Induction of dendritic cell-mediated T-cell activation by modified but not native low-density lipoprotein in humans and inhibition by annexin a5: involvement of heat shock proteins.

OBJECTIVE: Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E(-/-) mice. Here, we focus on the LDLx effects on human DCs and T cells. APPROACH AND RESULTS: Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-γ, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-ß and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-ß, and cell-cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. CONCLUSIONS: Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.

A follow-up study of a genome-wide association scan identifies a susceptibility locus for venous thrombosis on chromosome 6p24.1.

To identify genetic susceptibility factors conferring increased risk of venous thrombosis (VT), we conducted a multistage study, following results of a previously published GWAS that failed to detect loci for developing VT. Using a collection of 5862 cases with VT and 7112 healthy controls, we identified the HIVEP1 locus on chromosome 6p24.1 as a susceptibility locus for VT. Indeed, the HIVEP1 rs169713C allele was associated with an increased risk for VT, with an odds ratio of 1.20 (95% confidence interval 1.13-1.27, p = 2.86 x 10(-9)). HIVEP1 codes for a protein that participates in the transcriptional regulation of inflammatory target genes by binding specific DNA sequences in their promoter and enhancer regions. The current results provide the identification of a locus involved in VT susceptibility that lies outside the traditional coagulation/fibrinolysis pathway.

Circulating levels of secretory- and lipoprotein-associated phospholipase A2 activities: relation to atherosclerotic plaques and future all-cause mortality.

AIMS: Secretory- and lipoprotein-associated phospholipases A2 (sPLA2 and Lp-PLA2) are enzymes both suggested to be of importance for atherosclerosis. We investigated relationships between the activities of these enzymes in the circulation and atherosclerosis as well as future clinical events. METHODS AND RESULTS: The population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study included 1016 randomly selected subjects, all aged 70. The prevalence of carotid artery plaques was recorded by ultrasound (n= 954), and arterial stenosis was assessed by whole-body magnetic resonance angiography (WBMRA, n= 302). Secretory-associated phospholipase A2 [odds ratio 1.23 for 1 SD increase, 95% confidence interval (CI): 1.05-1.44, P= 0.007], but not Lp-PLA2 (P= 0.26), activity was significantly related to carotid atherosclerosis and to the amount of stenosis at WBMRA (P= 0.006) following adjustment for multiple risk factors (waist circumference, serum triglycerides, body mass index, C-reactive protein, high density lipoprotein-C, low density lipoprotein-C, triglycerides, GFR, fasting glucose, blood pressure, statin use, and exercise habits). Secretory-associated phospholipase A2 [hazard ratio (HR) 1.45 for 1 SD increase, 95% CI: 1.15-1.84, P= 0.001], but not Lp-PLA2 (HR 0.95, P= 0.55), activity was a significant risk factor for all-cause mortality (114 had died) during 7.0 years follow-up after adjustment for the risk factors described above. In a sample of 1029 post-myocardial infarction (MI) patients (French registry of Acute ST-elevation and non-ST-elevation Myocardial Infarction), sPLA2 (adjusted HR 1.32 for 1 unit increase, 95% CI: 1.02-1.71, P= 0.036), but not Lp-PLA2 (HR 1.03, P= 0.90), activity predicted death or recurrent MI during 1-year follow-up (n= 136 cases). CONCLUSION: sPLA2 activity was related to atherosclerosis and predicted all-cause mortality in a sample of elderly subjects, as well as death or MI in post-MI patients.

Inflammatory biomarkers, physical activity, waist circumference, and risk of future coronary heart disease in healthy men and women.

AIMS: The aim of this study was to determine the contribution of physical activity and abdominal obesity to the variation in inflammatory biomarkers and incident coronary heart disease (CHD) in a European population. METHODS AND RESULTS: In a prospective case-control study nested in the European Prospective Investigation into Cancer and Nutrition-Norfolk cohort, we examined the associations between circulating levels or activity of C-reactive protein, myeloperoxidase (MPO), secretory phospholipase A2 (sPLA2), lipoprotein-associated phospholipase A2 (Lp-PLA2), fibrinogen, adiponectin, waist circumference, physical activity, and CHD risk over a 10-year period among healthy men and women (45-79 years of age). A total of 1002 cases who developed fatal or non-fatal CHD were matched to 1859 controls on the basis of age, sex, and enrolment period. Circulating levels of C-reactive protein, sPLA2 (women only), fibrinogen, and adiponectin were linearly associated with increasing waist circumference and decreasing physical activity levels. After adjusting for waist circumference, physical activity, smoking, diabetes, systolic blood pressure, low-density lipoprotein and high-density lipoprotein cholesterol levels, and further adjusted for hormone replacement therapy in women, C-reactive protein, MPO (men only), sPLA2, fibrinogen, but not Lp-PLA2 and adiponectin were associated with an increased CHD risk. CONCLUSION: Inactive participants with an elevated waist circumference were characterized by deteriorated levels of inflammatory markers. However, several inflammatory markers were associated with an increased CHD risk, independent of underlying CHD risk factors such as waist circumference and physical activity levels.

PLA2G7 genotype, lipoprotein-associated phospholipase A2 activity, and coronary heart disease risk in 10 494 cases and 15 624 controls of European Ancestry.

BACKGROUND: Higher lipoprotein-associated phospholipase A(2)(Lp-PLA2) activity is associated with increased risk of coronary heart disease (CHD), making Lp-PLA2 a potential therapeutic target. PLA2G7 variants associated with Lp-PLA2 activity could evaluate whether this relationship is causal. METHODS AND RESULTS: A meta-analysis including a total of 12 studies (5 prospective, 4 case-control, 1 case-only, and 2 cross-sectional studies; n=26 118) was undertaken to examine the association of the following: (1) Lp-PLA2 activity versus cardiovascular biomarkers and risk factors and CHD events (2 prospective studies; n=4884); (2) PLA2G7 single-nucleotide polymorphisms and Lp-PLA2 activity (3 prospective, 2 case-control, 2 cross-sectional studies; up to n=6094); and (3) PLA2G7 single-nucleotide polymorphisms and angiographic coronary artery disease (2 case-control, 1 case-only study; n=4971 cases) and CHD events (5 prospective, 2 case-control studies; n=5523). Lp-PLA2 activity correlated with several CHD risk markers. Hazard ratios for CHD events for the top versus bottom quartile of Lp-PLA2 activity were 1.61 (95% confidence interval, 1.31 to 1.99) and 1.17 (95% confidence interval, 0.91 to 1.51) after adjustment for baseline traits. Of 7 single-nucleotide polymorphisms, rs1051931 (A379V) showed the strongest association with Lp-PLA2 activity, with VV subjects having 7.2% higher activity than AAs. Genotype was not associated with risk markers, angiographic coronary disease (odds ratio, 1.03; 95% confidence interval, 0.80 to 1.32), or CHD events (odds ratio, 0.98; 95% confidence interval, 0.82 to 1.17). CONCLUSIONS: Unlike Lp-PLA2 activity, PLA2G7 variants associated with modest effects on Lp-PLA2 activity were not associated with cardiovascular risk markers, coronary atheroma, or CHD. Larger association studies, identification of single-nucleotide polymorphisms with larger effects, or randomized trials of specific Lp-PLA2 inhibitors are needed to confirm or refute a contributory role for Lp-PLA2 in CHD.

Phospholipolyzed LDL induces an inflammatory response in endothelial cells through endoplasmic reticulum stress signaling.

Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2alpha, IRE1alpha, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.

Increased PAFAH and oxidized lipids are associated with inflammation and atherosclerosis in hypercholesterolemic pigs.

OBJECTIVE: To study the association of PAF-acetyl hydrolase (PAFAH) activity with inflammation, oxidative stress, and atherosclerosis in hypercholesterolemic swine. METHODS AND RESULTS: Cholesterol-rich diet feeding of miniature pigs was associated with an increase in PAFAH activity and an increase of the PAFAH to PON1 ratio. PLA2G7 RNA (coding for PAFAH) expression was increased in blood monocytes and plaque macrophages. Increased PAFAH activity was associated with higher plasma lysophosphatidylcholine and correlated with oxidized LDL. In THP1 monocytes and macrophages and in human blood-derived macrophages, oxidized LDL induced PLA2G7 RNA expression. Atherogenic diet feeding induced the accumulation of macrophages and oxidized LDL in the arterial wall leading to atherosclerosis. PAFAH activity correlated positively with plaque size and TNFalpha expression in plaque macrophages. CONCLUSIONS: We demonstrated that an increase in PAFAH activity was associated with increased levels of lysophosphatidylcholine, oxidized LDL, and inflammation, resulting in accelerated atherosclerosis in hypercholesterolemic minipigs. The significant correlation between PLA2G7 RNA expression in plaque macrophages and plasma PAFAH activity suggests that the latter is a consequence, rather than a cause of macrophage accumulation. Our cell experiments suggest that oxidized LDL can induce PAFAH, resulting in accumulation of lysophosphatidylcholine that increases the inflammatory action of oxidized LDL.

Effect of milk fat on LDL cholesterol and other cardiovascular risk markers in healthy humans: the INNOVALAIT project.

BACKGROUND: Milk has a specific saturated fatty acid profile and its calcium content may change the kinetics of fat absorption. OBJECTIVE: The goal of this study was to compare the effect on LDL Cholesterol and other risk markers of four isolipidic diets differing by their fat food source, vegetable fat, spring milk fat, winter milk fat or winter milk fat supplemented with calcium, in healthy moderately hypercholesterolemic humans. INDIVIDUALS AND METHODS: This double-blind, randomized trial with four parallel arms included 172 healthy adults with plasma LDL cholesterol (LDL-C) from 130 to 220 mg/dL and triglycerides below 300 mg/dL. Individual meal plans ensured a stable energy intake. In the three diets containing milk fat, milk fat provided 38% of energy. Vegetable fat and spring milk fat diets provided the same amount of saturated fatty acids while the winter milk fat diets were slightly richer in saturated fatty acids. Vegetable fat diet and winter milk fat diets provided the same amount of palmitic acid (7.0% EI), while the spring milk fat diet was slightly poorer in this fatty acid (5.1% EI). Cardiovascular risk markers were analyzed after 8 weeks of dietary intervention. RESULTS: There was no significant difference in LDL-C and other markers, except total cholesterol (TC), apo C3 and CRP. TC was significantly higher with spring milk fat than with vegetable fat. CONCLUSIONS: In this trial, the chosen vegetable fat did not have a significant beneficial effect on LDL-C compared to dairy fat. However, sub-group analysis showed differences in TC, apo C3 and CRP. These results need confirmation and long-term studies aiming at cardiovascular endpoints are warranted.

High pressure promotes monocyte adhesion to the vascular wall.

Hypertension is a known risk factor for the development of atherosclerosis. To assess how mechanical factors contribute to this process, mouse carotid arteries were maintained in organ culture at normal (80 mm Hg) or high (150 mm Hg) intraluminal pressure for 1, 6, 12, or 24 hours. Thereafter, fluorescent human monocytic cells (U937) were injected intraluminally and allowed to adhere for 30 minutes before washout. U937 adhesion was increased in vessels kept at 150 mm Hg 12 hours (23.5+/-5.7 versus 9.9+/-2.2 cells/mm at 80 mm Hg; P<0.05) or 24 hours (26.7+/-5.7 versus 8.8+/-1.5 cells/mm; P<0.05). At 24 hours, high pressure was associated with increased mRNA expression of monocyte chemoattractant protein-1, interleukin-6, keratinocyte-derived chemokine, and vascular cell adhesion molecule-1 (6.9+/-2.1, 4.4+/-0.1, 9.8+/-2.8, and 2.4+/-0.1-fold respectively; P<0.05), as assessed by quantitative RT-PCR and corroborated by immunohistochemistry, which also revealed an increase in intracellular adhesion molecule-1 expression. Nuclear factor kappaB inhibition using SN50 peptide abolished the overexpression of chemokines and adhesion molecules and reduced U937 adhesion in vessels at 150 mm Hg. Moreover, treatment of vessels and cells with specific neutralizing antibodies established that monocyte chemoattractant protein-1, interleukin-6, and keratinocyte-derived chemokine released from vessels at 150 mm Hg primed the monocytes, increasing their adhesion to vascular cell adhesion molecule-1 but not intracellular adhesion molecule-1 via alpha4beta1 integrins. The additive effect of chemokines on the adhesion of U937 cells to vascular cell adhesion molecule-1 was confirmed by in vitro assay. Finally, pressure-dependent U937 adhesion was blunted in arteries from mice overexpressing endothelial NO synthase. Hence, high intraluminal pressure induces cytokine and adhesion molecule expression via nuclear factor kappaB, leading to monocytic cell adhesion. These results indicate that hypertension may directly contribute to the development of atherosclerosis through nuclear factor kappaB induction.

The Tumor Suppressor SASH1 Interacts With the Signal Adaptor CRKL to Inhibit Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation. Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set. Results: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy. Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.

Increased oxidative stress in scavenger receptor BI knockout mice with dysfunctional HDL.

OBJECTIVE: In the current study the effect of disruption of SR-BI, a prominent regulator of HDL metabolism, on the activity of the HDL-associated antioxidant enzymes PON1 and PAF-AH as well as in vivo oxidative stress were investigated. METHODS AND RESULTS: SR-BI deficiency resulted in 1.4-fold (P<0.001) and 1.6-fold (P<0.01) lower serum paraoxonase and arylesterase activity of PON1, respectively. Furthermore, a trend to slightly lower PAF-AH activity was observed. In vivo oxidative stress was evaluated by measuring isoprostane F2alpha-VI (iPF2alpha-VI) and protein carbonyls. Compared with wild-type animals, SR-BI knockouts had 1.4-fold (P<0.05) higher levels of plasma iPF2alpha-VI, whereas urinary excretion was increased 2-fold (P<0.0001). Plasma carbonyls were 1.5-fold (P<0.05) higher in SR-BI knockout animals. Furthermore, iPF2alpha-VI and carbonyl levels were 2.1-fold (P<0.01) and 1.4-fold (P<0.01), respectively, increased in livers of SR-BI knockout mice, and in reaction to the increased oxidative stress the expression of several endogenous antioxidant systems was upregulated. On challenging the SR-BI knockout mice with an atherogenic Western-type diet, a further increase in oxidative stress in these animals was observed. CONCLUSION: SR-BI deficiency results in a reduced activity of the antioxidant enzyme PON1 and a significant increase in oxidative stress, potentially contributing to the proatherogenic effect of SR-BI deficiency.

Plasma PAF-acetylhydrolase: an unfulfilled promise?

Plasma Platelet-activating-Factor (PAF)-acetylhydrolase (PAF-AH also named lipoprotein-PLA(2) or PLA(2)G7 gene) is secreted by macrophages, it degrades PAF and oxidation products of phosphatidylcholine produced upon LDL oxidation and/or oxidative stress, and thus is considered as a potentially anti-inflammatory enzyme. Cloning of PAF-AH has sustained tremendous promises towards the use of PAF-AH recombinant protein in clinical situations. The reason for that stems from the numerous animal models of inflammation, atherosclerosis or sepsis, where raising the levels of circulating PAF-AH either through recombinant protein infusion or through the adenoviral gene transfer showed to be beneficial. Unfortunately, neither in human asthma nor in sepsis the recombinant PAF-AH showed sufficient efficacy. One of the most challenging questions nowadays is as to whether PAF-AH is pro- or anti-atherogenic in humans, as PAF-AH may possess a dual pro- and anti-inflammatory role, depending on the concentration and the availability of potential substrates. It is equally possible that the plasma level of PAF-AH is a diagnostic marker of ongoing atherosclerosis.

The proinflammatory mediator Platelet Activating Factor is an effective substrate for human group X secreted phospholipase A2.

Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.

Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function.

Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in the pathophysiology of atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has the highest catalytic activity toward phosphatidylcholine, one of the major phospholipid species of cell membranes and low-density lipoprotein (LDL). Our study examined the presence of hGX sPLA2 in human atherosclerotic lesions and investigated the ability of hGX modified LDL to alter human endothelial cell (HUVEC) function. Our results show that hGX sPLA2 is present in human atherosclerotic lesions and that the hydrolysis of LDL by hGX sPLA2 results in a modified particle that induces lipid accumulation in human monocyte-derived macrophages. Acting on endothelial cells, hGX-modified LDL activates the MAP kinase pathway, which leads to increased arachidonic acid release, increased expression of adhesion molecules on the surface of HUVEC, and increased adhesion of monocytes to HUVEC monolayers. Together, our data suggest that LDL modified by hGX, rather than hGX itself may have strong proinflammatory and proatherogenic properties, which could play an important role in the propagation of atherosclerosis.

Human platelets secrete the plasma type of platelet-activating factor acetylhydrolase primarily associated with microparticles.

OBJECTIVE: Platelet-activating factor acetylhydrolase (PAF-AH) expresses a Ca2+-independent phospholipase A2 activity and hydrolyzes platelet-activating factor as well as oxidized phospholipids. Two major types of PAF-AH have been described: the plasma type, which is associated with lipoproteins, and the intracellular type II PAF-AH. METHODS AND RESULTS: We investigated the type(s) of PAF-AH expressed in human platelets as well as the mechanism and the enzyme type secreted from platelets during activation. The majority of the enzyme activity (75.1+/-14.3% of total) is found in the cytosol, whereas 24.9+/-7.3% is associated with the membranes. Immunofluorescence microscopy studies and Western blotting analysis showed that platelets contain the plasma type as well as the intracellular type II PAF-AH. Furthermore, platelets contain high levels of the mRNA of plasma PAF-AH, whereas only a small quantity of the type II PAF-AH mRNA was detected. On activation, platelets secrete the plasma type of PAF-AH mainly associated with platelet-derived microparticles (PMPs). The enzyme activity was also detected on circulating PMPs in plasma from normolipidemic healthy subjects. CONCLUSIONS: This is the first indication that in addition to lipoproteins, PAF-AH in human plasma is carried by PMPs, suggesting that the PMP-associated PAF-AH may play a role in the dissemination of biological activities mediated by these particles.

Oxidized phospholipid: POVPC binds to platelet-activating-factor receptor on human macrophages. Implications in atherosclerosis.

Atherosclerosis as a chronic inflammatory disease resulting from the imbalance of the pro- and anti-inflammatory factors in the vessel wall. PAF and PAF-like oxidized phospholipids generated upon LDL oxidation in the intima of the arteries may interact with infiltrated monocytes/macrophages and lead to the alteration of gene expression patterns accompanied by an impaired production of chemokines, interleukins and proteolytic and lipolytic enzymes. The aim of this study was to evaluate the binding capacity of the major component of PAF-like oxidized phospholipids, namely the 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) to PAF-receptor (PAF-R) on the surface of human monocytes/macrophages and to further characterize the gene expression induced by such binding. We show that, POVPC binds to cultured human macrophages via PAF-R and transduces the signals leading to the intracellular Ca(2+) fluxes and modifies the transcription levels of numerous pro-inflammatory and pro-atherogenic genes. Although a some similarity of the gene expression patterns was observed when macrophages were activated with POVPC versus PAF, we observed that only POVPC treatment induced a several-fold activation of IL-8 gene. In turn, only PAF activated PAF-R, matrix metalloproteinase-13 and 15-lipoxygenase mRNA accumulation. Thus, we suggest, that POVPC signals in mature macrophages only in part through the PAF-R, a part of its effects may involve other receptors.

Platelet-activating factor increases VE-cadherin tyrosine phosphorylation in mouse endothelial cells and its association with the PtdIns3'-kinase.

Platelet-activating factor (PAF), a potent inflammatory mediator, is involved in endothelial permeability. This study was designed to characterize PAF receptor (PAF-R) expression and its specific contribution to the modifications of adherens junctions in mouse endothelial cells. We demonstrated that PAF-R was expressed in mouse endothelial cells and was functionally active in stimulating p42/p44 MAPK and phosphatidylinositol 3-kinase (PtdIns3'-kinase)/Akt activities. Treatment of cells with PAF induced a rapid time- and dose-dependent (10(-7) to 10(-10) M) increase in tyrosine phosphorylation of a subset of proteins ranging from 90 to 220 kDa, including the VE-cadherin, the latter effect being prevented by the tyrosine kinase inhibitors herbimycin A and bis-tyrphostin. We demonstrated that PAF promoted formation of multimeric aggregates of VE-cadherin with PtdIns3'-kinase, which was also inhibited by herbimycin and bis-tyrphostin. Finally, we show by immunostaining of endothelial cells VE-cadherin that PAF dissociated adherens junctions. The present data provide the first evidence that treatment of endothelial cells with PAF promoted activation of tyrosine kinases and the VE-cadherin tyrosine phosphorylation and PtdIns3'-kinase association, which ultimately lead to the dissociation of adherens junctions. Physical association between PtdIns3'-kinase, serving as a docking protein, and VE-cadherin may thus provide an efficient mechanism for amplification and perpetuation of PAF-induced cellular activation.

Role of lipid phosphate phosphatase 3 in human aortic endothelial cell function.

AIMS: Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase ß (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. METHODS AND RESULTS: To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. CONCLUSIONS: We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3.

Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis.

BACKGROUND: Obesity-associated dyslipidemia in humans is associated with increased low-density lipoprotein (LDL) oxidation. Mice with combined leptin and LDL receptor deficiency are obese and show severe dyslipidemia and insulin resistance. We investigated the association between oxidation of apolipoprotein B-containing lipoproteins, high-density lipoprotein (HDL) antioxidant defense, and atherosclerosis in these mice. METHODS AND RESULTS: LDL receptor knockout (LDLR-/-), leptin-deficient (ob/ob), double-mutant (LDLR-/-;ob/ob), and C57BL6 mice were fed standard chow. Double-mutant mice had higher levels of non-HDL (P<0.001) and HDL (P<0.01) cholesterol and of triglycerides (P<0.001). They also had higher oxidative stress, evidenced by higher titers of autoantibodies against malondialdehyde-modified LDL (P<0.001). C57BL6 and ob/ob mice had no detectable lesions. Lesions covered 20% of total area of the thoracic abdominal aorta in double-mutant mice compared with 3.5% in LDLR-/- mice (P<0.01). Higher macrophage homing and accumulation of oxidized apolipoprotein B-100-containing lipoproteins were associated with larger plaque volumes in the aortic root of double-mutant mice (P<0.01). The activity of the HDL-associated antioxidant enzymes paraoxonase and lecithin:cholesterol acyltransferase (LCAT) (ANOVA; P<0.0001 for both) was lower in double-mutant mice. Adenovirus-mediated LCAT gene transfer in double-mutant mice increased plasma LCAT activity by 64% (P<0.01) and reduced the titer of autoantibodies by 40% (P<0.01) and plaque volume in the aortic root by 42% (P<0.05) at 6 weeks. CONCLUSIONS: Dyslipidemia and insulin resistance in obese LDL receptor-deficient mice are associated with increased oxidative stress and impaired HDL-associated antioxidant defense, evidenced by decreased paraoxonase and LCAT activity. Transient LCAT overexpression was associated with a reduction of oxidative stress and atherosclerosis.

Weight-loss-associated induction of peroxisome proliferator-activated receptor-alpha and peroxisome proliferator-activated receptor-gamma correlate with reduced atherosclerosis and improved cardiovascular function in obese insulin-resistant mice.

BACKGROUND: Weight loss in obese insulin-resistant but not in insulin-sensitive persons reduces coronary heart disease risk. To what extent changes in gene expression are related to atherosclerosis and cardiovascular function is unknown. METHODS AND RESULTS: We studied the effect of diet restriction-induced weight loss on gene expression in the adipose tissue, the heart, and the aortic arch and on atherosclerosis and cardiovascular function in mice with combined leptin and LDL-receptor deficiency. Obesity, hypertriglyceridemia, and insulin resistance are associated with hypertension, impaired left ventricular function, and accelerated atherosclerosis in those mice. Compared with lean mice, peroxisome proliferator-activated receptors (PPAR)-alpha and PPAR-gamma expression was downregulated in obese double-knockout mice. Diet restriction caused a 45% weight loss, an upregulation of PPAR-alpha and PPAR-gamma, and a change in the expression of genes regulating glucose transport and insulin sensitivity, lipid metabolism, oxidative stress, and inflammation, most of which are under the transcriptional control of these PPARs. Changes in gene expression were associated with increased insulin sensitivity, decreased hypertriglyceridemia, reduced mean 24-hour blood pressure and heart rate, restored circadian variations of blood pressure and heart rate, increased ejection fraction, and reduced atherosclerosis. PPAR-alpha and PPAR-gamma expression was inversely related to plaque volume and to oxidized LDL content in the plaques. CONCLUSIONS: Induction of PPAR-alpha and PPAR-gamma in adipose tissue, heart, and aortic arch is a key mechanism for reducing atherosclerosis and improving cardiovascular function resulting from weight loss. Improved lipid metabolism and insulin signaling is associated with decreased tissue deposition of oxidized LDL that increases cardiovascular risk in persons with the metabolic syndrome.