Studies Of The Effect Of Proprotein Convertase Subtilisin/Kexin9on Atherogenesis And The Mechanisms Involved

Proprotein convertase subtilisin/kexin9(PCSK9), the ninth member of theproprotein convertase family, was discovered in2003and identified as a newcontributor to cholesterol homeostasis. Studies show that PCSK9serves a pivotalfunction in the degradation of low-density lipoprotein receptor (LDLR), whichcontributes to the decrease in hepatic cholesterol uptake and increase in circulatingLDL-C. Thus PCSK9has emerged as a popular target for the development of newcholesterol lowering drugs and therapeutic intervention of atherosclerosis. The currentstudies suggest that the process by which PCSK9enhances atherosclerosis isprimarily mediated through its action on the hepatic cholesterol metabolism. However,the direct action of PCSK9on the vasculature cannot be elucidated. PCSK9mightaccelerate atherosclerosis by mechanisms beyond the degradation of hepatic LDLR.The further study is needed to understand the potential new role and mechanisms ofPCSK9for the pathogenesis of atherosclerosis. This project is expected to reveal thenovel functions of PCSK9on atherosclerosis, which may provide new strategies foratherosclerosis prevention and treatment.The lipid accumulation and inflammatory response of vascular macrophages areknown to be the key events in the pathogenesis of atherosclerosis. The inhibition ofsuch events is becoming an attractive new approach for therapeutic intervention inatherosclerosis. Cholesterol fails to downregulate the expression of scavenger receptors, so macrophages expressing scavenger receptors can internalize substantialquantities of cholesterol ester from oxidized LDL. Such internalization results in foamcell formation and atherosclerotic lesion progression. Beyond the effects on hepaticlipid metabolism, data also suggest that PCSK9contributes to cholesterol homeostasisand lipid transport in intestinal epithelial cells. However, the functions andmechanism of PCSK9in macrophage cholesterol homeostasis remain unknown.In2010, Lan et al. discovered that PCSK9affects inflammation and stressresponse pathways beyond cholesterol metabolism in HepG2cells through microarrayanalysis. We previously demonstrated a positive relationship between levels of bloodPCSK9and IL-6and TNF-α in the patient with coronary heart disease. Macrophagesare the first inflammatory cells to invade atherosclerotic lesions, and they are the maincomponents of atherosclerotic plaques. More importantly, macrophages releaseproinflammatory cytokines, chemokines, and matrix metalloproteinases, which resultin lesions that are unstable and prone to rupture. The effect of PCSK9on macrophageinflammatory activation remains unknown.Therefore, we hypothesize that PCSK9increases lipid accumulation andinflammatory cytokine secretion in macrophages, thus promoting the development ofatherosclerosis directly. To test this hypothesis, we first assess the expression ofPCSK9and colocalization of macrophages and PCSK9in atherosclerotic lesions andthen examined the effect of oxLDL on PCSK9expression in macrophages in the firstphase. In the second phase, we construct a lentivirus vector carrying PCSK9cDNA orPCSK9shRNA to overexpress and silence PCSK9and then observe the effects ofPCSK9on macrophage lipid accumulation and inflammatory activation, as well asexplore its possible mechanisms. Finally, we evaluate the effects oflentiviral-mediated RNA interference of PCSK9on atherosclerotic lesion and plaqueinflammation, as well as lipid accumulation in atherosclerosis-prone ApoE-/-mice.We then investigate the possible function of the CD36and TLR4/NF-κB signalingpathways in this process. This project is expected to reveal the novel functions ofPCSK9on atherosclerosis, which may provide new strategies for atherosclerosisprevention and treatment. Part I: Expression of PCSK9in Macrophages of AtheroscleroticPlaques and Effect of oxLDL on PCSK9Expression in MacrophagesObjective: To determine the expression of PCSK9and colocalization ofmacrophages and PCSK9in atherosclerotic lesions, and to determine the effect ofoxLDL on PCSK9expression in macrophages.Methods: The expression and location of PCSK9in human and ApoE-/-mouseatherosclerotic lesions were examined by immunohistochemistry, through which thecolocalization of macrophages and PCSK9was detected by immunofluorescencedouble labeling. THP-1-derived macrophages and RAW264.7cells were cultured, andthe expression of PCSK9was assessed by immunofluorescence. Finally, the cellswere incubated with varied concentrations of oxLDL for different durations.Real-time quantitative PCR and Western blot analysis were conducted to detect thePCSK9protein and mRNA expression, respectively.Results: PCSK9was expressed in atherosclerotic lesions, particularly in intimaplaques, whereas the normal aorta showed no significant expression of PCSK9. Thelocation of PCSK9matched that of the CD68of macrophage markers, and PCSK9was found in both THP-1-derived macrophages and RAW264.7cells, mainly in thecytoplasm. Furthermore, PCSK9protein and mRNA expression was increased in adose-dependent and time-dependent manner by oxLDL in macrophages.Conclusion: PCSK9is expressed in atherosclerotic lesions and colocalizes withmacrophages. OxLDL upregulates PCSK9expression in macrophages in aconcentration-dependent and time-dependent manner. Part II: Effect and Mechanism of PCSK9on Macrophage LipidAccumulation and Inflammatory Cytokine productionInduced by OxLDLObjective: To observe the effects of PCSK9on lipid accumulation andinflammatory cytokine secretion induced by oxLDL in macrophage by overexpressingand silencing PCSK9, and to explore its possible mechanisms.Methods:1. The effective sequence of RNAi targeting PCSK9gene wasdesigned and synthesized. After annealing, double-stranded DNA was inserted intothe GV115lentivirus vector. The resulting lentiviral vector was called LV-PCSK9shRNA. The mouse PCSK9gene was amplified by real-time quantitative PCR andconnected to the lentiviral expression vector GV287to construct the lentiviral vectorLV-PCSK9. Colony PCR and DNA sequencing were used to verify the correction.The viral particles were generated by the co-transfection of293T cells with theLV-PCSK9shRNA or LV-PCSK9and two packaging vectors (pHelperl.0, pHelper2.0).The virus titer was determined by counting the percentage of GFP positive cells orRT-PCR. RNA interference was constructed in the same manner. LV-PCSK9shRNAand LV-PCSK9infected RAW264.7cells. PCSK9expression was examined byWestern blot assays and/or real-time quantitative PCR in RAW264.7cells.2. Lipidaccumulation and inflammatory cytokine secretion in macrophages treated withLV-PCSK9shRNA+oxLDL, LV-PCSK9+oxLDL, or oxLDL were evaluated by OilRed O staining, Filipin staining, and ELISA.3. Macrophages were transfected withLV-PCSK9shRNA or LV-PCSK9and then treated with or without oxLDL. Theexpressions of CD36, SR-AI, SR-BI, TLR4, and NF-κB were investigated by Westernblot and/or real-time quantitative PCR.Results:1. PCR and DNA sequencing demonstrated the successful constructionof LV-PCSK9shRNA and LV-PCSK9. The titers of LV-PCSK9shRNA andLV-PCSK9are5.0×108TU/mL and2.0×108TU/mL respectively. PCSK9expressionwas significantly inhibited by LV-PCSK9shRNA and increased by LV-PCSK9in RAW264.7macrophage PCSK9expression.2. Cellular cholesterol accumulationdecreased when treated by the LV-PCSK9shRNA compared with those treated byoxLDL alone, whereas LV-PCSK9significantly increased compared with thosetreated by oxLDL alone by Oil Red O and Filipin staining. TNF-α, IL-1β, IL-6, andMCP-1secretion were significantly decreased by LV-PCSK9shRNA treatmentcompared with those treated by oxLDL alone. Meanwhile, LV-PCSK9increasedinflammatory cytokine secretion.3. The expressions of SR-AI and SR-BI mRNAwere not obviously changed in cells treated with LV-PCSK9shRNA or LV-PCSK9, asdetected by real-time quantitative PCR. However, CD36and TLR4mRNAexpressions were significantly decreased by LV-PCSK9shRNA, but significantlyincreased by LV-PCSK9. The upregulation of SR-AI and SR-BI protein in RAW264.7macrophages induced by oxLDL was unaffected by LV-PCSK9shRNA or LV-PCSK9.However, LV-PCSK9shRNA suppressed the oxLDL-induced upregulation of CD36and TLR4proteins in RAW264.7macrophages, whereas LV-PCSK9exacerbated thiseffect. In addition, LV-PCSK9shRNA significantly inhibited oxLDL-induced p-IkBαexpression, IkBα degradation, and NF-κB nuclear translocation in cells, whereasLV-PCSK9significantly amplified this effect.Conclusion: PCSK9overexpression and interference lentiviral vectors aresuccessfully constructed. Overexpression of PCSK9increases the lipid accumulationand proinflammatory cytokine secretion of macrophages, whereas the downregulationof PCSK9decreases lipid accumulation and inflammatory cytokine secretion. PCSK9regulates lipid accumulation and inflammatory cytokine secretion through theupregulation of CD36expression and the activation of the TLR4/NF-κB-dependentpathway. Part III: Effect and Mechanism of LV-PCSK9shRNA on LipidAccumulation in Atherosclerotic Plaque and exprssion ofInflammatory factors in ApoE-/-MiceObjective: To observe the effect of LV-PCSK9shRNA on atherosclerotic lesionand plaque lipid accumulation and inflammation in ApoE-/-mice, and to investigateits possible mechanisms.Methods: Thirty male ApoE-/-mice aged six weeks were randomly allocated toeither the LV-PCSK9shRNA treatment group or control group (n=15). In theLV-PCSK9shRNA treatment group,4×107TU of viral suspension was injected intothe tail vein of the mice every six weeks. All mice were fed a high-fat diet. After12weeks, the animals were killed for the detection of lentivirus transfection efficiencyby using fluorescence microscopy. PCSK9expression in the aorta was examined byimmunohistochemistry, Western blot, and real-time quantitative PCR. Blood wascollected for the detection of plasma lipid by a by an autoanalyzer. Quantification oflesions in aorta and aortic sinus was tested by Sultan IV and HE staining. Lipidaccumulation in the aortic sinus was evaluated by Oil Red O, Masson, and Filipinstaining. Macrophage infiltration in lesions was tested by immunofluorescence andhistochemistry. TNF-α, IL-1β, and MCP-1expressions in lesions were investigated byimmunohistochemistry and real-time quantitative PCR. CD36, TLR4, and NF-κBexpression in lesions were examined by immunohistochemistry, Western blot assays,and real-time quantitative PCR, respectively.Results: After intravenous administration of LV-PCSK9shRNA, the GFP ofLV-PCSK9shRNA was detected in lesions of mice by using a fluorescencemicroscope. The mRNA and protein expression of PCSK9in aorta was decreased byLV-PCSK9shRNA. Although the plasma lipid exhibited no obvious change withLV-PCSK9shRNA treatment compared with the control group, the lesion area of theaorta was decreased in mice treated with LV-PCSK9shRNA. In ApoE-/-miceinjected with LV-PCSK9shRNA developed less advanced lesions in the aortic sinus and exhibited decreased lipid content, reduced macrophage accumulation, anddecreased mRNA levels and amount of inflammatory cytokines, such as TNF-α, IL-1β,and MCP-1. According to the results of immunohistochemistry, real-time PCR, andWestern blot analysis, the expressions of CD36and TLR4, as well as the translocationof NF-κB, were decreased in aortic sinus plaques and in the aorta of mice in theLV-PCSK9shRNA treatment group compared with the control group.Conclusion: LV-PCSK9shRNA has no effect on blood lipid levels, but stilldecreases the atherosclerotic lesion and plaque inflammation, as well as lipidaccumulation, in ApoE-/-mice. LV-PCSK9shRNA inhibits the progress ofatherosclerosis by decreasing CD36and TLR4expression and inhibiting NF-κBactivation in ApoE-/-mice.