Lipoprotein-associated Phospholipase A2 Expression Research Articles

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.

Lp-PLA2 silencing protects against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages

Atherosclerosis is a disease of the large- and medium-size arteries that is characterized by the formation of atherosclerotic plaques, in which foam cells are the characteristic pathological cells. However, the key underlying pathomechanisms are still not fully elucidated. In this study, we investigated the role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in ox-LDL-induced oxidative stress and cell apoptosis, and further, elucidated the potential machanisms in human THP1 macrophages. Flow cytometry and western blot analyses showed that both cell apoptosis and Lp-PLA2 expression were dose-dependently elevated after ox-LDL treatment for 24 h and also time-dependently increased after 50 mg/L ox-LDL incubation in THP1 macrophages. In addition, Lp-PLA2 silencing decreased ox-LDL-induced Lp-PLA2 and CD36 expression in THP1 macrophages. We also found that the levels of oil red O-staining, triglyceride (TG) and total cholesterol (TC) were significantly upregulated in ox-LDL-treated THP1 cells, but inhibited by Lp-PLA2 silencing. Furthermore, ox-LDL treatment resulted in significant increases of ROS and MDA but a marked decrease of SOD, effects that were reversed by Lp-PLA2 silencing in THP1 cells. Lp-PLA2 silencing reduced ox-LDL-induced cell apoptosis and caspase-3 expression in THP1 cells. Moreover, Lp-PLA2 siRNA transfection dramatically lowered the elevated levels of p-Akt and p-mTOR proteins in ox-LDL-treated THP1 cells. Both PI3K inhibitor LY294002 and mTOR inhibitor rapamycin decreased the augmented caspase-3 expression and TC content induced by ox-LDL, respectively. Taken together, these results revealed that Lp-PLA2 silencing protected against ox-LDL-induced oxidative stress and cell apoptosis via Akt/mTOR signaling pathway in human THP1 macrophages.

Expression of Lipoprotein associated phospholipase A2 enzyme in medical undergraduate students with metabolic syndrome

AimsMetabolic syndrome (MS) and atherosclerosis are chronic inflammatory conditions. Lipoprotein-associated phospholipase A2 (LpPLA2) is a circulatory marker of systemic inflammation and a risk predictor for cardiovascular diseases. This study aims to evaluate the expression of this enzyme in an effort to understand the underlying mechanism of atherosclerosis in MS. MethodsThis study included twenty patients of MS and same number of healthy controls. Anthropometry and clinical examination were carried out in both groups. Real time PCR was performed for LpPLA2 mRNA and relative expression was calculated using ΔΔCT method keeping β2 microglobin and β-actin as internal controls. ResultsLpPLA2 mRNA expression was higher in patients of MS. Fold change was 5.7 when β2 microglobin was used as normaliser and 4.97 when β-actin was used. mRNA levels of LpPLA2 correlated significantly with waist circumference (r=0.462, p=0.003) and systolic blood pressure (r=0.392, p=0.015) as well as high density lipoprotein cholesterol (r=−0.453, p=0.003). ConclusionsHigh expression of LpPLA2 mRNA indicates that systemic inflammation has role in pathogenesis of atherosclerosis in patients of MS. This is evident from its direct correlation with blood pressure. The study also suggests that expression of LpPLA2 may be associated with obesity. Therefore, LpPLA2 mRNA expression levels may develop as an important risk predictor for vascular complications in MS.

Overexpression of porcine lipoprotein-associated phospholipase A2 in swine

Lipoprotein-associated phospholipase A 2 (Lp-PLA2) is associated with the risk of vascular disease. It circulates in human blood predominantly in association with low-density lipoprotein cholesterol (LDL-C) and hydrolyses oxidized phospholipids into pro-inflammatory products. However, in the mouse circulation, it predominantly binds to high-density lipoprotein cholesterol (HDL-C) and exhibits anti-inflammatory properties. To further investigate the effects of Lp-PLA2 in the circulation, we generated over-expressed Lp-PLA2 transgenic swine. The eukaryotic expression plasmid of porcine Lp-PLA2 which driven by EF1α promoter was constructed and generate transgenic swine via SCNT. The expression and activity of Lp-PLA2 in transgenic swine were evaluated, and the total cholesterol (TC), HDL-C, LDL-C and triglyceride (TG) levels in the fasting and fed states were also assessed. Compared with wild-type swine controls, the transgenic swine exhibited elevated Lp-PLA2 mRNA levels and activities, and the activity did not depend on the feeding state. The TC, HDL-C and LDL-C levels were not significantly increased. There was no change in the TG levels in the fasting state between transgenic and control pigs. However, in the fed state, the TG levels of transgenic swine were slightly increased compared with the control pigs and were significantly elevated compared with the fasting state. In addition, inflammatory gene (interleukin [IL]-6, monocyte chemotactic protein [MCP]-1 and tumor necrosis factor [TNF]-α) mRNA levels in peripheral blood mononuclear cells (PBMCs) were significantly increased. The results demonstrated that Lp-PLA2 is associated with triglycerides which may be helpful for understanding the relationship of this protein with cardiovascular disease.

ω3-polyunsaturated fatty acids suppress lipoprotein-associated phospholipase A2 expression in macrophages and animal models.

ω3-polyunsaturated fatty acids (ω3-PUFAs) have beneficial effects on cardiovascular function, and lipoprotein-associated phospholipase A2 (Lp-PLA2 ) is associated with the risk of cardiovascular disease. Here, we investigated the effects of ω3-PUFAs on Lp-PLA2 expression in vitro and in vivo and explored the mechanisms involved. Human monocyticcells (THP-1) were induced into macrophages in an in vitro model. ω3-PUFAs suppressed Lp-PLA2 expression; the suppression induced by docosahexaenoic acid (DHA) was related to reduced inflammation. Tumor necrosis factor-α (TNF-α) was employed to stimulate the phosphorylation of p38 mitogen-activated protein kinase (MAPK), nuclear factor-κB (NF-κB) p65 and Lp-PLA2 expression in macrophages. The stimulation was inhibited by DHA and the anti-inflammatory drug sodium salicylate. Moreover, the stimulation of Lp-PLA2 expression by TNF-α could be suppressed by NF-κB and MAPK pathway inhibitors. Then, chronic inflammation was induced in an in vivo mouse model, resulting in an increase in Lp-PLA2 expression in peripheral blood mononuclear cells (PBMCs) and arteries. This increase was suppressed by ω3-PUFAs. Inhibition of Lp-PLA2 transcription in PBMCs was also observed in ω3-PUFA-enriched swine. Our results demonstrate that the protective effects of ω3-PUFAs against cardiovascular events may be related to the suppression of Lp-PLA2 levels.

Apolipoprotein CIII regulates lipoprotein-associated phospholipase A2 expression via the MAPK and NFκB pathways.

Apolipoprotein CIII (apo CIII), a small glycoprotein that binds to the surfaces of certain lipoproteins, is associated with inflammatory and atherogenic responses in vascular cells. Lipoprotein-associated phospholipase A2 (Lp-PLA2) has been proposed as an inflammatory biomarker and potential therapeutic target for cardiovascular disease (CVD). Here, we report that apo CIII increases Lp-PLA2 mRNA and protein levels in dose- and time- dependent manner in human monocytic THP-1 cells, and the increase can be abolished by MAPK and NFκB pathway inhibitors. Lp-PLA2 inhibitor, 1-linoleoyl glycerol attenuates the inflammation induced by apo CIII. In turn, exogenous Lp-PLA2 expression upregulates apo CIII and the upregulation can be inhibited by 1-linoleoyl glycerol in HepG2 cells. Moreover, plasma Lp-PLA2 level is correlated with apo CIII expression in pig liver. In vivo, Lp-PLA2 expression in monocytes and its activity in serum were significantly increased in human apo CIII transgenic porcine models compared with wild-type pigs. Our results suggest that Lp-PLA2 and apo CIII expression level is correlated with each other in vitro and in vivo.

Nitro-oleic acid downregulates lipoprotein-associated phospholipase A2 expression via the p42/p44 MAPK and NFκB pathways.

Nitro-oleic acid (OA-NO2), acting as anti-inflammatory signaling mediators, are involved in multiple signaling pathways. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is well known as a cardiovascular risk biomarker. Our results showed that OA-NO2 downregulated the expression of Lp-PLA2 in a time- and dose-dependent manner, whereas native OA had no such effect. Furthermore, OA-NO2 could repress Lp-PLA2 expression in the peripheral blood mononuclear cells of apo CIII-transgenic (apo CIII TG) pigs, which exhibited higher Lp-PLA2 expression and activity than did wild-type (WT) pigs. OA-NO2 inhibited Lp-PLA2 expression in macrophages, independent of nitric oxide formation and PPARγ-activation. However, OA-NO2 downregulates Lp-PLA2 by inhibiting the p42/p44 mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) pathways. When used to mediate anti-inflammatory signaling, the regulation of inflammatory cytokines and SOD by OA-NO2 might be associated with the reduction of Lp-PLA2. These results suggested that OA-NO2 might exert a vascular-protective effect partially via Lp-PLA2 inhibition.

Lysophosphatidylcholine and Carotid Intima-Media Thickness in Young Smokers: A Role for Oxidized LDL-Induced Expression of PBMC Lipoprotein-Associated Phospholipase A2?

BackgroundAlthough cigarette smoking has been associated with carotid intima-media thickness (CIMT) the mechanisms are yet not completely known. Lysophosphatidylcholine (lysoPC), a main product of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity, appears to be a major determinant of the pro-atherogenic properties of oxidized LDL (oxLDL) and to induce proteoglycan synthesis, a main player in intimal thickening. In this study we assessed whether cigarette smoking-induced oxidative stress may influence plasma Lp-PLA2 and lysoPC and Lp-PLA2 expression in peripheral blood mononuclear cells (PBMC), as well as the relationship between lysoPC and CIMT.Methods/Results45 healthy smokers and 45 age and sex-matched subjects participated in this study. Smokers, compared to non-smokers, showed increased plasma concentrations of oxLDL, Lp-PLA2 and lysoPC together with up-regulation of Lp-PLA2 (mRNA and protein) expression in PBMC (P<0.001). Plasma Lp-PLA2 positively correlated with both lysoPC (r=0.639, P<0.001) and PBMC mRNA Lp-PLA2 (r=0.484, P<0.001) in all subjects. Moreover CIMT that was higher in smokers (P<0.001), positively correlated with lysoPC (r=0.55, P<0.001). Then in in vitro study we demonstrated that both oxLDL (at concentrations similar to those found in smoker’s serum) and oxidized phospholipids contained in oxLDL, were able to up-regulate mRNA Lp-PLA2 in PBMC. This effect was likely due, at least in part, to the enrichment in oxidized phospholipids found in PBMC after exposure to oxLDL. Our results also showed that in human aortic smooth muscle cells lysoPC, at concentrations similar to those found in smokers, increased the expression of biglycan and versican, two main proteoglycans.ConclusionsIn smokers a further effect of raised oxidative stress is the up-regulation of Lp-PLA2 expression in PBMC with subsequent increase of plasma Lp-PLA2 and lysoPC. Moreover the correlation between lysoPC and CIMT together with the finding that lysoPC up-regulates proteoglycan synthesis suggests that lysoPC may be a link between smoking and intimal thickening.

Lp‐PLA2 is associated with structural valve degeneration of bioprostheses

In this study, we sought to determine the metabolic markers associated with structural valve degeneration (SVD). Structural valve degeneration (SVD) is the major cause of bioprosthetic valve failure leading to bioprostheses (BPs) stenosis or regurgitation. We hypothesized that lipoprotein-associated phospholipase A2 (Lp-PLA2) is involved in the SVD of BPs. We included 197 patients who underwent aortic valve replacement with a bioprosthetic valve and had echocardiographic follow-up to evaluate valve function. Moreover, explanted BPs (n=39) were analysed by immunohistochemistry for the expression of Lp-PLA2. After a mean follow-up of 7·9±0·2years, forty-one patients (21%) were identified as developing SVD. Patients with SVD had significantly higher plasma level of Lp-PLA2 mass (151·8±9·2ng/mL vs. 133·2±3·4ng/mL, P=0·03) and activity (27·6±0·9nmol/min/mL vs. 25·0±0·4nmol/min/mL, P=0·005). Multivariate analysis revealed that Lp-PLA2 activity (OR: 1·09, 95% CI: 1·01-1·18; P=0·03) was the strongest independent predictor of SVD. Immunohistochemistry studies of explanted BP showed that 77% of explanted BPs had the expression of Lp-PLA2, which correlated with the density of macrophages (CD68), and ox-LDL levels in bioprosthetic tissues. Increased blood plasma activity of Lp-PLA2 is associated with higher prevalence of SVD. These findings open new avenues for the identification of patients at risk for SVD and for the development of pharmacotherapy aiming at the prevention of SVD.

Elevated Expression of Lipoprotein-Associated Phospholipase A2 in Calcific Aortic Valve Disease: Implications for Valve Mineralization

This study sought to document the presence and role of lipoprotein-associated phospholipase A2 (Lp-PLA2) in calcific aortic valve disease (CAVD). CAVD is a chronic disorder characterized by pathological mineralization and remodeling. Studies have indicated that human CAVD tissues are infiltrated by lipids and that inflammation may play a role in the pathobiology. We hypothesized that Lp-PLA2 (encoded by the PLA2G7 gene) is expressed in CAVD and may play a role in the mineralization of valve interstitial cells. We have documented the expression of the phospholipase A2 family of genes in aortic valves by using a transcriptomic assay. Messenger ribonucleic acid and protein expression were confirmed in aortic valves explanted from 60 patients by quantitative polymerase chain reaction and immunohistochemistry, respectively. The effect of lysophosphatidylcholine, the product of Lp-PLA2 activity, was documented on the mineralization of valve interstitial cell cultures. Transcriptomic analyses of CAVD and control nonmineralized aortic valves revealed that Lp-PLA2 was increased by 4.2-fold in mineralized aortic valves. Higher expression of Lp-PLA2 in stenotic aortic valves was confirmed by quantitative polymerase chain reaction, immunohistochemistry, and enzymatic Lp-PLA2 activity. The number of Lp-PLA2 transcripts correlated with several indexes of tissue remodeling. Invitro, lysophosphatidylcholine increased the expression of alkaline phosphatase, the ectonucleotide pyrophosphatase/phosphodiesterase 1 enzyme, sodium-dependent phosphate cotransporter 1 (encoded by the SLC20A1 gene), and osteopontin. We then showed that lysophosphatidylcholine-induced mineralization involved ectonucleotidase enzyme as well as apoptosis through a protein-kinase-A-dependent pathway. Together, these results demonstrated that Lp-PLA2 is highly expressed in CAVD, and it plays a role in the mineralization of valve interstitial cells. Further work is necessary to document whether Lp-PLA2 could be considered as a novel target in CAVD.

Everolimus therapy is associated with reduced lipoprotein-associated phospholipase A2 (Lp-Pla2) activity and oxidative stress in heart transplant recipients

BackgroundSeveral studies demonstrated decreased severity and incidence of cardiac allograft vasculopathy (CAV) in heart transplant recipients receiving immunosuppressive therapy with everolimus. However, data regarding the influence of everolimus on risk factors predisposing to CAV are hitherto limited. We here systematically evaluated cardiovascular risk factors in heart transplanted patients, who underwent conversion to everolimus or were maintained on conventional therapy with calcineurin inhibitors (CNI). Methods50 Patients receiving everolimus and 91 patients receiving CNI in addition to mycophenolate mofetil and low-dosed steroids were included in the study. CAV risk factors were determined in plasma or urine using standard enzymatic or immunochemical methods. ResultsNo significant differences were observed between both groups with regard to lipid (total, LDL- and HDL-cholesterol), metabolic (glucose, insulin), inflammatory (C-reactive protein, IL-6, myeloperoxidase) and cardiac (troponin I, NT-proBNP) risk factors. However, significantly lower activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) and a negative correlation between the Lp-PLA2 activity and the everolimus concentration were observed in plasmas from everolimus-treated patients. Conversion to everolimus significantly lowered Lp-PLA2 activity in heart transplant recipients. Studies in vitro revealed reduced Lp-PLA2 expression in hepatocytes and macrophages pre-exposed to everolimus. In addition, reduced plasma markers of oxidative stress including oxidized LDL, 8-iso-prostaglandin F2α and protein carbonyls were noted in heart transplant recipients receiving everolimus therapy. ConclusionOur results suggest that everolimus specifically lowers plasma activity and cellular production of Lp-PLA2 and thereby dampens oxidative stress. These effects may additionally contribute to the reduced CAV incidence observed in heart transplant recipients receiving everolimus therapy.

Serum amyloid A stimulates lipoprotein-associated phospholipase A2 expression in vitro and in vivo

ObjectivesAlthough lipoprotein-associated phospholipase A2 (Lp-PLA2) has been regarded as a biomarker and a causative agent for acute coronary events recently, the mechanism of the regulation of Lp-PLA2 has not been fully elucidated yet. This study was aimed to investigate the influence of serum amyloid A (SAA) on the expression of Lp-PLA2 in THP-1 cells and ApoE-deficient (ApoE−/−) mice. MethodsTHP-1 cells were stimulated by SAA and the mRNA and protein expression of Lp-PLA2 was detected. ApoE−/− mice were intravenously injected with murine SAA1 lentivirus. Formyl peptide receptor like-1 (FPRL1) agonist (WKYMVm) and inhibitor (WRW4), mitogen-activated protein kinases (MAPKs) inhibitors, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist and inhibitor were used to investigate the mechanism of regulation of Lp-PLA2. ResultsRecombinant SAA up-regulated Lp-PLA2 expression in a dose and time-dependent manner in THP-1 cells. Immunohistochemical analysis of aortic root of ApoE−/− mice also demonstrated that the expression of Lp-PLA2 was up-regulated significantly with SAA treatment. WRW4 decreased SAA-induced Lp-PLA2 production; while WKYMVm could induce Lp-PLA2 expression. ERK1/2, JNK1/2, and p38 inhibition reduced SAA-induced Lp-PLA2 production. Furthermore, the results suggested the activation of PPAR-γ played a crucial role in this process. ConclusionThese results demonstrate that SAA up-regulates Lp-PLA2 production significantly via a FPRL1/MAPKs./PPAR-γ signaling pathway.

Amelioration of atherosclerosis in apolipoprotein E-deficient mice by inhibition of lipoprotein-associated phospholipase A2

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is involved in the pathogenesis of atherosclerosis, especially in advanced plaques. In the present study, the abilities of darapladib, a selective Lp-PLA(2) inhibitor, and lentivirus-mediated Lp-PLA(2) silencing on inflammation and atherosclerosis in apolipoprotein E-deficient mice were compared. Apolipoprotein E-deficient mice were fed on a high-fat diet and a constrictive collar was placed around the left carotid artery to induce plaque formation. The mice were randomly divided into control, negative control (NC), darapladib and RNA interference (RNAi) groups. Eight weeks after surgery, lentivirus-mediated RNAi construct or darapladib were used to decrease the expression of Lp-PLA(2). Plaques were collected five weeks later for histological analysis. Inflammatory gene expression in the atherosclerotic lesions were then determined at the mRNA and protein level. The expression of pro-inflammatory cytokines was significantly reduced in the treatment group, compared to nontreatment group, whereas the plasma concentration of anti-inflammatory cytokines increased markedly. Moreover, our results demonstrated a significant reduction in plaque lipid content, as well as a rise in collagen content following Lp-PLA(2) inhibition. Interestingly, when comparing the two methods of Lp-PLA(2) inhibition, animals treated with Lp-PLA(2) RNAi were found to exhibit lower plaque areas and enhanced improvement of plaque stability as compared with animals treated with darapladib. Darapladib had no attenuating effect on atherosclerotic plaque area. These therapeutic effects were independent of plasma lipoprotein levels. Lp-PLA(2) inhibition by darapladib or lentivirus-mediated RNAi ameliorated inflammation and atherosclerosis in apolipoprotein E-deficient mice. The effect was more prominent in the RNAi group.

Lentiviral-mediated RNA interference of lipoprotein-associated phospholipase A2 ameliorates inflammation and atherosclerosis in apolipoprotein E-deficient mice

Lipoprotein associated phospholipaseA2 (Lp-PLA2) overexpression is implicated in athero-sclerosis. In the present study, we evaluated the effects of lentiviral-mediated RNA interference (RNAi) of Lp-PLA2 on inflammation and atherosclerosis in apolipoproteinE-deficient mice. ApolipoproteinE-deficient mice were randomly allocated to control and experimental groups, and constrictive collars were used to induce plaque formation. Eight weeks after surgery, the lentiviral-mediated RNAi construct was used to silence expression of Lp-PLA2. Control and experimental lentivirus was transfected directly into carotid plaques or administered systemically. Tissues were collected for analysis 7weeks after transfection. Inflammatory gene expression in the plasma and atherosclerotic lesions was then determined at the mRNA and protein levels. We observed no differences in body weight and plasma lipid levels at the end of the investigation. However, the expression levels of Lp-PLA2 and pro-inflammatory cytokines were significantly reduced in the RNAi groups, compared to the controls, whereas the plasma concentration of anti-inflammatory cytokines was markedly increased. Moreover, our results demonstrated a significant reduction in plaque area and lipid content, as well as a rise in collagen content following RNAi treatment. Importantly, when comparing the two methods of viral delivery, we found that transluminal local transfection exhibited enhanced improvement of plaque stability as compared to systemic administration. Inhibition of Lp-PLA2 by lentiviral-mediated RNAi ameliorates inflammation and atherosclerosis in apolipoproteinE-deficient mice. In addition, transluminal local delivery of Lp-PLA2 shRNA is superior to systemic administration for stabilizing atherosclerotic plaques.

Regression of atherosclerosis in apolipoprotein E-deficient mice by lentivirus-mediated gene silencing of lipoprotein-associated phospholipase A2

Overexpression of lipoprotein-associated phospholipase A2 (Lp-PLA2) is implicated in atherosclerosis. We tested the hypothesis that lentivirus-mediated Lp-PLA2 silencing could inhibit atherosclerosis in apolipoprotein E-deficient mice. Sixty eight apolipoprotein E-deficient mice were fed a high-fat diet and a constrictive collar was placed around the left carotid artery to induce plaque formation. The mice were randomly divided into control, negative control (NC) and RNA interference (RNAi) groups. Lp-PLA2 RNAi or scrambled NC lentivirus viral suspensions were constructed and transfected into the carotid plaques 8weeks after surgery; the control group was administered saline. The carotid plaques were assessed 7weeks later using hematoxylin and eosin, Masson’s trichrome and oil red O staining; plasma and lesion inflammatory gene expression were examined using ELISAs and real-time PCR. Seven weeks after transfection, the serum concentration and plaque mRNA expression of Lp-PLA2 was significantly lower in the RNAi group, and lead to reduced local and systemic inflammatory gene expression. Lp-PLA2 RNAi also ameliorated plaque progression, reduced the plaque lipid content and increased the plaque collagen content. The effects of Lp-PLA2 RNAi were independent of serum lipoprotein levels, as the triglyceride and total cholesterol levels of the control, NC and RNAi groups were not significantly different. These findings support the hypothesis that lentivirus-mediated Lp-PLA2 gene silencing has therapeutic potential to inhibit atherosclerosis and increase plaque stability, without altering the plasma lipoprotein profile.

Abstract 347: Early Drug-Induced Inhibition of Proatherogenic Genes in Coronary Regions of Low Endothelial Shear Stress in Diabetic Hyperlipidemic Juvenile Swine

Introduction: Low endothelial shear stress (ESS) activates pro-inflammatory pathways and is a powerful instigator of atherogenesis. Angiotensin receptor blockers and statins have been associated with anti-inflammatory actions in advanced plaques. However, their effect on the earliest pathobiologic manifestations of atherosclerosis has not been studied. We tested the hypothesis that valsartan (V) or V plus simvastatin (V/S) exerts an early vasculoprotective effect in coronary regions exposed to low ESS in a porcine model of human-like atherosclerosis. Methods: Twelve diabetic-hyperlipidemic swine (age: 3 mo) were grouped into controls (n=4), and those treated with V (320 mg; n=4) or V/S (320/40 mg; n=4). 3D reconstruction of coronary arteries by angiography and intravascular ultrasound was performed in vivo at 4 (baseline) and 8 (follow-up) wks post-induction. Baseline local ESS was calculated by computational fluid dynamics and 3 mm segments with low (≤1.2 Pa; n=46) or higher (&gt;1.2 Pa; n=66) ESS were identified. Coronary arteries were harvested at follow-up. qRT-PCR was used for assessing the expression of intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), LDL receptor and lipoprotein-associated phospholipase-A 2 (LpPLA 2 ). Results: The upregulation of ICAM-1, MCP-1, LDL receptor (p&lt;0.05) and LpPLA 2 (p&lt;0.1) expression in low ESS segments was inhibited in the V and V/S groups compared to controls (Figure). Conclusion: V and V/S attenuate the proatherogenic effects of low ESS within only 8 wks. These results suggest a drug-induced mechanism of regional atheroprotection early in the natural history of coronary artery disease.

Simvastatin Reduces Lipoprotein-associated Phospholipase A2 in Lipopolysaccharide-stimulated Human Monocyte–derived Macrophages Through Inhibition of the Mevalonate–Geranylgeranyl Pyrophosphate–RhoA-p38 Mitogen-activated Protein Kinase Pathway

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), which is produced primarily by macrophages and is predominately found in the blood and in atherosclerotic plaques, represents a potentially promising target for combating atherosclerosis. Although statins are known to decrease the levels and activity of circulating and plaque Lp-PLA(2) during atherosclerosis, little is known regarding the mechanisms underlying inhibition of Lp-PLA(2) by statins. Therefore, the aim of this study was to explore the molecular mechanisms responsible for inhibition of Lp-PLA(2) by statins. Our results showed that treatment with simvastatin inhibited lipopolysaccharide (LPS)-induced increases in Lp-PLA(2) expression and secreted activity in human monocyte–derived macrophages in a dose- and time-dependent manner. These effects could be reversed by treatment with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not by treatment with squalene or farnesyl pyrophosphate. Treatment with the Rho inhibitor C3 exoenzyme also inhibited LPS-induced increases in Lp-PLA(2) expression and secreted activity, mimicking the effects of simvastatin. In addition, treatment with simvastatin blocked LPS-induced activation of RhoA, which could be abolished by treatment with GGPP. Inhibition of p38 mitogen-activated protein kinase (MAPK), but not extracellular signal regulated kinase 1/2 or Jun N-terminal kinase, suppressed LPS-induced increases in Lp-PLA(2) expression and secreted activity, similar to the effects of simvastatin. Treatment of human monocyte–derived macrophages with either simvastatin or C3 exoenzyme prevented LPS-induced activation of p38 MAPK, which could be abolished by treatment with GGPP. Together, these results suggest that simvastatin reduces Lp-PLA(2) expression and secreted activity in LPS-stimulated human monocyte–derived macrophages through the inhibition of the mevalonate–GGPP–RhoA-p38 MAPK pathway. These observations provide novel evidence that statins have pleiotropic effects and suggest that inhibition of Lp-PLA(2) via this mechanism may account, at least in part, for the clinical benefit of statins in combating atherosclerosis.

Pleiotropic Effects of Atorvastatin on Monocytes in Atherosclerotic Patients

The objective of this study was to investigate the gene expression signature of monocyte/macrophages and the pleiotropic effects of atorvastatin on monocytes in atherosclerotic patients. Forty patients with coronary heart diseases were randomly assigned to double-blind therapy with either 20 or 80 mg per day of atorvastatin. Follow-up visits occurred at weeks 6 and 12, including complete chemistry and lipid analyses and quantification of 14 target genes in monocytes. After 12 weeks of therapy, both groups gained beneficial alterations in lipid profiles. Both groups experienced significant reductions in gene expression of lipoprotein-associated phospholipase A2, CD13, leptin receptor, matrix metalloproteases-1, legumain, and prolyl oligopeptidase after 12 weeks of therapy. Only tumor protein 53 was increased in the atorvastatin 80-mg group. Moreover, nonsignificant interactions between dosage and duration of therapy were found. The pleiotropic effects of statins in atherosclerotic patients include increased expression of genes involved in apoptosis of monocyte/macrophage, inhibition of inflammatory responses, antioxidant properties, prevention of foam cell formation, and stabilization of atherosclerotic plaques. This property fuels potential clinical significance.

Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis

Plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA(2) is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA(2) and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA(2) protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA(2) mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA(2) were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA(2) were negatively correlated (r = - 0.578). The ratio of Lp-PLA(2) to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA(2) existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA(2) epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA(2) and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.

Expression of lipoprotein-associated phospholipase A2 in carotid artery plaques predicts long-term cardiac outcome

The aim was to test the hypothesis that carotid artery plaque expression of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) predicts cardiac events. Prospective cohort study of 162 consecutive patients undergoing elective carotid endarterectomy. Lipoprotein-associated phospholipase A(2) content was quantified by immunoblotting and lysophosphatidylcholine (lysoPC) by liquid chromatography tandem mass spectrometry. Additional biomolecular profiling by immunoblotting included C-reactive protein, p67phox, and matrix metalloproteinase-2 and -9. Macrophage plaque content was determined by quantitative immunostaining, plaque collagen content by quantitative Sirius red staining. Follow-up for cardiac death and non-fatal acute myocardial infarction was accomplished over a period of 48 +/- 14 months. Expression of Lp-PLA(2) and lysoPC was higher in carotid plaques of patients with than without cardiac events [median 1.6 (25th, 75th percentile 0.9, 2.5) vs. 0.8 (0.5, 2.0), P = 0.01 and 413 (281, 443) vs. 226 (96, 351) mmol/L, P = 0.03]. Smoking and point increase in carotid Lp-PLA(2) expression but no other traditional cardiovascular risk factor, histological or molecular marker remained predictive of cardiac events in the multivariate Cox proportional hazard analyses [HR 3.65 (1.36-9.83), P = 0.01 and HR 1.34 (1.01-1.77), P = 0.039]. Carotid plaque Lp-PLA(2) expression above the median constituted a more than three times higher risk for cardiac events [HR 3.39 (1.13-10.17), P = 0.03]. Lipoprotein-associated phospholipase A(2) expression in carotid artery plaques is a predictor of long-term cardiac outcome. The current study supports the concept of atherosclerosis as a systemic disease with multi-focal complications and personalized medicine.