Lipoprotein-associated Phospholipase A2 Expression Research Articles

Lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells is inhibited by microRNA-494-3p via targeting lipoprotein-associated phospholipase A2.

Gram-negative bacterial lipopolysaccharide (LPS) is a major component of inflammation and plays a key role in the pathogenesis of sepsis. According to our previous study, the expression of lipoprotein-associated phospholipase A2 (Lp-PLA2) is significantly upregulated in septic patients and is positively correlated with the severity of this disease. Herein, we investigated the potential roles of Lp-PLA2-targeting microRNAs (miRNAs) in LPS-induced inflammation in murine mononuclear macrophages (RAW264.7 cells). In LPS-stimulated RAW264.7 cells, Lp-PLA2 was confirmed to be expressed during the inflammatory response. The function of microRNA-494-3p (miR-494-3p) in the LPS-induced inflammatory response of RAW264.7 cells was determined by the transfection of a miR-494-3p mimic or inhibitor in vitro. Compared to the control, LPS induced a significant increase in the Lp-PLA2 level, which was accompanied by the release of inflammatory mediators. The bioinformatics and qRT‒PCR results indicated that the miR-494-3p level was associated with Lp-PLA2 expression in the LPS-induced inflammatory response of RAW264.7 cells. Dual-luciferase reporter assay results confirmed that the 3'-UTR of Lp-PLA2 was a functional target of microRNA-494-3p. During the LPS-induced inflammatory response of RAW264.7 cells, targeting Lp-PLA2 and transfecting miR-494-3p mimics significantly upregulated the expression of miR-494-3p, leading to a reduction in the release of inflammatory factors and conferring a protective effect on LPS-stimulated RAW264.7 cells. By targeting Lp-PLA2, miR-494-3p suppresses Lp-PLA2 secretion, thereby alleviating LPS-induced inflammation, which indicates that miR-494-3p may be a potential target for sepsis treatment.

Melatonin suppresses atherosclerosis by ferroptosis inhibition via activating NRF2 pathway.

Melatonin (MLT), a conserved small indole compound, exhibits anti-inflammatory and antioxidant properties, contributing to its cardioprotective effects. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with atherosclerosis disease risk, and is known as an atherosclerosis risk biomarker. This study aimed to investigate the impact of MLT on Lp-PLA2 expression in the atherosclerotic process and explore the underlying mechanisms involved. Invivo, ApoE-/- mice were fed a high-fat diet, with or without MLT administration, after which the plaque area and collagen content were assessed. Macrophages were pretreated with MLT combined with ox-LDL, and the levels of ferroptosis-related proteins, NRF2 activation, mitochondrial function, and oxidative stress were measured. MLT administration significantly attenuated atherosclerotic plaque progression, as evidenced by decreased plaque area and increased collagen. Compared with those in the high-fat diet (HD) group, the levels of glutathione peroxidase 4 (GPX4) and SLC7A11 (xCT, a cystine/glutamate transporter) in atherosclerotic root macrophages were significantly increased in the MLT group. Invitro, MLT activated the nuclear factor-E2-related Factor 2 (NRF2)/SLC7A11/GPX4 signaling pathway, enhancing antioxidant capacity while reducing lipid peroxidation and suppressing Lp-PLA2 expression in macrophages. Moreover, MLT reversed ox-LDL-induced ferroptosis, through the use of ferrostatin-1 (a ferroptosis inhibitor) and/or erastin (a ferroptosis activator). Furthermore, the protective effects of MLT on Lp-PLA2 expression, antioxidant capacity, lipid peroxidation, and ferroptosis were decreased in ML385 (a specific NRF2 inhibitor)-treated macrophages and in AAV-sh-NRF2 treated ApoE-/- mice. MLT suppresses Lp-PLA2 expression and atherosclerosis processes by inhibiting macrophage ferroptosis and partially activating the NRF2 pathway.

Lp-PLA2 silencing ameliorates inflammation and autophagy in nonalcoholic steatohepatitis through inhibiting the JAK2/STAT3 pathway.

Nonalcoholic steatohepatitis (NASH), a common cause of liver-related morbidity and mortality worldwide, is characterized by inflammation and hepatocellular injury. Our research focuses on lipoprotein-associated phospholipase A2 (Lp-PLA2), an inflammation-related biomarker that has recently garnered interest in the context of NASH due to its potential roles in disease pathogenesis and progression. We established a NASH mouse model using a high-fat diet (HFD) and treated it with sh-Lp-PLA2 and/or rapamycin (an mTOR inhibitor). Lp-PLA2 expression in NASH mice was detected by qRT-PCR. Serum levels of liver function parameters and inflammatory cytokines were detected using corresponding assay kits. We examined pathological changes in liver using hematoxylin-eosin, oil red O, and Masson staining, and observed autophagy through transmission electron microscopy. The protein levels of Lp-PLA2, mTOR, light chain 3 (LC3) II/I, phosphorylated Janus kinase 2 (p-JAK2)/JAK2, and phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/STAT3 were determined by western blotting. Kupffer cells extracted from C57BL/6J mice were treated to replicate NASH conditions and treated with sh-Lp-PLA2, rapamycin, and/or a JAK2-inhibitor to further verify the roles and mechanisms of Lp-PLA2 in NASH. Our data indicate an upregulation of Lp-PLA2 expression in HFD-induced NASH mice. Silencing Lp-PLA2 in NASH mice reduced liver damage and inflammation markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), triglycerides (TG), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6)), while increasing IL-10 levels, an anti-inflammatory cytokine. Additionally, Lp-PLA2 silencing decreased lipid and collagen accumulation and promoted autophagy. The beneficial effects of sh-Lp-PLA2 on NASH were enhanced by rapamycin. Furthermore, Lp-PLA2 silencing resulted in the downregulation of the expression of p-JAK2/JAK2 and p-STAT3/STAT3 in NASH mice. Similar results were observed in Kupffer cells treated under NASH conditions; Lp-PLA2 silencing promoted autophagy and repressed inflammation, effects which were potentiated by the addition of rapamycin or a JAK2-inhibitor. Our findings suggest that silencing Lp-PLA2 promotes autophagy via deactivating the JAK2/STAT3 signaling pathway, thereby restraining NASH progression. This highlights the potential therapeutic value of targeting Lp-PLA2, adding a new dimension to our understanding of NASH pathogenesis and treatment strategies.

DNA methylome profiling reveals epigenetic regulation of lipoprotein-associated phospholipase A2 in human vulnerable atherosclerotic plaque

BackgroundAtherosclerotic plaque vulnerability is a key feature of atheroprogression and precipitating acute cardiovascular events. Although the pivotal role of epigenetic regulation in atherosclerotic plaque destabilization is being recognized, the DNA methylation profile and its potential role in driving the progression and destabilization of atherosclerotic cardiovascular disease remains largely unknown. We conducted a genome-wide analysis to identify differentially methylated genes in vulnerable and non-vulnerable atherosclerotic lesions to understand more about pathogenesis.ResultsWe compared genome-wide DNA methylation profiling between carotid artery plaques of patients with clinically symptomatic (recent stroke or transient ischemic attack) and asymptomatic disease (no recent stroke) using Infinium Methylation BeadChip arrays, which revealed 90,368 differentially methylated sites (FDR < 0.05, |delta beta|> 0.03) corresponding to 14,657 annotated genes. Among these genomic sites, 30% were located at the promoter regions and 14% in the CpG islands, according to genomic loci and genomic proximity to the CpG islands, respectively. Moreover, 67% displayed hypomethylation in symptomatic plaques, and the differentially hypomethylated genes were found to be involved in various aspects of inflammation. Subsequently, we focus on CpG islands and revealed 14,596 differentially methylated sites (|delta beta|> 0.1) located at the promoter regions of 7048 genes. Integrated analysis of methylation and gene expression profiles identified that 107 genes were hypomethylated in symptomatic plaques and showed elevated expression levels in both advanced plaques and ruptured plaques. The imprinted gene PLA2G7, which encodes lipoprotein-associated phospholipase A2 (Lp-PLA2), was one of the top hypomethylated genes with an increased expression upon inflammation. Further, the hypomethylated CpG site at the promoter region of PLA2G7 was identified as cg11874627, demethylation of which led to increased binding of Sp3 and expression of Lp-PLA2 through bisulfate sequencing, chromatin immunoprecipitation assay and enzyme-linked immunosorbent assay. These effects were further enhanced by deacetylase.ConclusionExtensive DNA methylation modifications serve as a new and critical layer of biological regulation that contributes to atheroprogression and destabilization via inflammatory processes. Revelation of this hitherto unknown epigenetic regulatory mechanism could rejuvenate the prospects of Lp-PLA2 as a therapeutic target to stabilize the atherosclerotic plaque and reduce clinical sequelae.

Everolimus reduces atherosclerosis and lipoprotein-associated phospholipase A2 (LP-PLA2) secretion in apolipoprotein e knockout mice

Background and Aims: Several epidemiological studies have shown that lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent risk factor for coronary artery disease. Lp-PLA2 is secreted by proinflammatory cells and is highly expressed in the atherosclerotically changed arterial wall. Recently, lower activity of Lp-PLA2 has been observed in plasmas from everolimus-treated patients. In vitro studies revealed reduced Lp-PLA2 expression in hepatocytes and macrophages pre-exposed to everolimus, while antiatherosclerotic activity of everolimus has been shown in animal models of disease.

Lipoprotein-Associated Phospholipase A2: A Novel Contributor in Sjögren's Syndrome-Related Lymphoma?

BackgroundB-cell non-Hodgkin’s lymphoma (B-NHL) is one of the major complications of primary Sjögren’s syndrome (SS). Chronic inflammation and macrophages in SS minor salivary glands have been previously suggested as significant predictors for lymphoma development among SS patients. Lipoprotein-associated phospholipase A2 (Lp-PLA2)—a product mainly of tissue macrophages—is found in the circulation associated with lipoproteins and has been previously involved in cardiovascular, autoimmune, and malignant diseases, including lymphoma.ObjectiveThe purpose of the current study was to investigate the contributory role of Lp-PLA2 in B-NHL development in the setting of primary SS.MethodsLp-PLA2 activity in serum samples collected from 50 primary SS patients with no lymphoma (SS-nL), 9 primary SS patients with lymphoma (SS-L), and 42 healthy controls (HC) was determined by detection of [3H]PAF degradation products by liquid scintillation counter. Moreover, additional sera from 50 SS-nL, 28 SS-L, and 32 HC were tested for Lp-PLA2 activity using a commercially available ELISA kit. Lp-PLA2 mRNA, and protein expression in minor salivary gland (MSG) tissue samples derived from SS-nL, SS-L patients, and sicca controls (SC) were analyzed by real-time PCR, Western blot, and immunohistochemistry.ResultsSerum Lp-PLA2 activity was significantly increased in SS-L compared to both SS-nL and HC by two independent methods implemented [mean ± SD (nmol/min/ml): 62.0 ± 13.4 vs 47.6 ± 14.4 vs 50.7 ± 16.6, p-values: 0.003 and 0.04, respectively, and 19.4 ± 4.5 vs 15.2 ± 3.3 vs 14.5 ± 3.0, p-values: <0.0001, in both comparisons]. ROC analysis revealed that the serum Lp-PLA2 activity measured either by radioimmunoassay or ELISA has the potential to distinguish between SS-L and SS-nL patients (area under the curve [AUC]: 0.8022, CI [95%]: 0.64–0.96, p-value: 0.004 for radioimmunoassay, and AUC: 0.7696, CI [95%]: 0.66–0.88, p-value: <0.0001, for ELISA). Lp-PLA2 expression in MSG tissues was also increased in SS-L compared to SS-nL and SC at both mRNA and protein level. ROC analysis revealed that both MSG mRNA and protein Lp-PLA2 have the potential to distinguish between SS-nL and SS-L patients (area under the curve [AUC] values of 0.8490, CI [95%]: 0.71–0.99, p-value: 0.0019 and 0.9444, CI [95%]: 0.79–1.00, p- value: 0.0389 respectively). No significant difference in either serum Lp-PLA2 activity or MSG tissue expression was observed between SS-nL and HC.ConclusionsLp-PLA2 serum activity and MSG tissue mRNA/protein expression could be a new biomarker and possibly a novel therapeutic target for B-cell lymphoproliferation in the setting of SS.

Radiation-induced lipoprotein-associated phospholipase A2 increases lysophosphatidylcholine and induces endothelial cell damage

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1β, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.

DNA Methylome Profiling Reveals Epigenetic Regulation of Lipoprotein-Associated Phospholipase A &lt;sub&gt;2&lt;/sub&gt; in Human Vulnerable Atherosclerotic Plaque

Background: Atherosclerotic plaque vulnerability is a key feature of atheroprogression and precipitating acute cardiovascular events. The DNA methylation profile and its potential role in driving the progression and destabilization of atherosclerotic cardiovascular disease remains largely unknown. We conducted a genome-wide analysis to identify differentially methylated genes in vulnerable and non-vulnerable atherosclerotic lesions to understand more about pathogenesis. Methods: Carotid artery plaques were obtained from patients with clinically symptomatic disease (recent stroke or transient ischemic attack) and asymptomatic disease. Genome-wide DNA methylation was profiled using Infinium Methylation BeadChip arrays. Gene ontology analysis was performed to reveal pathophysiological functions of differentially methylated genes. The hypomethylated genes with increased expression in atherosclerotic plaques were identified by an integrated analysis of DNA methylome with transcriptomic data. The methylation status of the CpG sites at the promoter of the top hypomethylated genes was delineated by bisulfite sequencing. Findings: Genome-wide DNA methylation profiling of symptomatic and asymptomatic plaques identified 90,368 differentially methylated sites ( p <0.05) corresponding to 14,657 annotated genes. Among these genomic sites, 30% located at promoter regions and 14% located in CpG islands, according to genomic loci and genomic proximity to the CpG islands, respectively. Moreover, 67% displayed hypomethylation in symptomatic plaques, and the differentially hypomethylated genes were found to be involved in various aspects of inflammation. The imprinted gene PLA2G7, which encodes the lipoprotein-associated phospholipase A2 (Lp-PLA2), was one of the top hypomethylated genes with an increased expression upon inflammation. Further, the hypomethylated CpG site at the promoter region of PLA2G7 was identified as cg11874627, demethylation of which increased binding of Sp3 and expression of Lp-PLA2. Interpretation: Extensive DNA methylation modifications serve as a new and critical layer of biological regulation that contributes to atheroprogression and destabilization via inflammatory processes. Revelation of this hitherto unknown epigenetic regulatory mechanism could rejuvenate the prospects of Lp-PLA2 as a therapeutic target to stabilize the atherosclerotic plaque and reduce clinical sequelae. Funding: National Natural Science Foundation of China (81770359, 81270276, and 81500365); Beijing Health Technologies Promotion Program (BHTPP202004); Open Project Fund of State Key Laboratory of Molecular Developmental Biology of China (2021-MDB-KF-18 and 2020-MDB-KF-17); and Beijing Natural Science Foundation (7172040). Declaration of Interest: The authors declare no conflict of interest relevant to the present manuscript. Ethical Approval: All procedures involving humans were approved by the Institutional Ethics Review Board of China-Japan Friendship Hospital (No. 2019-85-K56) and conformed to the standards of the Declaration of Helsinki. All participants provided written informed consent before enrollment in this study.

The role of lipoprotein-associated phospholipase A2 in acute kidney injury of septic mice.

BackgroundThis experiment aimed to investigate the role and mechanism of lipoprotein-associated phospholipase A2 (Lp-PLA2) in kidney injury in septic mice induced by cecal ligation and perforation (CLP).MethodsMale BALB/c mice were randomly divided into two groups: sham-operation group (Sham group) and septic group (CLP group). The septic model was simulated by cecal ligation and puncture method, but only cecal ligation was used for the sham operation group. The whole serum and renal tissue samples of the mice were collected 24 hours after modeling in both groups. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, the renal injury score was recorded, and the creatinine (Cr) and blood urea nitrogen (BUN) levels were detected by automatic biochemical analyzer, while the serum Lp-PLA2 level was detected by enzyme-linked immunosorbent assay (ELISA). The 7-day survival rate and the survival curve of the two groups were statistically analyzed.ResultsCompared with the Sham group, the pathological score of renal injury in the CLP Group was higher, the level of Lp-PLA2 in serum was significantly increased (all P<0.01), and the expression of Lp-PLA2 in renal tissue was significantly elevated (all P<0.01). Furthermore, the 7-day survival rate of the Sham group was 90%, while that of CLP group was 25%.ConclusionsThe expression level of Lp-PLA2 in blood and kidney tissue of septic mice was increased and correlated with prognosis. However, the predictive value of Lp-PLA2 for prognosis in septic mice needs further study.

Association of Carotid Plaque and Serum Lipoprotein-Associated Phospholipase A2 (LP-PLA2) with Postoperative Delirium in Geriatric Patients Undergoing Hip Replacement: A Prospective Cohort Study.

BackgroundThe aim of this study was to investigate the relationships among carotid plaque (CP), serum lipoprotein-associated phospholipase (LP-PLA2), and POD in elderly patients.Material/MethodsSixty-two elderly patients undergoing hip replacement with spinal-epidural anesthesia were divided into CP and non-CP groups based on the preoperative presence or absence of carotid atherosclerotic plaques, as assessed by ultrasound. POD was diagnosed by means of the Confusion Assessment Method (CAM). Blood samples were collected (preoperatively, postoperatively, and postoperative day 2) for the assessment of serum LP-PLA2 by enzyme-linked immunosorbent assay. The CP group was further divided into POD and no-POD subgroups based on the occurrence of POD.ResultsThe incidence of POD was higher in the CP group than in the non-CP group (P<0.05). While the LP-PLA2 level did not significantly differ between CP and non-CP groups preoperatively (P>0.05), it was higher in the CP group than in the non-CP group postoperatively and on postoperative day 2 (P<0.05). In the CP group, the LP-PLA2 level did not significantly differ between the subgroups preoperatively or postoperatively (P>0.05), but was significantly higher in the POD subgroup than in the no-POD subgroup on postoperative day 2 (P<0.05). Furthermore, the LP-PLA2 level on postoperative day 2 was an independent risk factor for POD (odds ratio: 1.03, 95% confidence interval: 1.00–1.07).ConclusionsThe preoperative presence of carotid plaque is closely associated with a higher incidence of POD. The potential mechanism may involve the increased expression of LP-PLA2 in the serum, which can lead to plaque destabilization and subsequent inflammatory cascades.

Interleukin-4, hemopexin, and lipoprotein-associated phospholipase A2 are significantly increased in patients with unstable carotid plaque

AbstractObjectiveThis study aimed to compare the plasma levels of lipoprotein-associated phospholipase A2 (Lp-PLA2), hemopexin (Hpx), and interleukin-4 (IL-4) in patients with carotid artery atherosclerosis based on neurological symptoms and plaque histopathology and to find association between plaque stability and neurological symptoms. This single-center study included patients treated surgically for significant stenosis of the internal carotid artery. Serum levels of biomarkers were determined, and a histopathological analysis of the carotid plaques was performed. Within 70 patients, 40 asymptomatic and 30 symptomatic; 38 patients (54.3%) were diagnosed with unstable carotid plaque and 32 patients (45.7%) had a stable carotid plaque. Significantly higher incidence of unstable carotid plaque was detected in symptomatic patients (p &lt;0.001). Compared to asymptomatic patients, higher expression of Lp-PLA2 (285.30 ± 2.05 μg/l), Hpx (0.38 ± 0.01 ng/l), and IL-4 (65.77 ± 3.78 ng/l) in plasma were detected in symptomatic patients. Subsequently, higher expression of Lp-PLA2 (297.34 ± 2.3 μg/l), Hpx (0.41 ± 0.02 ng/l), and IL-4 (64.74 ± 4.47 ng/l) in plasma was observed in patients with unstable plaques (n=38). Statistically significant (p &lt;0.001) differences in expression of Lp-PLA2, Hpx, and IL-4 between patients with unstable and stable plaques were detected. Moreover, only the differences between symptomatic and asymptomatic patients in the expression of Lp-PLA2 and IL-4 in plasma were statistically significant (p &lt;0.001). This study showed that Lp-PLA2, IL-4, and Hpx levels are significantly increased in patients with an unstable carotid plaque.

Effects of dl-3-n-butylphthalide on serum lipoprotein-associated phospholipase A2 and hypersensitive C-reactive protein levels in acute cerebral infarction.

ObjectiveThis study aims to explore the curative effect of dl‐3‐n‐butylphthalide (NBP) on patients with acute cerebral infarction (ACI) and its effects on serum lipoprotein‐associated phospholipase A2 (Lp‐PLA2) and hypersensitive C‐reactive protein (hs‐CRP) levels.MethodsA total of 136 ACI patients treated in our hospital, who met the criteria, were selected and randomly divided into two groups: control group (n = 60, including 28 males and 32 females) and treatment group (n = 76, including 32 males and 44 females). Patients in the control group were treated with routine drug therapy, while patients in the treatment group were treated with NBP on this basis. A dose of 100 ml was administered by intravenous injection for 2 times/day, for 14 days. The curative effect was evaluated using the National Institute of Health Stroke Scale (NIHSS) and Barthel index (BI) self‐care ability. The levels of the two factors in serum were measured using enzyme‐linked immunosorbent assay, and the changes in levels of these two factors in serum at different time points before and after treatment were compared between the two groups.Results(a) Lp‐PLA2 and hs‐CRP levels in the treatment group after treatment were significantly lower than those before treatment and those in the control group after treatment (p < .05). (b) The NIHSS and BI scores in the treatment group were significantly lower after treatment than before treatment and those in the control group after treatment (p < .05).ConclusionDl‐3‐n‐butylphthalide can improve the expression of Lp‐PLA2 and hs‐CRP in serum in ACI patients. Furthermore, NBP has significant efficacy in inhibiting inflammation and improving neurological symptoms.

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by combined RNA interference of lipoprotein-associated phospholipase A2 and YKL-40.

To test the hypothesis that combined RNA interference (RNAi) of lipoprotein-associated phospholipase A2 (Lp-PLA2) and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis. A total of 120 apolipoprotein E-deficient mice (apoE-/- mice) were randomly divided into five groups, including the vehicle alone, scrambled RNAi, Lp-PLA2 RNAi, YKL-40 RNAi, and combined Lp-PLA2 and YKL-40 RNAi groups. Constrictive collars were used to induce plaque formation. Lp-PLA2 RNAi and YKL-40 RNAi viral suspensions were transduced into carotid plaques of the mice. Carotid plaques were harvested for histological analysis four weeks after viral vector transduction. Inflammatory gene expression in the plasma and atherosclerotic plaques was determined by ELISA and real-time PCR. Four weeks after RNAi, the serum concentration and plaque mRNA expression of Lp-PLA2 and YKL-40 were remarkably attenuated, leading to reduced inflammatory gene expression. Plaques from the Lp-PLA2 or YKL-40 RNAi group showed lower lipid content, higher collagen content, increased fibrous cap thickness, and lower mRNA expressions of MCP-1 and MMP-8 than than those in the vehicle and scramble groups. When compared with the isolated Lp-PLA2 or YKL-40 RNAi group, the combined Lp-PLA2 and YKL-40 RNAi group exhibited higher collagen content and fibrous cap thickness, and lower lipid content and local inflammation. The beneficial effects of RNAi were independent of the plasma lipoprotein profile. Combined RNAi of Lp-PLA2 and YKL-40 is superior to RNAi of Lp-PLA2 or YKL-40 alone in ameliorating atherosclerosis.

Interrelationship between nuclear factor-erythroid-2-related factor 2, NADPH quinone oxidoreductase and lipoprotein-associated phospholipase A2 expression in young patients of metabolic syndrome

Metabolic syndrome (MS) is associated with inflammation and oxidative stress (OS). Keap1/Nrf2/ARE is a cytoprotective pathway induced by OS and inflammation. This study aims to evaluate the expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) and its downstream target gene NADPH quinone oxidoreductase-1 (NQO-1) in MS. Since lipoprotein-associated phospholipase A2(LpPLA2) is an important inflammatory marker believed to have a role in complications of MS, the association of its expression with that of Nrf2 and NQO-1 was also studied. Medical students (n = 26) were categorised in two groups according to NCEP ATP III criteria with WHO criteria for obesity for South Asian region: patients of MS (n = 13) and controls (n = 13). mRNA expression of Nrf2, NQO-1 and LpPLA2 genes was evaluated by qPCR in blood using specific primers. Fold change was calculated by 2–ΔΔcT method keeping β-actin as internal control. Expression of NQO-1 and LpPLA2 was found to be higher in MS. However, Nrf2 expression was low in patients who had hypertriglyceridemia when compared with patients with normal triglyceride levels. A significant correlation was observed in expression of LpPLA2, with Nrf2 and NQO-1. Our data suggests that there may be compensatory activation of antioxidant defence mechanism in young patients of MS. Further evidence is provided by higher expression of LpPLA2 and its correlation with Nrf2 and NQO-1 in MS which suggests that inflammatory stress may induce expression of genes of cytoprotective pathways. Additionally, this study, for the first time, indicates that Nrf2 may have some role in regulating triglyceride (TG) concentration.

Abstract TP120: Elevated Lipoprotein-Associated Phospholipase A2 is Differentially Associated With Symptomatic Intracranial Atherosclerosis

Background and Objective: Intracranial atherosclerotic disease (ICAD) is a common etiology of stroke in China. Lipoprotein-associated phospholipaseA2 (Lp-PLA2), an inflammatory enzyme expressed in atherosclerotic plaques, has been linked to an increased risk of ischemic stroke. Relatively few studies have explored the relationship between Lp-PLA2 and various ischemic stroke subtypes. We aimed to determine the association between the serum Lp-PLA2 mass and the main types of symptomatic large vessel atherosclerosis. Methods: In this cross-sectional study, we reviewed data on 126 eligible acute ischemic stroke patients consecutively admitted to a single medical center in China from March 2014 to February 2016. Clinical symptoms, signs and traditional risk factors of cerebrovascular diseases were recorded. The level of serum lipids, homocysteine and glucose etc. were collected. Serum Lp-PLA2 and high-sensitivity C-reactive protein (hs-CRP) were measured. According to the results of CT angiography, patients were divided into four different groups: 1) no artery stenosis (N), 2) both intracranial and extracranial artery stenosis (B), 3) intracranial artery stenosis (I), 4) extracranial artery stenosis (E). The association of serum Lp-PLA2 and other inflammatory markers to ICAD were analyzed with Pearson correlation test. Results: Lp-PLA2 expression in the ICAD group was higher than other three groups (I vs. N vs. B vs. E: 112.22±66.79 ug/l vs. 81.70±38.50 ug/l vs.89.25±52.15 ug/l vs. 106.11± 57.76 ug/l, p=0.025). When Lp-PLA2 was above the median level, the presence of ICAD was higher than other three groups (Q3: 16[50.0%] vs. 1[3.1%] vs. 9[28.1%] vs. 6[18.8%], P=0.002; Q4: 15[48.4%] vs. 5[16.1%] vs. 8[25.8%] vs. 3[9.7%], P=0.014). In ICAD group, Lp-PLA2 mass was positively correlated with hs-CRP (r=0.899, P &lt;0.001), and inversely correlated with total cholesterol (r=-0.307, P =0.028) and apoliprotein B (r=-0.320, P =0.022). Conclusions: Lp-PLA2 is differentially associated with ICAD compared to extracranial artery stenosis or no stenosis, which suggests that inflammation may play a greater role in the pathophysiology of ICAD versus these other symptomatic stroke categories.

Maternal Gestational Diabetes Mellitus increases placental and foetal lipoprotein-associated Phospholipase A2 which might exert protective functions against oxidative stress

Increased Lipoprotein associated phospholipase A2 (LpPLA2) has been associated with inflammatory pathologies, including Type 2 Diabetes. Studies on LpPLA2 and Gestational Diabetes Mellitus (GDM) are rare, and have focused mostly on maternal outcome. In the present study, we investigated whether LpPLA2 activity on foetal lipoproteins is altered by maternal GDM and/or obesity (a major risk factor for GDM), thereby contributing to changes in lipoprotein functionality. We identified HDL as the major carrier of LpPLA2 activity in the foetus, which is in contrast to adults. We observed marked expression of LpPLA2 in placental macrophages (Hofbauer cells; HBCs) and found that LpPLA2 activity in these cells was increased by insulin, leptin, and pro-inflammatory cytokines. These regulators were also increased in plasma of children born from GDM pregnancies. Our results suggest that insulin, leptin, and pro-inflammatory cytokines are positive regulators of LpPLA2 activity in the foeto-placental unit. Of particular interest, functional assays using a specific LpPLA2 inhibitor suggest that high-density lipoprotein (HDL)-associated LpPLA2 exerts anti-oxidative, athero-protective functions on placental endothelium and foetus. Our results therefore raise the possibility that foetal HDL-associated LpPLA2 might act as an anti-inflammatory enzyme improving vascular barrier function.

Resveratrol suppresses lipoprotein-associated phospholipase A2 expression by reducing oxidative stress in macrophages and animal models.

Resveratrol is a naturally occurring polyphenolic compound with known cardioprotective, anti-inflammatory, and antioxidant properties. Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of resveratrol on Lp-PLA2 expression in vitro and in vivo and explored the underlying mechanisms. Human monocytic cells (THP-1) were induced to differentiate into macrophages for an in vitro experimental model. Resveratrol suppressed Lp-PLA2 expression and reduced inflammation; lipopolysaccharide (LPS, 1 μg/mL), tumor necrosis factor-α (TNF-α, 10 ng/mL) and reactive oxygen species (ROS) were employed to stimulate an increase in Lp-PLA2 expression and ROS levels, and the stimulation was inhibited by resveratrol (50 μM) and other antioxidants. The inhibition of resveratrol was inversed partially by sirtuin 1 (SIRT1) inhibitors (Nicotinamide, 1-10 mM) (p<0.05). Next, a chronic inflammation mouse model induced by a HFD (high fat diet) supplemented with resveratrol 100 mg/kg/day orally for 12 weeks, resulted in resveratrol-induced decreases in the Lp-PLA2 levels in the plasma and liver and increases in the superoxide dismutase 2 (SOD2) expression in the liver (p<0.05). Based on our results, the protective effects of resveratrol on cardiovascular events may be related to its ability to suppress Lp-PLA2 expression.

AMP-activated protein kinase mediates the effects of lipoprotein-associated phospholipase A2 on endothelial dysfunction in atherosclerosis.

The present study aimed to investigate the effects of lipoprotein-associated phospholipase A2 (Lp-PLA2) on endothelial dysfunction in an in vitro cell model of atherosclerosis, and to determine whether AMP-activated protein kinase (AMPK) mediates the effects of Lp-PLA2 on endothelial dysfunction. A total of 392 patients with coronary artery disease (CAD), including various sub-conditions, were recruited, and the plasma concentrations of Lp-PLA2 were evaluated. In addition, an in vitro model of atherosclerosis was established by exposing human umbilical vein endothelial cells (HUVECs) to oxidized low-density lipoprotein (oxLDL). SB-435495 was used to inhibit Lp-PLA2, and compound C was used to suppress AMPK expression. Lp-PLA2, AMPKα and phosphorylated-AMPKα (T172) expression in HUVECs were evaluated using western blot analysis. The concentrations of nitric oxide (NO), endothelin 1 (ET-1), intercellular adhesion molecule 1 (ICAM-1) and platelet/endothelial cell adhesion molecule 1 (PECAM-1) in cell culture supernatant were determined using commercially available ELISA kits. MTT assays were employed to indicate changes in cell viability. The current study found the plasma Lp-PLA2 levels were elevated in the CAD patients with stable angina pectoris, unstable angina pectoris, acute coronary syndromes and acute myocardial infarction, compared with a healthy control population. In addition, the in vitro results showed that Lp-PLA2 expression levels were elevated in oxLDL-exposed HUVECs. Lp-PLA2 suppression could increase cell viability, induce the production of NO and decrease the secretion of ET-1, in addition to suppressing the expression of cell adhesion molecules, including ICAM-1 and PECAM-1 in oxLDL-exposed HUVECs. The expression of AMPKα and phosphorylated-AMPKα (T172) was regulated by Lp-PLA2, and AMPK suppression was able to reverse the effects of Lp-PLA2 with regard to cell viability, endothelial vasorelaxation capacity and the secretion of adhesion molecules in oxLDL-exposed HUVECs. In conclusion, the present study provides initial evidence that Lp-PLA2 is able to cause endothelial dysfunction in an in vitro model of atherosclerosis, and the effects of Lp-PLA2 on endothelial dysfunction was at least partially a result of the downregulation of AMPKα, thus contributing to the progression of atherosclerosis.

Differential Expression of Lipoprotein-Associated Phospholipase A2 in Monocyte Subsets: Impact of Uremia and Atherosclerosis

Background: Monocytic products, such as lipoprotein-associated phospholipase A2 (Lp-PLA2), may participate in the development of atherosclerosis. Heterogeneity of monocytes is widely acknowledged. Classical, intermediate, and non-classical subsets can be discerned. Recently, an inflammatory, pro-atherogenic monocyte population could be identified in hemodialysis patients. In this study, we investigated the expression of Lp-PLA2 on leucocytes and different monocyte subpopulations and their possible role in uremia, inflammation, and atherosclerosis. Methods: Chronic kidney disease stage 5-D (CKD5-D; n = 57), healthy control subjects with hs-C-reactive protein (CRP) levels ≤1 mg/L (CO-N, n = 22) and a control group with inflammatory activation (CRP levels >1 mg/L, CO-I, n = 29) were enrolled in this cross-sectional observation. The CKD5-D cohort was dichotomized into patients with (A+) and without subclinical atherosclerosis (A-) by carotid artery ultrasound measurement. Lp-PLA2 activity was determined in plasma samples, Lp-PLA2 mRNA expression analysis in leucocytes, and sorted monocyte subsets. Effects of Lp-PLA2 overexpression were studied in classical vs. intermediate and non-classical subsets. Results: The classical monocytes expressed the highest Lp-PLA2 mRNA levels as compared to other subpopulations. CKD5-D patients revealed significantly higher Lp-PLA2 transcripts, as well as higher Lp-PLA2 plasma activity as compared to healthy and “inflammatory” controls. In vitro data confirmed that uremia significantly contributes to Lp-PLA2 mRNA upregulation. Non-classical monocytes of A+ patients revealed significant higher Lp-PLA2 mRNA compared to A-. Conclusion: Uremic environment but not inflammation per se increases plasma Lp-PLA2 activity and upregulates monocytic Lp-PLA2 mRNA expression. The highest Lp-PLA2 levels were found in the classical and not in the inflammatory subsets. Atherosclerosis also contributes to a subset-specific increase in Lp-PLA2 mRNA expression.

Elaborate evaluation of serum and tissue oxidized LDL level with darapladib therapy: A feasible diagnostic marker for early atherogenesis

ObjectiveTo compare oxidized low density lipoprotein (oxLDL) levels in serum and vascular wall of Sprague-Dawley rats, identify their patterns in 8 weeks and 16 weeks of dyslipidemia induced by high fat diet, compare foam cells in aorta of each group and investigate lipoprotein-associated phospholipase A2 (Lp-PLA2) role in atherosclerosis by darapladib administration. MethodsThis study generated in twenty-four Sprague-Dawley rats. Rats were divided into 6 groups, which were received normal diet (normal group), high fat diet and high fat diet plus darapladib therapy for both 8 weeks and 16 weeks. Surgeries were performed at Week 8 and Week 16 to take the blood serum and aortic tissue. Level of oxLDL in serum, oxLDL aortic tissue, foam cell amount in aortic tissue, and Lp-PLA2 expression in aortic tissue were measured. ResultsThere were significant differences in oxLDL level in serum, aortic tissue and foam cell amount (P < 0.05). There was no significant difference in Lp-PLA2 expression in aortic tissue. OxLDL in serum and aortic tissue had a very strong correlation (r2 > 0.9, P < 0.05). This study also composed an equation for oxLDL level in aortic tissue prediction. Factorial ANOVA found that there was a significant difference of oxLDL level in the interactions between duration and location, location and treatment, and also duration, location and treatment (P < 0.01). Administration of darapladib was able to reduce levels of oxLDL in serum, aortic tissue and foam cell significantly (P < 0.05, P < 0.05 and P < 0.01, subsequently). ConclusionsOxLDL level is location-dependent and duration-dependent. As a feasible early diagnosis, we can predict oxLDL level in aortic tissue by its level in serum. Though Lp-PLA2 expression was unsignificant, Lp-PLA2 inhibition by darapladib can reduce oxidative stress and inflammation in atherogenesis.