Metabolic syndrome associates with left atrial dysfunction.

BACKGROUND AND AIMS: Obesity and metabolic syndrome (MetS) are risk factors of atrial fibrillation (AF), but limited data exist on their effect on left atrial (LA) function. The aim of the study was to evaluate the effects of cardiac, hepatic and intra-abdominal ectopic fat depots and cardiometabolic risk factors on LA function in non-diabetic male subjects. METHODS AND RESULTS: Myocardial and hepatic triglyceride contents were measured with 1.5T 1H-magnetic resonance spectroscopy and LA and left ventricular function, visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), epicardial and pericardial fat by magnetic resonance imaging (MRI) in 33 men with MetS and 40 men without MetS. LA volumes were assessed using a novel three-chamber orientation based MRI approach. LA ejection fraction (EF) was lower in MetS patients than in the control group (44 ± 7.7% in MetS vs. 49 ± 8.6% in controls, p = 0.013) without LA enlargement, indicating LA dysfunction. LA EF correlated negatively with waist circumference, body mass index, SAT, VAT, fasting serum insulin, and homeostasis model assessment of insulin resistance index, and positively with fasting serum high-density lipoprotein cholesterol. VAT was the best predictor of reduced LA EF. CONCLUSIONS: MetS associates with subclinical LA dysfunction. Multiple components of MetS are related to LA dysfunction, notably visceral obesity and insulin resistance. Further studies are needed to elucidate the role of mechanical atrial remodeling in the development of AF.

Biomarkers and prediction of myocardial triglyceride content in non-diabetic men.

BACKGROUND AND AIMS: Lipid oversupply to cardiomyocytes or decreased utilization of lipids leads to cardiac steatosis. We aimed to examine the role of different circulating metabolic biomarkers as predictors of myocardial triglyceride (TG) content in non-diabetic men. METHODS AND RESULTS: Myocardial and hepatic TG contents were measured with 1.5 T magnetic resonance (MR) spectroscopy, and LV function, visceral adipose tissue (VAT), abdominal subcutaneous tissue (SAT), epicardial and pericardial fat by MR imaging in 76 non-diabetic men. Serum concentration of circulating metabolic biomarkers [adiponectin, leptin, adipocyte-fatty acid binding protein 4 (A-FABP 4), resistin, and lipocalin-2] including ß-hydroxybuturate (ß-OHB) were measured. Subjects were stratified by tertiles of myocardial TG into low, moderate, and high myocardial TG content groups. Concentrations of ß-OHB were lower (p = 0.003) and serum levels of A-FABP 4 were higher (p < 0.001) in the group with high myocardial TG content compared with the group with low myocardial TG content. ß-OHB was negatively correlated with myocardial TG content (r = -0.316, p = 0.006), whereas A-FABP 4 was not correlated with myocardial TG content (r = 0.192, p = 0.103). In multivariable analyses ß-OHB and plasma glucose levels were the best predictors of myocardial TG content independently of VAT and hepatic TG content. The model explained 58.8% of the variance in myocardial TG content. CONCLUSION: Our data showed that ß-OHB and fasting glucose were the best predictors of myocardial TG content in non-diabetic men. These data suggest that hyperglycemia and alterations in lipid oxidation may be associated with cardiac steatosis in humans.

Electrocardiographic changes associated with insulin resistance.

BACKGROUND AND AIM: Cardiac steatosis has been related to increased risk of heart disease. We investigated the association between cardiac steatosis, electrocardiographic (ECG) abnormalities, and individual components of the metabolic syndrome (MetS). METHODS AND RESULTS: A 12-lead ECG and laboratory data were examined in 31 men with the MetS and in 38 men without the MetS. Myocardial triglyceride (MTG) content was measured with 1.5 T magnetic resonance (MR) spectroscopy and epicardial and pericardial fat by MR imaging. MTG content, epicardial and pericardial fat depots were higher in men with the MetS compared with subjects without the MetS (p < 0.001). The heart rate was increased (p < 0.001), the PR interval was longer (p < 0.044), the frontal plane QRS axis shifted to the left (p < 0.001), and the QRS voltage (p < 0.001) was lower in subjects with the MetS. The frontal plane QRS axis and the QRS voltage were inversely correlated with MTG content, waist circumference (WC), body mass index (BMI), TGs, and fasting blood glucose. High-density lipoprotein cholesterol correlated positively and measures of insulin resistance negatively with the QRS voltage. MTG content and hypertriglyceridemia were determinants of the frontal plane QRS and WC and hyperglycemia were predictors of the QRS voltage. CONCLUSION: The MetS and cardiac steatosis appear to associate with multiple changes on 12-lead ECG. The frontal plane QRS axis is shifted to the left and the QRS voltage is lower in subjects with the MetS. Standard ECG criteria may underestimate the presence of left ventricular hypertrophy in obese subjects with cardiometabolic risk factors.

Outcome of ST-elevation myocardial infarction versus non-ST-elevation acute coronary syndrome treated with titanium-nitride-oxide-coated versus everolimus-eluting stents: insights from the BASE-ACS trial.

AIM: The BASE-ACS trial demonstrated an outcome of titanium-nitride-oxide-coated bioactive stents (BAS) that was statistically non-inferior to that of everolimus-eluting stents (EES) at 12-month follow-up, in patients presenting with acute coronary syndrome (ACS) who underwent early percutaneous coronary intervention (PCI). We explored a post-hoc analysis of the 12-month outcome of the BASE-ACS trial in the subgroup of patients with ST-elevation myocardial infarction (STEMI) versus non-ST-elevation ACS (non-STEACS). METHODS: A total of 827 patients with ACS (321 STEMI) were randomly assigned to receive either BAS or EES. The primary endpoint was a composite of cardiac death, non-fatal myocardial infarction (MI) and ischemia-driven target lesion revascularization (TLR) at 12-month follow-up. RESULTS: The 12-month cumulative incidence of the primary endpoint was similar between the two subgroups (9% versus 9.5%, in STEMI versus non-STEACS patients respectively, P=0.90). The 12-month rate of cardiac death was significantly higher in the STEMI subgroup as compared with the non-STEACS subgroup (2.8 versus 0.6%, respectively, P=0.01). However, the rates of non-fatal MI, ischemia-driven TLR, definite stent thrombosis, and non-cardiac death were all statistically matched between the two subgroups (P>0.05 for all). CONCLUSION: In the current post-hoc analysis of the BASE-ACS trial based on the infarction type, the 12-month outcome of patients who underwent early PCI for ACS was slightly worse in the setting of STEMI as compared with non-STEACS, as reflected by a significantly higher rate of cardiac death.

Myeloperoxidase and hypochlorite, but not copper ions, oxidize heparin-bound LDL particles and release them from heparin.

A key factor in atherosclerosis is the retention of low density lipoprotein (LDL) in the extracellular matrix of the arterial intima, where it binds to the negatively charged glycosaminoglycan chains of proteoglycans. Oxidation may lead to modification of the lysine residues of apolipoprotein B-100 of LDL, which normally mediate the binding of LDL to glycosaminoglycans. Here, we studied whether various modes of oxidation can release LDL from heparin, a glycosaminoglycan with a strong negative charge, in vitro. We found that copper ions were unable to oxidize heparin-bound LDL particles because of their redox inactivation by the glycosaminoglycans. In contrast, myeloperoxidase and hypochlorite, a product of myeloperoxidase, were able to oxidize heparin-bound LDL, and this oxidation led to the release of the oxidized particles from heparin. When the released LDL particles were compared with the residual heparin-bound LDL particles, the released particles were more electronegative and contained more modified lysine residues than did the particles that remained bound. Because human atherosclerotic lesions contain catalytically active myeloperoxidase and (lipo)proteins modified by hypochlorite, the results suggest that myeloperoxidase-secreting monocytes/macrophages in the arterial intima can oxidize and extract LDL from the extracellular matrix with ensuing uptake by the macrophages of the oxidized and released LDL, with eventual formation of foam cells.

TNFalpha down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis.

The cytokine TNFalpha is known to be secreted by testicular germ cells. However, its effect on maturing germ cells is unknown, and its role in the regulation of spermatogenesis is unclear. Here we aimed at characterizing the effects of TNFalpha on germ cell survival in the human testis. We found that TNFalpha effectively and dose-dependently inhibited germ cell apoptosis, which was induced in vitro by incubating segments of human seminiferous tubules under serum-free culture conditions. EMSAs indicated increased activity of nuclear factor kappaB in seminiferous tubules cultured under apoptosis-inducing conditions. However, we did not observe any significant effect of TNFalpha on the activation of this transcription factor, which is often considered to be a mediator of TNFalpha-induced survival signals. As the expression of the TNF receptor protein in the human seminiferous epithelium was predominantly found in the Sertoli cells, the antiapoptotic effect of TNFalpha is probably mediated via these somatic cells. Interestingly, expression of the Fas ligand, a known inductor of testicular apoptosis, was down-regulated by TNFalpha. Thus, in the seminiferous tubules, germ cell-derived TNFalpha may regulate the level of the Fas ligand and thereby control physiological germ cell apoptosis.

Lipolysis of LDL by human secretory phospholipase A(2) induces particle fusion and enhances the retention of LDL to human aortic proteoglycans.

The first morphological sign of atherogenesis is the accumulation of extracellular lipid droplets in the proteoglycan-rich subendothelial layer of the arterial intima. Secretory nonpancreatic phospholipase A(2) (snpPLA(2)), an enzyme capable of lipolyzing LDL particles, is found in the arterial extracellular matrix and in contact with the extracellular lipid droplets. We have recently shown that in the presence of heparin, lipolysis of LDL with bee venom PLA(2) induces aggregation and fusion of the particles. Here, we studied the effect of human snpPLA(2) on the integrity of LDL particles and on their interaction with human aortic proteoglycans. In addition, the capacity of the proteoglycans to retain PLA(2)-lipolyzed LDL particles was tested in a microtiter well assay. We found that lipolysis of LDL induced fusion of proteoglycan-bound LDL particles, which increased their binding strength to the proteoglycans. Moreover, lipolysis of LDL with snpPLA(2) under physiological salt and albumin concentrations induced a 3-fold increase in the amount of LDL bound to proteoglycans. The results imply a role for PLA(2) in the retention and accumulation of LDL to the proteoglycan matrix in atherosclerosis.

Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles.

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH(3))(3) resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.

Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions.

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.

Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL.

Low density lipoprotein (LDL) particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. In the process of atherogenesis these particles are modified and they accumulate in the arterial wall. Although the composition and overall structure of the LDL particles is well known, the fundamental molecular interactions and their impact on the structure of LDL particles are not well understood. Here, the existing pieces of structural information on LDL particles are combined with computer models of the individual molecular components to give a detailed structural model and visualization of the particles. Strong evidence is presented in favor of interactions between LDL lipid constituents that lead to specific domain formation in the particles. A new three-layer model, which divides the LDL particle into outer surface, interfacial layer, and core, and which is capable of explaining some seemingly contradictory interpretations of molecular interactions in LDL particles, is also presented. A new molecular interaction model for the beta-sheet structure and phosphatidylcholine headgroups is introduced and an overall view of the tertiary structure of apolipoprotein B-100 in the LDL particles is presented. This structural information is also utilized to understand and explain the molecular characteristics and interactions of modified, atherogenic LDL particles.

Modified LDL - trigger of atherosclerosis and inflammation in the arterial intima.

Atherosclerosis is characterized by chronic inflammation of an injured intima. The pathological processes are initiated by accumulation of morphologically distinct, modified forms of LDL, and followed by cellular infiltration and foam cell formation. Activated intimal cells secrete enzymes and agents capable of modifying LDL, and the modified lipids of LDL, in turn, are able to activate intimal cells and to trigger various inflammatory signals. These processes can initiate and maintain a vicious circle in the intima and lead to lesion progression. In this review, we focus on the LDL modifications relevant to the initial lipid accumulation and discuss their pro-inflammatory effects.

Lipoprotein lipase (LPL) strongly links native and oxidized low density lipoprotein particles to decorin-coated collagen. Roles for both dimeric and monomeric forms of LPL.

Low density lipoprotein (LDL) and oxidized LDL are associated with collagen in the arterial intima, where the collagen is coated by the small proteoglycan decorin. When incubated in physiological ionic conditions, decorin-coated collagen bound only small amounts of native and oxidized LDL, the interaction being weak. When decorin-coated collagen was first allowed to bind lipoprotein lipase (LPL), binding of native and oxidized LDL increased dramatically (23- and 7-fold, respectively). This increase depended on strong interactions between LPL that was bound to the glycosaminoglycan chains of the collagen-bound decorin and native and oxidized LDL (kDa 12 and 5.9 nM, respectively). To distinguish between binding to monomeric (inactive) and dimeric (catalytically active) forms of LPL, affinity chromatography on heparin columns was conducted, which showed that native LDL bound to the monomeric LPL, whereas oxidized LDL, irrespective of the type of modification (Cu(2+), 2, 2'-azobis(2-amidinopropane)hydrochloride, hypochlorite, or soybean 15-lipoxygenase), bound preferably to dimeric LPL. However, catalytic activity of LPL was not required for binding to oxidized LDL. Finally, immunohistochemistry of atherosclerotic lesions of human coronary arteries revealed specific areas in which LDL, LPL, decorin, and collagen type I were present. The results suggest that LPL can retain LDL in atherosclerotic lesions along decorin-coated collagen fibers.

Decorin links low-density lipoproteins (LDL) to collagen: a novel mechanism for retention of LDL in the atherosclerotic plaque.

A process central to the initiation of atherosclerosis is retention of plasma-derived low-density lipoprotein (LDL) particles in the extracellular matrix of the arterial intima. In this process, the apolipoprotein B-100 component of LDL binds to various components of the extracellular matrix, notably the negatively charged proteoglycans. In addition to proteoglycans, the intimal matrix contains large amounts of collagen. LDL also accumulates in collagen-rich areas of the arterial intima. The mechanism of this accumulation has remained obscure, because experiments in vitro have shown that LDL binds poorly to collagen. Our recent data provide evidence that the ability of collagen to bind LDL in vitro is greatly enhanced by decorin, a collagen-binding small proteoglycan that also is present in atherosclerotic lesions. This result provides a novel mechanism for retention of LDL in collagen-rich regions of the arterial intima.

Detection of low density lipoprotein particle fusion by proton nuclear magnetic resonance spectroscopy.

Recent evidence suggests that fusion of low density lipoprotein (LDL) particles is a key process in the initial accumulation of lipid in the arterial intima. In order to gain a better understanding of this early event in the development of atherosclerosis, it would thus be necessary to characterize the process of LDL fusion in detail. Such studies, however, pose severe methodological difficulties, such as differentiation of particle fusion from aggregation. In this paper we describe the use of novel methodology, based on 1H NMR spectroscopy, to study lipoprotein particle fusion. To test the methodology, we chose proteolytic fusion of LDL particles, an in vitro model that has been well characterized in our laboratory. The spectroscopic data suggested that proteolysis of LDL with alpha-chymotrypsin induced slow initiation of fusion, which was followed by particle fusion at an increased rate. Moreover, 1H NMR spectroscopic data on different kinds of LDL interactions, for example, when LDL formed aggregates with antibodies against human apolipoprotein B-100, were obtained and compared with the electron microscopic characteristics of these preparations. An important finding was that limited aggregation of LDL particles did not disturb the 1H NMR spectroscopic parameters used for the detection of particle fusion and preserved the physico-chemical information on the particles. The 1H NMR methodology developed is sensitive to and specific for low density lipoprotein (LDL) fusion and may also allow for studies of the fate of LDL particles in other in vitro preparations that mimic the arterial interactions in vivo.

Human arterial proteoglycans increase the rate of proteolytic fusion of low density lipoprotein particles.

Low density lipoprotein (LDL) particles can undergo fusion in the arterial intima, where they are bound to proteoglycans. Here we studied the effect of human arterial proteoglycans on proteolytic fusion of LDL in vitro. For this purpose, an assay was devised based on fluorescence resonance energy transfer that allowed continuous monitoring of fusion of proteoglycan-bound LDL particles. We found that addition of human arterial proteoglycans markedly increased the rate of proteolytic fusion of LDL. The glycosaminoglycans isolated from the proteoglycans also increased the rate of fusion, demonstrating that this effect was produced by the negatively charged sulfated polysaccharides in the proteoglycans. Furthermore, heparin, chondroitin 6-sulfate, and dextran sulfate, three commercially available sulfated polysaccharides, also increased the rate of LDL fusion, with heparin and chondroitin 6-sulfate being as effective as and dextran sulfate more effective than human proteoglycans. The ability of the sulfated polysaccharides to increase the rate of proteolytic fusion of LDL depended critically on their ability to form insoluble complexes with LDL, which, in turn, resulted in an increased rate of LDL proteolysis and, in consequence, in an increased rate of LDL fusion. The results reveal a novel mechanism regulating LDL fusion and point to the potentially important role of arterial proteoglycans in the generation of LDL-derived lipid droplets in the arterial intima during atherogenesis.

Oxidation of low density lipoprotein particles decreases their ability to bind to human aortic proteoglycans. Dependence on oxidative modification of the lysine residues.

Oxidation of low density lipoprotein (LDL) leads to its rapid uptake by macrophages in vitro, but no detailed studies have addressed the effect of oxidation on the binding of LDL to proteoglycans. We therefore treated LDL with various substances: copper sulfate, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH), soybean lipoxygenase, and mouse peritoneal macrophages, and determined the extent to which the oxidatively modified LDL bound to human aortic proteoglycans in an affinity column. Oxidation of LDL with copper, AAPH, or macrophages, all of which increased its electrophoretic mobility, was associated with reduced binding to proteoglycans, until strongly oxidized LDL was totally unable to bind to them. After treatment of LDL with soybean lipoxygenase, the change in electrophoretic mobility was small, and the amount of binding to proteoglycans was only slightly decreased. The increased electrophoretic mobility of oxidized LDL reflects modification of the lysine residues of apolipoprotein B-100 (apoB-100). To mimic the oxidative modification of lysines, we treated LDL with malondialdehyde. This treatment also totally prevented the binding of LDL to proteoglycans. In contrast, if the lysine residues of apoB-100 were methylated to shield them against oxidative modification, subsequent treatment of LDL with copper sulfate failed to reduce the degree of LDL binding to proteoglycans. Finally, the active lysine residues in the oxidized LDL particles, which are thought to be involved in this binding, were quantified with NMR spectroscopy. In oxidized LDL, the number of these residues was found to be decreased. The present results show that, after modification of the lysine residues of apoB-100 during oxidation, the binding of LDL to proteoglycans is decreased, and suggest that oxidation of LDL tends to lead to intracellular rather than extracellular accumulation of LDL during atherogenesis.

The proteoglycan decorin links low density lipoproteins with collagen type I.

Decorin is a small dermatan sulfate-rich proteoglycan which binds to collagen type I in vitro and in vivo. In atherosclerotic lesions the contents of low density lipoprotein (LDL), decorin, and collagen type I are increased, and ultrastructural studies have suggested an association between LDL and collagen in the lesions. To study interactions between LDL, decorin, and collagen type I, we used solid phase systems in which LDL was coupled to a Sepharose column, or in which LDL, decorin, or collagen type I was attached to microtiter wells. The interaction between LDL and decorin in the fluid phase was evaluated using a gel mobility shift assay. We found that LDL binds to decorin by ionic interactions. After treatment with chondroitinase ABC, decorin did not bind to LDL, showing that the glycosaminoglycan side chain of decorin is essential for LDL binding. Acetylated and cyclohexanedione-treated LDL did not bind to decorin, demonstrating that both lysine and arginine residues of apoB-100 are necessary for the interaction. When collagen type I was attached to the microtiter plates, only insignificant amounts of LDL bound to the collagen. However, if decorin was first allowed to bind to the collagen, binding of LDL to the decorin-collagen complexes was over 10-fold higher than to collagen alone. Thus, decorin can link LDL with collagen type I in vitro, which suggests a novel mechanism for retention of LDL in collagen-rich areas of atherosclerotic lesions.

Aggregation and fusion of modified low density lipoprotein.

In atherogenesis, low density lipoprotein (LDL, diameter 22 nm) accumulates in the extracellular space of the arterial intima in the form of aggregates of lipid droplets (droplet diameter up to 400 nm). Here we studied the effects of various established in vitro LDL modifications on LDL aggregation and fusion. LDL was subjected to vortexing, oxidation by copper ions, proteolysis by alpha-chymotrypsin, lipolysis by sphingomyelinase, and nonenzymatic glycosylation, and was induced to form adducts with malondialdehyde or complexes with anti-apoB-100 antibodies. To assess the amount of enlarged LDL-derived structures formed (due to aggregation or fusion), we measured the turbidity of solutions containing modified LDL, and quantified the proportion of modified LDL that 1) sedimented at low-speed centrifugation (14,000 g), 2) floated at an increased rate at high-speed centrifugation (rate zonal flotation at 285,000 gmax), 3) were excluded in size-exclusion column chromatography (exclusion limit 40 MDa), or 4) failed to enter into 0.5%. Fast Lane agarose gel during electrophoresis. To detect whether particle fusion had contributed to the formation of the enlarged LDL-derived structures, particle morphology was examined using negative staining and thin-section transmission electron microscopy. We found that 1) aggregation was induced by the formation of LDL-antibody complexes, malondialdehyde treatment, and glycosylation of LDL; 2) fusion of LDL was induced by proteolysis of LDL by alpha-chymotrypsin; and 3) aggregation and fusion of LDL were induced by vortexing, oxidation by copper ions, and lipolysis by sphingomyclinase of LDL. The various modifications of LDL differed in their ability to induce aggregation and fusion.