A novel NanoBiT-based assay monitors the interaction between lipoprotein lipase and GPIHBP1 in real time.

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.

Chylomicrons stimulate incretin secretion in mouse and human cells.

AIMS/HYPOTHESIS: Lipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release. Therefore, we investigated the effect of chylomicrons on GLP-1 and GIP secretion in vitro. METHODS: The effect of chylomicrons on incretin secretion was investigated using GLUTag cells and duodenal cultures of both murine and human origin. The role of lipoprotein lipase (LPL) and FFA1 in GLUTag cells was assessed by pharmacological inhibition and small (short) interfering RNA (siRNA)-mediated knockdown. The effect of chylomicrons on intracellular calcium concentration ([Ca2+]i) was determined by imaging GLUTag cells loaded with Fura-2. In the primary setting, the contributions of FFA1 and GPR119 were investigated using L cell-specific Gpr119 knockout cultures treated with the FFA1 antagonist GW1100. RESULTS: Chylomicrons stimulated GLP-1 release from GLUTag cells, and both GLP-1 and GIP secretion from human and murine duodenal cultures. Chylomicron-triggered GLP-1 secretion from GLUTag cells was largely abolished following lipase inhibition with orlistat or siRNA-mediated knockdown of Lpl. In GLUTag cells, both GW1100 and siRNA-mediated Ffar1 knockdown reduced GLP-1 secretion in response to chylomicrons, and, consistent with FFA1 Gq-coupling, chylomicrons triggered an increase in [Ca2+]i. However, LPL and FFA1 inhibition had no significant effect on chylomicron-mediated incretin secretion in murine cultures. Furthermore, the loss of GPR119 had no impact on GLP-1 secretion in response to chylomicrons, even in the presence of GW1100. CONCLUSIONS/INTERPRETATION: Chylomicrons stimulate incretin hormone secretion from GLUTag cells as well as from human and murine duodenal cultures. In GLUTag cells, the molecular pathway was found to involve LPL-mediated lipolysis, leading to the release of lipid species that activated FFA1 and elevated intracellular calcium.

[Effect of metabolic drugs on the secretion of acylation stimulating protein in 3T3-L1 adipocytes].

AIM: To develop a 3T3-L1 pre-adipocyte model for evaluating the secretion of acylation stimulating protein (ASP) and an ELISA assay for measuring ASP production and investigate the effects and related potential mechanisms of metabolic drugs on the secretion of ASP, and on the complement C3, triglyceride (TG) mass, nonesterified fatty acids (NEFA) release and fatty acid (FA) uptake into adipocytes. METHODS: After differentiated, 3T3-L1 pre-adipocytes were treated with chylomicrons, metformin, rosiglitazone, rimonabant for 48 h. ASP and C3 were measured using a sandwich ELISA. NEFA levels were measured using enzymatic colorimetric kits. FA uptake was measured in a bottom-reading fluorescent microplate reader. TG mass and protein levels were determined using enzymatic colorimetric assay and the Bradford assay, respectively. RESULTS: Chylomicrons increased ASP production (up to 411%±133%, P<0.05). Rosiglitazone and rimonabant decreased ASP production (-53% to -85%, P<0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P<0.01). By contrast, metformin also decreased ASP (-54% to -100%, P<0.05), but with no change in precursor protein C3. In addition, metformin decreased TG mass (maximum -60%, P<0.05) and real-time FA uptake (maximum -75%, P<0.05). CONCLUSION: 3T3-L1 pre-adipocyte model is successfully developed, which can be used for evaluating the effects of metabolic drugs on the secretion of ASP. ELISA assay for measuring ASP production is also developed.

Acute hypertriglyceridemia promotes intestinal lymphatic lipid and drug transport: a positive feedback mechanism in lipid and drug absorption.

Elevated systemic levels of triglyceride-rich lipoproteins (TRL) are a risk factor for the development of atherosclerosis. In patients with metabolic syndrome (MetS), intestinal TRL overproduction contributes to high systemic TRL levels, and recent studies suggest that systemic changes in MetS such as increases in plasma fatty acids and insulin resistance stimulate intestinal TRL production. The current study has examined whether increases in systemic TRL influence intestinal lipid transport and lipoprotein assembly pathways and evaluates the impact of these changes on the absorption and lymphatic transport of lipids and a model lipophilic drug (halofantrine). Mesenteric lymph-duct or bile-duct cannulated rats were administered IV saline or (14)C-labeled chylomicron (CM) (to increase systemic TRL) and intraduodenal (3)H lipids and drug. Changes to biliary lipid output and lymphatic lipid and drug transport were subsequently examined. Increasing systemic TRL concentrations stimulated a significant increase in lymphatic lipid and drug transport. The increased lipids in lymph were not derived from bile or the intestinal blood supply (fatty acid or IV infused (14)C-CM). Rather, an increase in lymphatic transport of duodenally sourced lipids was evident. Increasing plasma levels of TRL therefore stimulated lipid absorption and lymphatic transport via a positive feedback process. The data also suggest that the changes to intestinal TRL formation that result from raised systemic TRL levels may impact on the absorption of highly lipophilic drugs and therefore the reproducibility of drug treatments.

Evaluation of chylomicron effect on ASP production in 3T3-L1 adipocytes.

In the past few years, there has been increasing interest in the production and physiological role of acylation-stimulating protein (ASP), identical to C3adesArg, a product of the alternative complement pathway generated through C3 cleavage. Recent studies in C3 (-/-) mice that are ASP deficient have demonstrated a role for ASP in postprandial triglyceride clearance and fat storage. The aim of the present study was to establish a cell model and sensitive ELISA assay for the evaluation of ASP production using 3T3-L1 adipocytes. 3T3-L1 preadipocytes were differentiated into adipocytes, then cultured in different media such as serum-free (SF), Dulbecco's modified Eagle's medium (DMEM)/F12 + 10% fetal calf serum (FBS), and at varying concentrations of chylomicrons and insulin + chylomicrons up to 48 h. ASP production in SF and DMEM/F12 + 10% FBS was compared. Chylomicrons stimulated ASP production in a concentration- and time-dependent manner. By contrast, chylomicron treatment had no effect on the production of C3, the precursor protein of ASP, which was constant over 48 h. Addition of insulin (100 nM) to a low-dose of chylomicrons (100 µg TG/ml) significantly increased ASP production compared with chylomicrons alone at 48 h (P < 0.001). Furthermore, addition of insulin significantly increased C3 secretion at both 18 and 48 h of incubation (P < 0.05, P < 0.001, respectively). Overall, the proportion of ASP to C3 remained constant, indicating no change in the ratio of C3 cleaved to generate ASP. This study demonstrated that 3T3-L1 adipocyte is a useful model for the evaluation of C3 secretion and ASP production by using a sensitive mouse-specific ELISA assay. The stimulation of ASP production with chylomicrons demonstrates a physiologically relevant response, and provides a strategy for further studies on ASP production and function.

Chylomicrons combined with endotoxin moderate microvascular permeability.

Triglyceride-rich lipoprotein-bound endotoxin (CM-LPS) inhibits the host innate immune response to sepsis by attenuating the hepatocellular response to pro-inflammatory cytokine stimulation. This 'cytokine tolerance' in hepatocytes is a transient, receptor-dependent process that correlates with internalization of CM-LPS via low density lipoprotein (LDL) receptors. Since endothelial cells are integral to the immune response and similarly express LDL receptors, we hypothesized that CM-LPS could be internalized and ultimately attenuate the deleterious effects of pro-inflammatory molecules like tumor necrosis factor-α (TNF-α) and platelet activating factor (PAF) on endothelial permeability. Here, we show that CM-LPS complexes induce cytokine tolerance in endothelial cells. In rats, TNF-α increased hydraulic conductivity 2.5-fold over baseline and PAF increased it 5-fold; but, pretreatment with CM-LPS or an attenuated analog (CM-LPS*) inhibited these changes. Nuclear/cytoplasmic levels of p65 were reduced after TNF-α-stimulation in endothelial cell monolayers pretreated with CM-LPS, a finding consistent with inhibition of nuclear factor (NF)-κB translocation. Also consistent with inhibition was stabilized intercellular adhesion, as illustrated with antibody to VE-cadherin using confocal microscopy. These results provide additional support for the integral role of lipoproteins in the innate immune response to infection and lend further credence to developing lipid-based therapy for Gram-negative sepsis.

The role of free fatty acids, pancreatic lipase and Ca+ signalling in injury of isolated acinar cells and pancreatitis model in lipoprotein lipase-deficient mice.

AIM AND METHODS: Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia (HTG) often seen in patients carrying various gene mutations in lipoprotein lipase (LPL). This study investigates a possible pathogenic mechanism of cell damage in isolated mouse pancreatic acinar cells and of pancreatitis in LPL-deficient and in wild type mice. RESULTS: Addition of free fatty acids (FFA) or of chylomicrons to isolated pancreatic acinar cells caused stimulation of amylase release, and at higher concentrations it also caused cell damage. This effect was decreased in the presence of the lipase inhibitor orlistat. Surprisingly, pancreatic lipase whether in its active or inactive state could act like an agonist by inducing amylase secretion, increasing cellular cGMP levels and converting cell damaging sustained elevations of [Ca(2+)](cyt) to normal Ca(2+) oscillations. Caerulein increases the levels of serum amylase and caused more severe inflammation in the pancreas of LPL-deficient mice than in wild type mice. CONCLUSION: We conclude that high concentrations of FFA as present in the plasma of LPL-deficient mice and in patients with HTG lead to pancreatic cell damage and are high risk factors for the development of acute pancreatitis. In addition to its enzymatic effect which leads to the generation of cell-damaging FFA from triglycerides, pancreatic lipase also prevents Ca(2+) overload in pancreatic acinar cells and, therefore, counteracts cell injury.

Chylomicron-bound LPS selectively inhibits the hepatocellular response to proinflammatory cytokines.

BACKGROUND/AIMS: Pretreatment of rodent hepatocytes with chylomicron-bound lipopolysaccharide (CM-LPS) renders these cells unresponsive to subsequent stimulation by proinflammatory cytokines. We sought to test the selectivity of this response. METHODS: Cellular responses to hypoxia, oxidative stress, apoptosis, and heat-shock response, and thermotolerance induced in CM-LPS pretreated hepatocytes were compared with responses in non-pretreated cells. RESULTS: CM-LPS inhibited the hepatocellular response to proinflammatory cytokines without affecting the response to the other cellular stressors. It did not affect the response to oxidative stress, as measured by mitochondrial activity after hydrogen peroxide was added, or protein induction before or after stimulation with cobalt chloride. Also, induction of heat shock proteins did not differ between the CM-LPS pretreated cells and non-pretreated cells. CM-LPS did not interfere with the adoption of the thermotolerant phenotype, as shown by similar mitochondrial activity between pretreated and non-pretreated cells. Although stimulation with tumor necrosis factor-alpha and actinomycin D increased activity of the apoptotic enzymes, there were no differences between cells pretreated with CM-LPS and non-pretreated hepatocytes. CONCLUSION: When the response to proinflammatory cytokines is inhibited, hepatocellular responses to hypoxia, oxidative stress, heat shock, and apoptosis remain intact after pretreatment with CM-LPS. CM-LPS may have a specific anti-inflammatory effect on hepatocytes.

Spontaneous atherosclerosis in aged lipoprotein lipase-deficient mice with severe hypertriglyceridemia on a normal chow diet.

Large-scale epidemiological studies have revealed a strong association between hypertriglyceridemia and coronary arteriosclerotic disease. However, there are conflicting reports whether the severe hypertriglyceridemia caused by lipoprotein lipase (LPL) deficiency is pro- or antiatherogenic. To determine the effect of LPL deficiency on atherosclerosis, we pursued long-term observation of the development of atherosclerotic lesions in an LPL gene deficient mouse model. At 4 months of age, homozygous LPL-deficient mice exhibited severe hypertriglyceridemia but no signs of aortic atherosclerotic lesions. At >15 months of age, these mice developed foam cell-rich atherosclerotic lesions at the aortic root, whereas wild-type and heterozygous mice were lesion-free at the same age. Further investigation revealed that plasma malondialdehyde levels in >15-month-old LPL-deficient mice were significantly higher than those of heterozygous and wild-type mice. Electron spin resonance analysis showed a marked increase in oxidative susceptibility of chylomicrons from the aged LPL-deficient mice. Incubation of chylomicrons from >15-month-old LPL-deficient mice with cultured human umbilical vein endothelial cells showed significantly increased upregulation of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, markers of enhanced endothelial activation, and enhanced adherence of human THP-1 mononuclear cells. These results clearly demonstrate the occurrence of spontaneous atherosclerosis in aged LPL-deficient mice mediated by the oxidation of chylomicrons and the activation of vascular endothelial cells.

Lipolysis of emulsion models of triglyceride-rich lipoproteins is altered in male patients with abdominal aorta aneurysm

Disorders of the lipid metabolism may play a role in the genesis of abdominal aorta aneurysm. The present study examined the intravascular catabolism of chylomicrons, the lipoproteins that carry the dietary lipids absorbed by the intestine in the circulation in patients with abdominal aorta aneurysm. Thirteen male patients (72 ± 5 years) with abdominal aorta aneurysm with normal plasma lipid profile and 13 healthy male control subjects (73 ± 5 years) participated in the study. The method of chylomicron-like emulsions was used to evaluate this metabolism. The emulsion labeled with 14C-cholesteryl oleate and ³H-triolein was injected intravenously in both groups. Blood samples were taken at regular intervals over 60 min to determine the decay curves. The fractional clearance rate (FCR) of the radioactive labels was calculated by compartmental analysis. The FCR of the emulsion with ³H-triolein was smaller in the aortic aneurysm patients than in controls (0.025 ± 0.017 vs 0.039 ± 0.019 min-1; P < 0.05), but the FCR of14C-cholesteryl oleate of both groups did not differ. In conclusion, as indicated by the triglyceride FCR, chylomicron lipolysis is diminished in male patients with aortic aneurysm, whereas the remnant removal which is traced by the cholesteryl oleate FCR is not altered. The results suggest that defects in the chylomicron metabolism may represent a risk factor for development of abdominal aortic aneurysm.

Chylomicron remnants stimulate release of interleukin-1beta by THP-1 cells.

Recent findings suggest that the oxidative modification of low-density lipoproteins (LDL) and an increase in triglyceride-rich lipoprotein particles including chylomicron remnants contribute to the progression of atherosclerosis, as does the inflammatory response. We therefore examined whether and how these lipoproteins affected interleukin (IL)-1beta release and mRNA expression for IL-1beta and IL-18 in THP-1 cells, a human monocyte cell line. Chylomicron remnants increased IL-1beta release into the conditioned medium by THP-1 in a dose- and time-dependent manner. At concentrations up to 1 microg/ml, chylomicron remnants increased IL-1beta release by 4-fold compared with the control. Neither native LDL nor oxidized LDL (OxLDL) significantly increased IL-1beta release. Chylomicron remnants increased IL-1beta mRNA expression by 3 times. Native LDL or OxLDL did not increase IL-1beta mRNA, while neither these lipoproteins nor chylomicron remnants increased IL-18 mRNA. Chylomicron remnants also increased the activities of caspase-1 and nuclear factor (NF)-kappaB significantly, while native LDL or OxLDL did not. In conclusion, chylomicron remnants stimulated IL-1beta mRNA expression and IL-1beta protein production probably via caspase-1 and NF-kappaB activation in THP-1 cells.

Uptake of lipophilic drugs by plasma derived isolated chylomicrons: linear correlation with intestinal lymphatic bioavailability.

Association of a drug with chylomicrons in the enterocyte is an essential step in the lymphatic absorption pathway. In this article, the uptake of lipophilic compounds by chylomicrons ex vivo was compared to the corresponding intestinal lymphatic bioavailability reported in rats in order to elucidate the degree of correlation and to evaluate the utilization of this correlation as a predictive measurement of the lymphatic bioavailability potential of lipophilic drugs. Nine lipophilic compounds (Vitamin D(3), Vitamin E, halofantrine, probucol, diazepam, testosterone, cyclosporin A, benzo[a]pyrene and p,p'-DDT) at a concentration of 1.75 x 10(-6)M were incubated for 1h with chylomicron emulsion separated from rat blood. A strong linear correlation was found between the degree of association of compounds with chylomicrons ex vivo and the lymphatic transport reported in rats (r(2)=0.94, P<0.0001), whereas logP and solubility in long chain triglycerides showed only moderate correlation with lymphatic bioavailability. The linear correlation between the degree of uptake of compounds by isolated chylomicrons and intestinal lymphatic transport suggests that the two processes are governed by similar factors. Thus, the degree of association of lipophilic compounds with isolated chylomicrons can be used as a simple screening model for estimation of intestinal lymphatic transport potential of drug molecules. This approach is important in view of the practical difficulties in direct determination of the lymphatic bioavailability in vivo.

Chylomicron remnants regulate early growth response factor-1 in vascular smooth muscle cells.

Early growth response factor-1 (Egr-1) is a zinc-finger transcription factor that induces genes that promote atherosclerosis. The goal of the present study was to determine whether Egr-1 expression is modulated by atherogenic, triglyceride rich lipoproteins known as chylomicron remnants. Chylomicron remnants induced Egr-1 mRNA and protein expression in rat cultured vascular smooth muscle cells (VSMCs) and activated extracellular signal-regulated kinase (ERK) 1/2 in VSMCs. Further, chylomicron remnant-induced Egr-1 expression was inhibited by PD98059, a selective inhibitor of MAPK kinase (MEK), suggesting that the action of chylomicron remnants on Egr-1 was dependent on the ERK/MEK pathway. Chylomicron remnants also induced mRNA expression of the pro-inflammatory cytokines, IL-2 and IFN-gamma in VSMCs. We conclude that chylomicron remnants act as atherogenic lipoproteins via induction of Egr-1 expression and via cytokine-mediated inflammation.

Effects of cholesterol in chylomicron remnant models of lipid emulsions on apoE-mediated uptake and cytotoxicity of macrophages.

Chylomicron remnants have been suggested to be involved in the development of atherosclerosis. To investigate the mechanisms of chylomicron remnant-induced atherosclerosis, we prepared cholesterol (Chol)-containing emulsion particles as models for chylomicron remnants. Chol markedly increased the apolipoprotein E (apoE) binding maximum of emulsions without changing the binding affinity and thereby promoted emulsion uptake by J774 macrophages. Fluorescence measurements showed that Chol increased acyl chain order and head group hydration of the surface phospholipid (PL) layer of emulsions. The binding maximum of apoE was closely correlated with the hydration and the increase in the PL head group separation at the emulsion surface. From experiments using inhibitors for lipoprotein receptors, heparan sulfate proteoglycans and low density lipoprotein receptor-related protein were found to be the major contributors to the uptake of Chol-containing emulsions. Trypan blue dye exclusion revealed that the uptake of Chol-containing emulsions induced cytotoxicity to J774 macrophages. This study proposes a mechanism of atherosclerosis induced by chylomicron remnants.

Efflux of lipid from macrophages after induction of lipid accumulation by chylomicron remnants.

The fate of cholesterol and triacylglycerol taken up and accumulated by macrophages after exposure to chylomicron remnants was investigated using macrophages derived from the human monocyte cell line THP-1 and chylomicron remnant-like particles containing human apolipoprotein (apo) E (CRLPs) as the experimental model. In THP-1 macrophages lipid loaded with CRLPs and incubated with various cholesterol acceptors for 24 h, the mass of cholesterol and cholesteryl ester found in the cells was not changed by HDL, HDL3 or lipid-free ApoA-I, although it was decreased by 38% by ApoA-I-phosphatidylcholine vesicles (ApoA-I-PC). After loading of the macrophages with [3H]cholesterol-labelled CRLPs, only about 5% of the label was effluxed in 24 h in the absence of cholesterol acceptors, and this increased to about 10% with ApoA-I or PC only, and to about 30% with apoA-I-PC. In similar experiments with [3H]triolein, only about 4% of the labelled triacylglycerol taken up by the cells was released into the medium in 24 h, and a large (>60%) and consistent proportion of the intracellular radioactivity remained associated with the triacylglycerol throughout this period. These results suggest that cholesterol and triacylglycerol derived from chylomicron remnants are not readily cleared from macrophages, and this is likely to contribute to the atherogenicity of the remnant lipoproteins.

Effect of remnant-like particle on shear-induced platelet activation and its inhibition by antiplatelet agents.

Remnant-like particles (RLPs) have been reported to promote atherosclerosis and to have effects on platelet function. We studied the effects of RLP on shear-induced platelet activation and their inhibition by antiplatelet agents in vitro. RLP were separated using two monoclonal antibodies, anti apo B-100 and anti apo A-I. These RLP fractions were added to whole blood (WB) or platelet-rich plasma (PRP) in serial dilution of 1, 10 or 100 microg RLP triglyceride (TG) per ml of total sample volume. These samples were incubated, and then stimulated with a high shear stress of 108 dyn/cm(2). Shear-induced platelet aggregation (SIPA) was calculated from the percentage of single platelet loss. P-selectin expression on platelet surface and platelet-derived microparticle (PMP) generation were measured before and after stimulation with shear stress using flow cytometer. SIPA was significantly enhanced by RLP in WB but not in PRP. This enhancing effect was not dose-dependent and was greatest at 10 microg TG/ml. P-selectin expression induced by shear stress was only enhanced by RLP at a concentration of 100 microg TG/ml in both WB and PRP, while generation of PMP induced by shear stress was only enhanced by RLP at a concentration of 100 microg TG/ml in WB. Aspirin inhibited only the enhancement of SIPA by RLPs, while cilostazol inhibited the enhancement of not only SIPA but also p-selectin expression and PMP generation by RLPs.

Chylomicron remnants and oxidised low density lipoprotein have differential effects on the expression of mRNA for genes involved in human macrophage foam cell formation.

The effects of chylomicron remnants (non-oxidised or oxidised) and oxidised low density lipoprotein (oxLDL) on the expression of mRNA for a wide range of genes believed to play a role in macrophage foam cell formation were compared using macrophages derived from the human monocyte cell line THP-1. Chylomicron remnant-like particles (CMR-LPs), oxidised CMR-LPs (oxCMR-LPs) and oxLDL were incubated with THP-1 macrophages, and the relative abundance of mRNA transcripts for genes involved in lipoprotein uptake, intracellular lipid metabolism, transport and storage and cholesterol efflux from macrophages was determined. The results show that CMR-LPs and oxLDL differ markedly in their effects on the expression of mRNA for a number of the genes tested. OxLDL increased mRNA levels for the scavenger receptors CD36 (x3.2) and lectin-like oxLDL receptor 1 (x2.1), and peroxisome proliferator-activated receptor gamma while CMR-LPs did not. In contrast, the expression of mRNA for the LDL receptor-like protein was raised by CMR-LPs (x1.8) but not oxLDL. Furthermore, down-regulation of mRNA levels for the ATP-binding cassette transporter (ABC) A1 was observed with CMR-LPs (x0.6), compared to the up-regulation found with oxLDL (x4.4). In addition, a number of significant differences were found between the effects of CMR-LPs and oxCMR-LPs, with the oxidised particles causing a striking rise in mRNA expression for the multi-drug resistance 1 gene (x13.7), but otherwise showing pattern more similar to that seen with oxLDL. These findings provide evidence to indicate that chylomicron remnants cause lipid accumulation in macrophages by influencing the expression of genes which regulate lipid metabolism at the transcriptional level, and that the mechanisms involved differ in important respects from those triggered by oxLDL.

Remnant lipoprotein particles induce apoptosis in endothelial cells by NAD(P)H oxidase-mediated production of superoxide and cytokines via lectin-like oxidized low-density lipoprotein receptor-1 activation: prevention by cilostazol.

BACKGROUND: Remnant lipoprotein particles (RLPs), products of lipolytic degradation of triglyceride-rich lipoprotein derived from VLDL, exert atherogenesis. In this study, we observed how RLPs induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) and cilostazol prevented cell death. METHODS AND RESULTS: RLPs were isolated from the plasma of hyperlipidemic patients by use of an immunoaffinity gel mixture of anti-apolipoprotein A-1 and anti-apolipoprotein B-100 monoclonal antibodies. RLPs (50 microg/mL) significantly increased superoxide formation in HUVECs associated with elevated gp91phox mRNA and protein expression and Rac1 translocation, accompanied by increased production of tumor necrosis factor (TNF)-alpha and interleukin-1beta, DNA fragmentation, and cell death. Cilostazol (1 to 100 micromol/L) significantly suppressed not only NAD(P)H oxidase-dependent superoxide production but also TNF-alpha and interleukin-1beta release and restored viability. RLPs activated a lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which was not inhibited by cilostazol. Treatment of HUVECs with monoclonal antibody for LOX-1 attenuated RLP-mediated production of superoxide, TNF-alpha, and interleukin-1beta and DNA fragmentation. CONCLUSIONS: RLPs stimulated NAD(P)H oxidase-dependent superoxide formation and induction of cytokines in HUVECs via activation of LOX-1, consequently leading to reduction in cell viability with DNA fragmentation, and cilostazol exerts a cell-protective effect by suppressing these variables.

Chylomicron-remnant-like particles modify production of vasoactive mediators by endothelial cells.

Endothelial-cell dysfunction is a critical initiating event in the pathogenesis of atherosclerosis. Although there is evidence to suggest that chylomicron remnants (CMRs), lipoproteins derived from the diet, influence endothelial-cell function to generate a pro-atherogenic phenotype, the mechanisms involved remain undefined. We have examined the effects of CMR-like particles (CMR-LPs) on human endothelial-cell function, focusing on the cyclo-oxygenase (COX) and nitric oxide synthase (NOS) pathways. CMR-LPs strongly enhanced the expression of the inducible cyclo-oxygenase COX-2 and increased prostacyclin synthesis in a biphasic manner. Studies with the COX-2-selective inhibitor NS-398 confirmed the COX-2 dependency of the later increase in prostanoid production. Pre-incubation with CMR-LPs reduced basal and thrombin-stimulated cGMP generation, whereas expression of endothelial NOS was not modified by remnant treatment.

Chylomicron remnants induce monocyte chemoattractant protein-1 expression via p38 MAPK activation in vascular smooth muscle cells.

Chylomicron remnants, major lipoproteins at postprandial hyperlipidemia, have been considered to be proatherogenic lipoproteins. However, the mechanisms by which chylomicron remnants enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of chylomicron remnants on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs). We prepared chylomicrons from the lymph of gastrostomized rats fed with egg solution and obtained chylomicron remnants from the plasma of hepatectomized rats which were injected with chylomicrons. Treatment of VSMC with chylomicron remnants resulted in a significant increase of the expression of MCP-1 mRNA and protein in a time-and a dose-dependent manner. Further, chylomicron remnants activated p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2). Pretreatment of VSMCs with p38 MAPK inhibitors, SB203580 and SB202190, resulted in a dose-dependent inhibition of chylomicron remnants-induced MCP-1 mRNA and protein expression, whereas a MAPK kinase inhibitor, PD98059, had no effect on these responses. MCP-1 secretion by chylomicron remnants was much more pronounced than those by chylomicrons, oxidized low-density lipoproteins, or lysophosphatidylcholine. These results indicated that chylomicron remnants stimulated MCP-1 expression in VSMCs, and suggested that chylomicron remnants might contribute to the formation of atherosclerosis through this proinflammatory effect.