Amelioration Of Atherosclerosis In Apolipoprotein E-deficient Mice By Inhibition Of Lipoprotein-associated Phospholipase A₂

Part I Lentivirus-mediated RNA interference of lipoproteinassociated phospholipase A2ameliorated the expression ofinflammatory genes in RAW264.7cellsBackgroundAtherosclerosis (AS) is a multi-factorial complex process intertwined withinflammation. Lipoprotein associated phospholipase A2(Lp-PLA2) is a novelinflammation factor and marker of atherosclerosis. Lp-PLA2can predict thevulnerability and rupture of atherosclerotic plaques as well as future cardiovascularevents. Lp-PLA2hydrolyzes oxidizes phospholipids, generating the bioactive lipidmediators lysophosphatidylcholine (LPC) and oxidized non-esterified fatty acid(oxNEFA), which both play a key role in atherosclerosis. Inhibiting inflammationfactors and gene therapy represents a novel approach to deal with AS in the future.RNAi is a clinically feasible method to down-regulate the expression of target genesefficiently and selectively. In the present study, RNA interference (RNAi) techniquewas adopted to reduce the expression of Lp-PLA2in mouse monocyte-macrophages (RAW264.7cells) and the expression of Lp-PLA2and inflammatory genes weredetected by ELLISA and real time polymerase chain reaction (RT-PCR).ObjectivesTo identify the effects of oxygenized low density lipoprotein (oxLDL) andlentivirus-mediated RNAi of Lp-PLA2on the expression of Lp-PLA2andinflammatory genes in RAW264.7cells.MethodsLp-PLA2RNAi or scrambled negative control (NC) lentivirus viral suspensionswere constructed, and the titers averaged1×109TU/ml. RAW264.7mousemacrophage cells were cultured in DMEM containing10%FBS. After oxLDLpretreatment, the expression Lp-PLA2increased sharply. We pretreated the cells with60μg/ml of oxLDL because the expression of Lp-PLA2reached the platform stageafter60μg/ml of oxLDL stimulation. Mouse RAW264.7cells (90%confluent) werethen untransfected or transfected with NC lentivirus or Lp-PLA2RNAi lentivirus(multiplicity of infection=50) to determine their silencing efficiency. Lp-PLA2andinflammatory gene expression were detected by ELLISA kit, and mRNA level ofLp-PLA2and inflammatory genes were detected by RT-PCR.Results1. The expression of Lp-PLA2was very low before ox-LDL stimulation, whileox-LDL upregulated the expression of Lp-PLA2in a concentration and timedependent manner. The expression of Lp-PLA2reached the platform stage after60μg/ml of oxLDL pretreatment. Therefore, we pretreated the cells with60μg/mloxLDL.2. Gene-silencing analysis demonstrated that the site A lentivirus mosteffectively blocked Lp-PLA2mRNA and protein expression.3. Lp-PLA2RNA interference could inhibit MCP-1、IL-6and MMP-8activityand its mRNA expression induced by oxLDL. ConclusionsLentivirus-mediated RNA interference of Lp-PLA2could inhibit the expressionof Lp-PLA2and inflammatory genes evoked by oxLDL. Part Ⅱ Regression of atherosclerosis in apolipoprotein E-deficientmice by lentivirus-mediated RNA interference oflipoprotein-associated phospholipase A2BackgroundDespite intensive management of the conventional atherosclerosis risk factors,many patients continue to experience the onset and recurrence of coronary events.Atherosclerosis (AS) is a multi-factorial complex process intertwined withinflammation. Overexpression of lipoprotein-associated phospholipase A2(Lp-PLA2)is implicated in atherosclerosis. Lp-PLA2hydrolyzes oxidizes phospholipids,generating the bioactive lipid mediators lysophosphatidylcholine (LPC) and oxidizednon-esterified fatty acid (oxNEFA), which both play a key role in atherosclerosis.Inhibiting inflammation factors and gene therapy represents a novel approach to dealwith AS in the future. RNAi has been shown to be quite efficacious in silencing targetgenes in both dividing and non-dividing cells. In the present study, we set up mousemodel of AS and RNA interference (RNAi) technique was adopted to reduce theexpression of Lp-PLA2, then we examined the morphology of plaques and theexpression of inflammatory genes to provide additional insight into the mechanismsinvolved in this process.ObjectivesWe tested the hypothesis that lentivirus-mediated Lp-PLA2silencing could inhibit atherosclerosis in apolipoprotein E-deficient mice.MethodsEighty six apolipoprotein E-deficient mice were fed a high-fat diet and aconstrictive collar was placed around the left carotid artery to induce plaqueformation. In brief, the common carotid arteries were dissected and a constrictivesilastic collar (0.30mm) was placed on the left common carotid artery by placementof3circumferential silk ties in all mice. The mice were randomly divided into control,negative control (NC) and RNA interference (RNAi) groups. Lp-PLA2RNAi orscrambled NC lentivirus viral suspensions were constructed and transfected into thecarotid plaques8weeks after surgery; the control group was administered saline. Thecarotid plaques were assessed seven weeks later using hematoxylin and eosin,Masson’s trichrome and oil red O staining; plasma and lesion inflammatory geneexpression were examined using ELISA and real-time PCR.Results1. Seven weeks after transfection, the serum concentration and plaque mRNAexpression of Lp-PLA2was significantly lower in the RNAi group, and lead toreduced local and systemic inflammatory gene expression.2. Lp-PLA2RNAi also ameliorated plaque progression, reduced the plaque lipidcontent and plaque area, as well as increased the plaque collagen content and fibrouscap thickness, indicating increased plaque stability.3. The effects of Lp-PLA2RNAi were independent of serum lipoprotein levels, asthe triglyceride and total cholesterol levels of the control, NC and RNAi groups werenot significantly different.ConclusionsThese findings support the hypothesis that lentivirus-mediated Lp-PLA2RNAinterference has therapeutic potential to inhibit atherosclerosis and increase plaquestability, without altering the plasma lipoprotein profile. Part Ⅲ Amelioration of atherosclerosis in apolipoproteinE-deficient mice by inhibition of lipoprotein-associatedphospholipase A2：a study comparing RNAinterference with selectiveLp-PLA2inhibitorBackgroundDespite major advances in treatment of atherosclerosis, a large number ofvictims of the disease who are apparently healthy die suddenly without priorsymptoms. This led us to identify novel therapeutic targets in the setting ofatherosclerosis. Lipoprotein-associated phospholipase A2(Lp-PLA2) is a novelinflammation factor and marker of atherosclerosis. Overexpression of Lp-PLA2isimplicated in atherosclerosis. Darapladib, a selective Lp-PLA2inhibitor, provides anattractive target for intervention to reduce atherosclerosis. Previous studiesdemonstrated that darapladib decreased intra-plaque LPC content, attenuated theexpression of inflammatory genes, and reduced necrotic core formation in animalmodels of atherosclerosis. However, darapladib did not reduce human plaque volumein a phase Ⅱclinical study. In summary, experimental and epidemiological evidenceis not equivocal about the effects of darapladib. RNAi has been shown to be quiteefficacious in silencing target genes in both dividing and non-dividing cells. In thepresent study, we employed the two methods (darapladib and Lp-PLA2RNAi) andcompared the distinct mechanisms involved in atherosclerosis.AimsLp-PLA2is involved in the pathogenesis of atherosclerosis, especially inadvanced plaques. In the present study, the abilities of darapladib, a selectiveLp-PLA2inhibitor, and lentivirus-mediated Lp-PLA2silencing on inflammation andatherosclerosis in apolipoprotein E-deficient mice were compared.MethodsApolipoprotein E-deficient mice were fed on a high-fat diet and a constrictivecollar was placed around the left carotid artery to induce plaque formation. In brief, the common carotid arteries were dissected and a constrictive silastic collar (0.30mm)was placed on the left common carotid artery by placement of3circumferential silkties in all mice. 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 theexpression of Lp-PLA2. The carotid plaques were assessed five weeks later forhistological analysis using hematoxylin and eosin, Masson’s trichrome and oil red Ostaining. Inflammatory gene expression in the atherosclerotic lesions were thendetermined at the mRNA and protein level.Results1. The expression of pro-inflammatory cytokines was significantly reduced in thetreatment group, compared to nontreatment group, whereas the plasma concentrationof anti-inflammatory cytokines increased markedly.2. Moreover, our results demonstrated a significant reduction in plaque lipidcontent, as well as a rise in collagen content following Lp-PLA2inhibition.Interestingly, when comparing the two methods of Lp-PLA2inhibition, animalstreated with Lp-PLA2RNAi were found to exhibit lower plaque areas and enhancedimprovement of plaque stability as compared with animals treated with darapladib.Darapladib had no attenuating effect on atherosclerotic plaque area.3. These therapeutic effects of Lp-PLA2inhibition were independent of serumlipoprotein levels, as the high-density lipoprotein cholesterol, low-density lipoproteincholesterol, triglyceride and total cholesterol levels of the control, NC, darapladib andLp-PLA2RNAi groups were not significantly different.ConclusionsLp-PLA2inhibition by darapladib or lentivirus-mediated RNAi amelioratedinflammation and atherosclerosis in apolipoprotein E-deficient mice. The effect wasmore prominent in the Lp-PLA2RNAi group.