Macrophage Produced Phospholipid Transfer Protein (PLTP)

描述（由申请人提供）：巨噬细胞（MF）通过脂质的积累和炎性细胞因子的产生在动脉粥样硬化形成中发挥核心作用。血浆磷脂转运蛋白（PLTP）与MF的发生有关，其表达受脂质负荷的影响。我们报道了在病变内产生的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抗炎和抗动脉粥样硬化的机制。第一个目的将测试MF PLTP破坏TLR 2/1质膜二聚化和信号传导，但不破坏内体中预先形成的TLR 3二聚体介导的信号传导的假设。我们将评估MF PLTP对PLTP- /-LDLr-/-小鼠中TLR 2/1和TLR 3信号传导的抗炎作用，所述PLTP- /-LDLr-/-小鼠经辐照并用来自PLTP+/+或PLTP-/-小鼠的骨髓（BM）重建。将研究细胞因子的产生和动脉粥样硬化。第二个目标将测试PLTP改变胆固醇依赖性膜结构域（脂筏）的假设。我们将确定野生型（WT）或突变型PLTP（结合ABCA 1但缺乏脂质转移活性）是否在体内和体外破坏脂筏和/或抑制TLR 2/1二聚化。第三个目的将测试减少肝脏产生的PLTP以降低血浆水平的PLTP将有利于细胞相关PLTP的有益作用的想法。我们将评估PLTP对LDLr-/-小鼠中TLR 2信号传导的抗炎作用，其中LDLr-/-小鼠的肝脏PLTP受到抑制，但未缺失。这将通过体内施用小鼠PLTP反义寡核苷酸（阿索）来实现。用于研究慢性炎症的新型靶向小鼠模型用于建立我们假设的生理意义，并将通过体外研究进行补充，以确定这些生理事件中涉及的机制。激光扫描共聚焦免疫荧光显微镜用于表征早期动脉粥样硬化进展。荧光共振能量转移（FRET）被用来表征响应于PLTP的脂筏的变化。最后，所提出的研究将使用反义寡核苷酸（阿索）来促进抗炎细胞PLTP的有益作用，同时减少循环血浆PLTP的不良影响。我们现在必须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。 公共卫生相关性：拟议的研究将显着改变PLTP的研究方向，提供了良好的证据，其在细胞内炎症通路的调节作用。我们的研究结果将导致对MF生理学和炎症的新的和更好的理解，从而为动脉粥样硬化，糖尿病和其他具有强烈炎症成分的慢性疾病的新治疗选择提供基础。

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.