effects of increasing concentrations of EP on platelet function, on the course of clot development, and on standard coagulation times. Additionally, clot ultrastructure using scanning electron microscopy was analysed. RESULTS: EP exerted significant antiplatelet actions: i) Impedance aggregometry amplitudes (11.7 ± 3.0 ohm, 0 µg/mL EP) dose dependently decreased (7.8 ± 3.1 ohm, 1000 µg/mL EP; -33.3%). ATP exocytosis (0.87 ± 0.24 nM, 0 µg/mL EP) measured by the luminiscent method dose-dependently decreased (0.56 ± 0.14 nM, 1000 µg/mL; -35.6%). ii) Closure times (104.4 ± 23.8 s, 0 µg/mL EP) using the Platelet function analyzer were dose-dependently prolonged (180.5 ± 82.5 s, 1000 µg/mL EP; +72.9%) using membranes coated with collagen/ADP. iii) Surface coverage (15.9 ± 5.1%, 0 µg/mL EP) dose-dependently decreased (9.0 ± 3.7%, 1000 µg/mL EP; -43.4%) using the Cone and Platelet analyzer. EP also exerted significant anticoagulant actions: Coagulation times (177.9 ± 37.8, 0 µg/mL EP) evaluated by means of thrombelastometry were dose-dependently prolonged (212.8 ± 57.7 s, 1000 µg/mL EP; +19.6%). Activated partial thromboplastin times (31.5 ± 1.8 s, 0 µg/mL EP) were dose-dependently prolonged (35.6 ± 2.3 s, 1000 µg/mL EP; +13.0%). Prothrombin times (0.94 ± 0.02 INR, 0 µg/mL EP) were dose-dependently prolonged (1.09 ± 0.04 INR, 1000 µg/mL EP; +16.0%). CONCLUSION: We found that EP possesses antiplatelet and anticoagulant properties in whole blood. Together with its proven anti-inflammatory and antioxidative properties, EP is a potentially antiatherogenic drug.

Endothelial lipase increases eNOS activating capacity of high-density lipoprotein.

Endothelial lipase (EL) changes structural and functional properties of high-density lipoprotein (HDL). HDL is a relevant modulator of endothelial nitric oxide synthase (eNOS) activity, but the effect of EL on HDL induced eNOS-activation has not yet been investigated. Here, we examined the impact of EL-modified HDL (EL-HDL) on eNOS activity, subcellular trafficking, and eNOS- dependent vasorelaxation. EL-HDL and empty virus (EV)-HDL as control were isolated from human serum incubated with EL-overexpressing or EV infected HepG2 cells. EL-HDL exhibited higher capacity to induce eNOS phosphorylation at Ser1177 and eNOS activity in EA.hy 926 cells, as well as eNOS-dependent vasorelaxation of mouse aortic rings compared to control HDL. As revealed by confocal and structured illumination-microscopy EL-HDL-driven induction of eNOS was accompanied by an increased eNOS-GFP targeting to the plasma membrane and a lower eNOS-GFP colocalization with Golgi and mitochondria. Widefield microscopy of filipin stained cells revealed that EL-HDL lowered cellular free cholesterol (FC) and as found by thin-layer chromatography increased cellular cholesterol ester (CE) content. Additionally, cholesterol efflux capacity, acyl-coenzyme A: cholesterol acyltransferase activity, and HDL particle uptake were comparable between EL-HDL and control HDL. In conclusion, EL increases eNOS activating capacity of HDL, a phenomenon accompanied by an enrichment of the plasma membrane eNOS pool, a decreased cell membrane FC and increased cellular CE content.

Simvastatin Efficiently Lowers Small LDL-IgG Immune Complex Levels: A Therapeutic Quality beyond the Lipid-Lowering Effect.

We investigated a polyethylene glycol non-precipitable low-density lipoprotein (LDL) subfraction targeted by IgG and the influence of statin therapy on plasma levels of these small LDL-IgG-immune complexes (LDL-IgG-IC). LDL-subfractions were isolated from 6 atherosclerotic subjects and 3 healthy individuals utilizing iodixanol density gradient ultracentrifugation. Cholesterol, apoB and malondialdehyde (MDA) levels were determined in each fraction by enzymatic testing, dissociation-enhanced lanthanide fluorescence immunoassay and high-performance liquid chromatography, respectively. The levels of LDL-IgG-IC were quantified densitometrically following lipid electrophoresis, particle size distribution was assessed with dynamic light scattering and size exclusion chromatography. The influence of simvastatin (40 mg/day for three months) on small LDL-IgG-IC levels and their distribution among LDL-subfractions (salt gradient separation) were investigated in 11 patients with confirmed coronary artery disease (CAD). We demonstrate that the investigated LDL-IgG-IC are small particles present in atherosclerotic patients and healthy subjects. In vitro assembly of LDL-IgG-IC resulted in particle density shifts indicating a composition of one single molecule of IgG per LDL particle. Normalization on cholesterol levels revealed MDA values twice as high for LDL-subfractions rich in small LDL-IgG-IC if compared to dominant LDL-subfractions. Reactivity of affinity purified small LDL-IgG-IC to monoclonal antibody OB/04 indicates a high degree of modified apoB and oxidative modification. Simvastatin therapy studied in the CAD patients significantly lowered LDL levels and to an even higher extent, small LDL-IgG-IC levels without affecting their distribution. In conclusion simvastatin lowers levels of small LDL-IgG-IC more effectively than LDL-cholesterol and LDL-apoB levels in atherosclerotic patients. This antiatherogenic effect may additionally contribute to the known beneficial effects of this drug in the treatment of atherosclerosis.

Increased phosphatidylethanolamine N-methyltransferase gene expression in non-small-cell lung cancer tissue predicts shorter patient survival.

Lipid mobilization is of great importance for tumor growth and studies have suggested that cancer cells exhibit abnormal choline phospholipid metabolism. In the present study, we hypothesized that phosphatidylethanolamine N-methyltransferase (PEMT) gene expression is increased in non-small-cell lung cancer (NSCLC) tissues and that increased gene expression acts as a predictor of shorter patient survival. Forty-two consecutive patients with resected NSCLC were enrolled in this study. Paired samples of lung cancer tissues and adjacent non-cancer lung tissues were collected from resected specimens for the estimation of PEMT expression. SYBR Green-based real-time polymerase chain reaction was used for quantification of PEMT mRNA in lung cancer tissues. Lipoprotein lipase (LPL) and fatty acid synthase (FASN) activities had already been measured in the same tissues. During a four-year follow-up, 21 patients succumbed to tumor progression. One patient did not survive due to non-cancer reasons and was not included in the analysis. Cox regression analysis was used to assess the prognostic value of PEMT expression. Our findings show that elevated PEMT expression in the cancer tissue, relative to that in the adjacent non-cancer lung tissue, predicts shorter patient survival independently of standard prognostic factors and also independently of increased LPL or FASN activity, the two other lipid-related predictors of shorter patient survival. These findings suggest that active phosphatidylcholine and/or choline metabolism are essential for tumor growth and progression.

In vitro oxidation of LDL by ozone.

Recent studies suggest that ozone is present in atherosclerotic lesions. Since these lesions are characterized by a dramatic accumulation of low-density lipoprotein (LDL), we aimed to investigate whether ozone is capable of oxidizing LDL, thereby rendering this lipoprotein atherogenic. Lipid hydroperoxide (LPO) concentrations and thiobarbituric acid reactive substances (TBARS) were measured to assess the oxidative status of the lipid part of LDL. Relative electrophoretic mobility (REM) and oxidation-specific immune epitopes were measured to assess the oxidative status of the protein part (apoB) of the LDL particle. Ozone turned out to be a potent oxidant of LDL. LPO concentrations, TBARS, REM, and oxidation-specific immune epitopes significantly increased upon ozonization. Our results suggest that ozonization of LDL may be a novel pathway which supports atherogenesis. Ozone is capable of oxidizing the lipid part of LDL, followed by immediate oxidation of the protein part of LDL, rendering the lipoprotein atherogenic.

Sequential synthesis and methylation of phosphatidylethanolamine promote lipid droplet biosynthesis and stability in tissue culture and in vivo.

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.

Role of hepatic phospholipids in development of liver injury in Mdr2 (Abcb4) knockout mice.

BACKGROUND/AIMS: Multidrug resistance protein 2 (Abcb4) gene knockout mice (Mdr2(-/-)) lack phosphatidylcholine (PC) excretion into bile and spontaneously develop sclerosing cholangitis, biliary fibrosis and hepatocellular carcinomas. We therefore aimed to test whether formation and hepatic retention of abnormal PC metabolites contribute to the pathogenesis of liver injury in Mdr2(-/-) mice. METHODS: Mdr2(-/-) mice were either fed a diet supplemented with soybean lecithin 2.5% w/w [phosphatidylcholine-enriched diet (PCD), to increase hepatic PC content] or a choline-deficient diet (CDD, to reduce hepatic PC content) for 4 weeks; controls received chow with energy and nutrient content equivalent to PCD and CDD. Serum liver tests, liver histology, markers of fibrosis, cholangiocyte activation, cell proliferation and thin-layer chromatography for phospholipid (PL) composition were carried out. RESULTS: PCD decreased serum alkaline phosphatase and total bilirubin levels compared with controls, while liver histology as well as hepatic hydroxyproline content as markers of liver fibrosis did not differ among groups. Both PCD and CDD decreased hepatocellular proliferation compared with controls. Hepatic, serum and biliary PLs remained unchanged despite dietary manipulations and no potentially toxic PL metabolites were detected. CONCLUSIONS: Mdr2(-/-) mice maintain stable hepatic, serum and biliary PL metabolism in response to dietary PC manipulations. Our findings therefore suggest that liver injury in Mdr2(-/-) mice is not due to formation of toxic PL metabolites.

Compound heterozygosity (G71R/R140H) in the lecithin:cholesterol acyltransferase (LCAT) gene results in an intermediate phenotype between LCAT-deficiency and fish-eye disease.

The esterification of free cholesterol (FC) in plasma, catalyzed by the enzyme lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), is a key process in lipoprotein metabolism. The resulting cholesteryl esters (CE) represent the main core lipids of low (LDL) and high density lipoproteins (HDL). Primary (familial) LCAT-deficiency (FLD) is a rare autosomal recessive genetic disease caused by the complete or near absence of LCAT activity. In fish-eye disease (FED), residual LCAT activity is still detectable. Here, we describe a 32-year-old patient with corneal opacity, very low LCAT activity, reduced amounts of CE (low HDL-cholesterol level), and elevated triglyceride (TG) values. The lipoprotein pattern was abnormal with regard to lipoprotein composition and concentration, but distinct lipoprotein classes were still present. Despite of typical features of glomerular proteinuria, creatinine clearance was normal. DNA sequencing and restiction fragment analyses revealed two separate mutations in the patient's LCAT gene: a previously described G to A transition in exon 4 converting Arg140 to His, inherited from his mother, and a novel G to C transversion in exon 2 converting Gly71 to Arg, inherited from his father, indicating that M.P. was a compound heterozygote. Determination of enzyme activities of recombinant LCAT proteins obtained upon transfection of COS-7 cells with plasmids containing G71R-LCAT or wild-type LCAT cDNA revealed very low alpha- and absence of beta-LCAT activity for the G71R mutant. The identification of the novel G71R LCAT mutation supports the proposed molecular model for the enzyme implying that the "lid" domain at residues 50-74 is involved in enzyme:substrate interaction. Our data are in line with the hypothesis that a key event in the etiology of FLD is the loss of distinct lipoprotein fractions.

Heterozygous missense mutations in BSCL2 are associated with distal hereditary motor neuropathy and Silver syndrome.

Distal hereditary motor neuropathy (dHMN) or distal spinal muscular atrophy (OMIM #182960) is a heterogeneous group of disorders characterized by an almost exclusive degeneration of motor nerve fibers, predominantly in the distal part of the limbs. Silver syndrome (OMIM #270685) is a rare form of hereditary spastic paraparesis mapped to chromosome 11q12-q14 (SPG17) in which spasticity of the legs is accompanied by amyotrophy of the hands and occasionally also the lower limbs. Silver syndrome and most forms of dHMN are autosomal dominantly inherited with incomplete penetrance and a broad variability in clinical expression. A genome-wide scan in an Austrian family with dHMN-V (ref. 4) showed linkage to the locus SPG17, which was confirmed in 16 additional families with a phenotype characteristic of dHMN or Silver syndrome. After refining the critical region to 1 Mb, we sequenced the gene Berardinelli-Seip congenital lipodystrophy (BSCL2) and identified two heterozygous missense mutations resulting in the amino acid substitutions N88S and S90L. Null mutations in BSCL2, which encodes the protein seipin, were previously shown to be associated with autosomal recessive Berardinelli-Seip congenital lipodystrophy (OMIM #269700). We show that seipin is an integral membrane protein of the endoplasmic reticulum (ER). The amino acid substitutions N88S and S90L affect glycosylation of seipin and result in aggregate formation leading to neurodegeneration.