Macrophage Produced Phospholipid Transfer Protein (PLTP)

描述（由申请人提供）：巨噬细胞（MF）通过脂质积累和炎症细胞因子的产生在动脉粥样硬化中起核心作用。血浆磷脂转移蛋白（PLTP）与病变中的MF相关，其表达因脂质负荷而上调。我们报道了病变内产生的PLTP具有动脉粥样硬化保护作用。该项目的长期目标是确定细胞相关PLTP抗炎和动脉粥样硬化保护的机制。第一个目的是验证MF PLTP破坏TLR2/1质膜二聚体和信号传导的假设，但不破坏TLR3二聚体介导的内体信号传导。我们将评估MF PLTP对PLTP+/+或PLTP-/-小鼠骨髓（BM）重建的PLTP-/- ldlr -/-小鼠TLR2/1和TLR3信号通路的抗炎作用。细胞因子的产生和动脉粥样硬化将被研究。第二个目标将检验PLTP改变胆固醇依赖膜结构域（脂筏）的假设。我们将在体内和体外确定野生型（WT）或突变型PLTP（与ABCA1结合但缺乏脂质转移活性）是否会破坏脂筏和/或抑制TLR2/1二聚化。第三个目标将测试这样一种观点，即减少肝脏PLTP的产生以降低血浆PLTP水平将有利于细胞相关PLTP的有益作用。我们将在肝脏PLTP抑制但未缺失的LDLr-/-小鼠中评估PLTP对TLR2信号的抗炎作用。这将通过在体内给药小鼠PLTP反义寡核苷酸（ASO）来完成。用于慢性炎症研究的新型靶向小鼠模型被用来确定我们假设的生理意义，并将通过体外研究来补充，以确定这些生理事件的机制。激光扫描共聚焦免疫荧光显微镜用于描述早期动脉粥样硬化的进展。荧光共振能量转移（FRET）是用来表征在响应PLTP脂筏的变化。最后，拟议的研究将使用反义寡核苷酸（ASO）来支持抗炎细胞PLTP的有益作用，同时减少循环血浆PLTP的不良影响。当务之急是我们现在知道是否应该追求细胞导向或血浆导向的PLTP动脉粥样硬化保护疗法用于人类心血管危险因素的治疗。

DESCRIPTION (provided by applicant): Macrophages (MF) play a central role in atherogenesis through accumulation of lipids and production of inflammatory cytokines. Plasma phospholipid transfer protein (PLTP) is associated with MF in lesions and its' expression is up-regulated by lipid loading. We reported that PLTP produced within lesions is atheroprotective. The long-term goal of this project is to identify mechanisms whereby cell associated PLTP is anti-inflammatory and atheroprotective. The first aim will test the hypothesis that MF PLTP disrupts TLR2/1 plasma membrane dimerization and signaling, but not preformed TLR3 dimer mediated signaling in endosomes. We will assess the anti-inflammatory effects of MF PLTP on TLR2/1 and TLR3 signaling in PLTP- /-LDLr-/- mice irradiated and reconstituted with bone-marrow (BM) from PLTP+/+ or PLTP-/- mice. Cytokine production and atherosclerosis will be studied. The second aim will test the hypothesis that PLTP alters cholesterol dependent membrane domains (lipid rafts). We will determine if wild-type (WT) or mutant PLTP (which binds ABCA1 but lacks lipid transfer activity) disrupts lipid rafts and/or inhibits TLR2/1 dimerization in vivo and in vitro. The third aim will test the idea that reduced hepatic production of PLTP to reduce plasma levels of PLTP will favor the beneficial effects of cell associated PLTP. We will assess the anti-inflammatory effects of PLTP on TLR2 signaling in LDLr-/- mice with suppressed, but not deleted, hepatic PLTP. This will be accomplished with in vivo administration of mouse PLTP antisense oligonucleotides (ASO). Novel, targeted mouse models for study of chronic inflammation are used to establish the physiological significance of our hypotheses and will be complemented by in vitro studies to identify the mechanisms involved in these physiological events. Laser scanning confocal immunofluorescent microscopy is used to characterize early atherosclerosis progression. Fluorescent resonance energy transfer (FRET) is used to characterize changes in lipid rafts in response to PLTP. Finally, the proposed studies will use antisense oligonucleotides (ASO) to favor the beneficial effects of anti-inflammatory cellular PLTP while reducing the undesirable affects of circulating plasma PLTP. It is imperative that we now know whether cell-directed or plasma-directed PLTP atheroprotective therapeutics should be pursued for human treatment of cardiovascular risk factors. PUBLIC HEALTH RELEVANCE: The proposed studies will significantly alter the direction of PLTP research by providing sound evidence of its role in the regulation of intracellular inflammatory pathways. The results of our studies will lead to a new and improved understanding of MF physiology and inflammation thus providing the bases for novel treatment options for atherosclerosis, diabetes and other chronic diseases that have a strong inflammatory component.