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.

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.

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.

Plasma PAFAH/PLA2G7 Genetic Variability, Cardiovascular Disease, and Clinical Trials.

PAFAH is specific for short acyl groups esterified at the sn-2 position of glycerol in phospholipids, and apart from PAF, it hydrolyzes oxidized phospholipids produced during LDL oxidation. As the majority of the plasma PAFAH activity is bound in humans to LDL, it is also called the lipoprotein-associated phospholipase A2 (Lp-PLA2), and it was associated with the proinflammatory processes in atherosclerosis. The epidemiological studies in Caucasian populations demonstrated that high PAFAH levels might be a risk factor for cardiovascular disease through generation of proinflammatory lysoPC/lysoPAF and oxidized fatty free acids and led to the development of darapladib, a reversible PAFAH inhibitor. In the preclinical study in diabetic and hypercholesterolemic pigs, darapladib decreased both plasma and in situ lesion PAFAH/Lp-PLA2 activity, reduced lesion lysoPC content, and also reduced the complex coronary lesion development by reducing the necrotic core. In the recently published double-blind trial with darapladib (STABILITY study), it was shown that darapladib did not affect the time to cardiovascular death, myocardial infarction, or stroke. Similarly, in the SOLID-TIMI 52 study, darapladib did not reduce the risk of major coronary events; for those reasons, the clinical trials with darapladib will probably definitely stop in this pathology. Finally, in the absence of a tangible effect of V279F loss-of-function mutation on the cardiovascular risk in Asiatic populations and no effect of A379V polymorphism which modifies PAFAH activity in Caucasians, combined with no effect of the anti-PAFAH/Lp-PLA2 drug darapladib in clinical trials, let us conclude that it is unlikely that PAFAH could be implicated in atherosclerosis per se. We rather believe that PAFAH/Lp-PLA2 is a biomarker of atherosclerosis.

SASH1, a new potential link between smoking and atherosclerosis.

OBJECTIVE: We have previously reported that SASH1 expression is increased in circulating human monocytes from smokers and was positively correlated with the number of carotid atherosclerotic plaques. The aim of this study was to further validate the link between smoking, SASH1 and atherosclerosis within the vascular wall and to assess the impact of SASH1 expression on endothelial cell functions. METHOD: Human carotids with atherosclerotic plaques were obtained from 58 patients (45 of them with known smoking status: smoker, non-smoker, ex-smokers), and were processed for gene expression analyses and immunostaining. To investigate its function, SASH1 was silenced in human aortic endothelial cells (HAECs) using two different siRNA and subcellular localization of SASH1 was determined by immunostaining and subcellular fractionation. Subsequently the transcriptomic analyses and functional experiments (wound healing, WST-1 proliferation or Matrigel assays) were performed to characterize SASH1 function. RESULTS: SASH1 was expressed in human vascular cells (HAECs, smooth muscle cells) and in monocytes/macrophages. Its tissue expression was significantly higher in the atherosclerotic carotids of smokers compared to non-smokers (p < 0.01). In HAECs, SASH1 was expressed mostly in the cytoplasm and SASH1 knockdown resulted in an increased cell migration, proliferation and angiogenesis. Transcriptomic and pathway analyses showed that SASH1 silencing results in a decreased CYP1A1 expression possibly through the inhibition of TP53 activity. CONCLUSION: We showed that SASH1 expression is increased in atherosclerotic carotids in smokers and its silencing affects endothelial angiogenic functions; therefore we provide a potential link between smoking and atherosclerosis through SASH1 expression.

Bariatric Surgery Induces Disruption in Inflammatory Signaling Pathways Mediated by Immune Cells in Adipose Tissue: A RNA-Seq Study.

BACKGROUND: Bariatric surgery is associated to improvements in obesity-associated comorbidities thought to be mediated by a decrease of adipose inflammation. However, the molecular mechanisms behind these beneficial effects are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed RNA-seq expression profiles in adipose tissue from 22 obese women before and 3 months after surgery. Of 15,972 detected genes, 1214 were differentially expressed after surgery at a 5% false discovery rate. Upregulated genes were mostly involved in the basal cellular machinery. Downregulated genes were enriched in metabolic functions of adipose tissue. At baseline, 26 modules of coexpressed genes were identified. The four most stable modules reflected the innate and adaptive immune responses of adipose tissue. A first module reflecting a non-specific signature of innate immune cells, mainly macrophages, was highly conserved after surgery with the exception of DUSP2 and CD300C. A second module reflected the adaptive immune response elicited by T lymphocytes; after surgery, a disconnection was observed between genes involved in T-cell signaling and mediators of the signal transduction such as CXCR1, CXCR2, GPR97, CCR7 and IL7R. A third module reflected neutrophil-mediated inflammation; after surgery, several genes were dissociated from the module, including S100A8, S100A12, CD300E, VNN2, TUBB1 and FAM65B. We also identified a dense network of 19 genes involved in the interferon-signaling pathway which was strongly preserved after surgery, with the exception of DDX60, an antiviral factor involved in RIG-I-mediated interferon signaling. A similar loss of connection was observed in lean mice compared to their obese counterparts. CONCLUSIONS/SIGNIFICANCE: These results suggest that improvements of the inflammatory state following surgery might be explained by a disruption of immuno-inflammatory cascades involving a few crucial molecules which could serve as potential therapeutic targets.

PLA2G10 Gene Variants, sPLA2 Activity, and Coronary Heart Disease Risk.

BACKGROUND: Observational studies report that secretory phospholipase A2 (sPLA2) activity is a marker for coronary heart disease (CHD) risk, and activity measures are thought to represent the composite activity of sPLA2-IIA, -V, and -X. The aim of this study was to use genetic variants of PLA2G10, encoding sPLA2-X, to investigate the contribution of sPLA2-X to the measure of sPLA2 activity and coronary heart disease (CHD) risk traits and outcome. METHODS AND RESULTS: Three PLA2G10 tagging single-nucleotide polymorphisms (rs72546339, rs72546340, and rs4003232) and a previously studied PLA2G10 coding single-nucleotide polymorphism rs4003228, R38C, were genotyped in a nested case: control cohort drawn from the prospective EPIC-Norfolk Study (2175 cases and 2175 controls). Meta-analysis of rs4003228 (R38C) and CHD was performed using data from the Northwick Park Heart Study II and 2 published cohorts AtheroGene and SIPLAC, providing in total an additional 1884 cases and 3119 controls. EPIC-Norfolk subjects in the highest tertile of sPLA2 activity were older and had higher inflammatory markers compared with those in the lowest tertile for sPLA2 activity. None of the PLA2G10 tagging single-nucleotide polymorphism nor R38C, a functional variant, were significantly associated with sPLA2 activity, intermediate CHD risk traits, or CHD risk. In meta-analysis, the summary odds ratio for R38C was odds ratio=0.97 (95% confidence interval, 0.77-1.22). CONCLUSIONS: PLA2G10 variants are not significantly associated with plasma sPLA2 activity or with CHD risk.

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.

Graphical modeling of gene expression in monocytes suggests molecular mechanisms explaining increased atherosclerosis in smokers.

Smoking is a risk factor for atherosclerosis with reported widespread effects on gene expression in circulating blood cells. We hypothesized that a molecular signature mediating the relation between smoking and atherosclerosis may be found in the transcriptome of circulating monocytes. Genome-wide expression profiles and counts of atherosclerotic plaques in carotid arteries were collected in 248 smokers and 688 non-smokers from the general population. Patterns of co-expressed genes were identified by Independent Component Analysis (ICA) and network structure of the pattern-specific gene modules was inferred by the PC-algorithm. A likelihood-based causality test was implemented to select patterns that fit models containing a path "smoking→gene expression→plaques". Robustness of the causal inference was assessed by bootstrapping. At a FDR ≤0.10, 3,368 genes were associated to smoking or plaques, of which 93% were associated to smoking only. SASH1 showed the strongest association to smoking and PPARG the strongest association to plaques. Twenty-nine gene patterns were identified by ICA. Modules containing SASH1 and PPARG did not show evidence for the "smoking→gene expression→plaques" causality model. Conversely, three modules had good support for causal effects and exhibited a network topology consistent with gene expression mediating the relation between smoking and plaques. The network with the strongest support for causal effects was connected to plaques through SLC39A8, a gene with known association to HDL-cholesterol and cellular uptake of cadmium from tobacco, while smoking was directly connected to GAS6, a gene reported to have anti-inflammatory effects in atherosclerosis and to be up-regulated in the placenta of women smoking during pregnancy. Our analysis of the transcriptome of monocytes recovered genes relevant for association to smoking and atherosclerosis, and connected genes that before, were only studied in separate contexts. Inspection of correlation structure revealed candidates that would be missed by expression-phenotype association analysis alone.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells.

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

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.

Human group X secreted phospholipase A2 induces dendritic cell maturation through lipoprotein-dependent and -independent mechanisms.

OBJECTIVE: Increased secreted phospholipase A(2) (sPLA(2)) activity has been documented in several inflammatory disorders. Among sPLA(2)s, the human group X (hGX)-sPLA(2) has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA(2) has been detected in human atherosclerotic lesions, indicating that sPLA(2)s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression. METHODS AND RESULTS: In this study, we show that hGX-sPLA(2)-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA(2) phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA(2) added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA(2)-phospholipolysed LDL. CONCLUSION: hGX-sPLA(2) secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.

Genetic and environmental regulation of inflammatory CVD biomarkers Lp-PLA2 and IgM anti-PC.

OBJECTIVE: We set out to investigate the relative contribution of genetic and environmental effect on two inflammatory CVD biomarkers; lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and anti-phosphorylcholine IgM (anti-PC). Their relationships and possible co-regulation with other established CVD biomarkers are also examined. METHODS: Lp-PLA(2) activity (N=1600) and anti-PC (N=2036) levels were measured in elderly Swedish twins. Correlation analyses and heritability estimation were conducted by structural equation modeling. RESULTS: We attribute 0.37 of the variance of Lp-PLA(2) and 0.40 of anti-PC variance to genetic variance. In addition, a bivariate heritability of 0.33, 0.35 and 0.36 could be detected for levels of Lp-PLA(2) together with ApoB, total cholesterol and LDL, respectively. Anti-PC was only weakly related to other biomarkers of CVD, which may suggest a more independent role of anti-PC as a biomarker. CONCLUSIONS: In this large sample, Lp-PLA(2) activity has lower heritability and higher environmental regulation than previously reported. Anti-PC levels are partly influenced by dominance genetics and appear to be regulated independently of more established CVD biomarkers.

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.

Antagonistic regulation of macrophage phenotype by M-CSF and GM-CSF: implication in atherosclerosis.

OBJECTIVES: We characterized the transcriptional profiles of GM-CSF- (GM-MØ) and M-CSF-induced macrophages (M-MØ) and investigated in situ a subset of differentially expressed genes in human and mouse atherosclerotic lesions. METHODS AND RESULTS: Using microarrays we identified a number of genes and biological processes differentially regulated in M-MØ vs GM-MØ. By varying in culture the M-CSF/GM-CSF ratio (0-10), a spectrum of macrophage phenotypes was explored by RT-QPCR. M-CSF (10 ng/ml) stimulated expression of several genes, including selenoprotein-1 (SEPP1), stabilin-1 (STAB1) and CD163 molecule-like-1 (CD163L1) which was inhibited by a low dose of GM-CSF (1 ng/ml); M-CSF inhibited the expression of pro-platelet basic protein (PPBP) induced by GM-CSF. Combining tissue microarrays/quantitative immunohistochemistry of human aortic lesions with RT-QPCR expression data either from human carotids vs mammary non-atherosclerotic arteries or from the apoE null mice normal and atherosclerotic aortas showed that, STAB1, SEPP1 and CD163L1 (M-CSF-sensitive genes) and PPBP (GM-CSF-sensitive gene) were expressed in both human arterial and apoE null mice atherosclerotic tissues. CONCLUSION: A balance between M-CSF vs GM-CSF defines macrophage functional polarisation and may contribute to the progression of atherosclerosis.

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.

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.

Extracellular phospholipases in atherosclerosis.

Phospholipases A2 (PLA2) are a family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids liberating lysophospholipids and free fatty acids; important second messengers involved in atherogenesis. Plasma PAF-acetylhydrolase (PAF-AH) or Lp-PLA2 is a Ca(2+)-independent PLA2 which is produced by monocyte-derived macrophages and by activated platelets, and circulates in plasma associated with lipoproteins. PAF-AH catalyzes the removal of the acetyl/short acyl group at the sn-2 position of PAF and oxidized phospholipids produced during inflammation and oxidative stress. In humans, PAF-AH is mainly associated with small dense LDL and to a lesser extent with HDL and with lipoprotein(a). PAF-AH is N-glycosylated prior to secretion which diminishes its association with HDL raising the question of its distribution between the proatherogenic LDL vs the antiatherogenic HDL. Hypercholesterolemic patients have higher plasma PAF-AH activity which is reduced upon hypolipidemic therapy. PAF-AH specific inhibitor darapladib stabilizes human and swine plaques, therefore challenging the antiatherogenic potential of PAF-AH shown in small animal models. Among secreted PLA2s (sPLA2), the group X sPLA2 (PLA2GX), due to its very high activity towards phosphatidylcholine the main phospholipid of LDL, became an attractive target in atherosclerosis. We showed that PLA2GX is present in human atherosclerotic lesions and that the PLA2GX-phospholipolysed LDL triggers human macrophage-foam cell formation. In contrast to other sPLA2s, including group IB, IIA and V, PLA2GX can efficiently hydrolyze PAF present in lipoproteins or vesicles indicating that PLA2GX may be a novel player in PAF regulation upon inflammatory processes. By a genetic approach we uncovered a relatively rare polymorphism (Arg38Cys) which produces a catalytically inactive PLA2GX; although no association was observed with cardiovascular risk factors in the AtheroGene study, this result should be replicated in cohorts of other inflammatory diseases. We anticipate that mores studies will be necessary to sort out the exact role of extracellular PLA2 family members in atherosclerosis initiation and progression.

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.