Eicosanoids and Endothelial Cell Dysfunction

描述（由申请人提供）：过度炎症被认为是增殖性心血管疾病（如动脉粥样硬化）发病的潜在因素。磷脂酶A2s （PLA2s）在炎症反应中起重要作用。花生四烯酸（AA）主要由PLA2s产生，通过环加氧酶（COX）、脂加氧酶（LOX）和细胞色素P450单加氧酶（CYP）途径代谢，分别产生前列腺素（pg）、氢过氧二十碳四烯酸（HPETEs）和环氧二十碳三烯酸（EETs）。AA的部分COX和LOX产物具有促炎作用，而CYP，尤其是AA的CYP2C8/9产物具有抗炎作用。粥样硬化动脉产生15(S)- hete作为AA的主要15- LOX产物。尽管15- lox1参与低密度脂蛋白氧化已被广泛研究，以了解其在动脉粥样硬化病理生物学中的作用，但其AA产物15(S)-HETE在动脉粥样硬化中的作用却知之甚少。在这方面，在该资助申请的前一个资助周期中，我们已经证明15(S)-HETE通过诱导成纤维细胞生长因子-2、血管内皮生长因子和基质金属蛋白酶-2的表达来调节血管生成，这是血管壁疾病的一个重要因素。在这些研究过程中，我们发现15(S)-HETE破坏内皮细胞（EC）屏障功能。由于EC的关键功能之一是维持血管壁完整性，因此EC屏障功能的扰动可能是EC功能障碍和炎症的起始点，这是动脉粥样硬化发病机制中的两个基本事件。基于这些新的观察结果，我们预测类二十烷，特别是AA的15- lox产物，即15(S)-HETE，通过其扰乱EC屏障功能的能力，促进炎症细胞向内皮下空间的细胞旁运动，并为动脉粥样硬化的发展奠定了基础。为了解决这一假设，我们建议测试以下具体目标：1。15-LOX1-15(S)-HETE轴通过酪氨酸磷酸化的紧密连接（TJ）蛋白破坏TJ，从而扰乱EC屏障功能；2. 非受体酪氨酸激酶Src和Pyk2介导15- LOX1-15(S)- hete诱导的TJ蛋白酪氨酸磷酸化及其从TJ蛋白上的分解导致EC屏障功能障碍；3. 丝裂原活化蛋白激酶（MAPKs）介导15-LOX1-15(S)- hete诱导的TJ蛋白丝氨酸/苏氨酸磷酸化及其从TJ上的分解导致EC屏障功能障碍。15-LOX1-15(S)-HETE轴通过破坏内皮屏障功能，促进炎症细胞向内皮下空间的细胞旁运动，促进高脂饮食喂养小鼠的炎症和动脉粥样硬化。因此，本次资助申请中提出的实验将提供关于15-LOX1-15(S)-HETE轴在导致炎症和动脉粥样硬化的内皮屏障功能障碍中的潜在作用的新信息，这可能对治疗药物的开发有用。

DESCRIPTION (provided by applicant): Inflammation, in excess, was believed to be an underlying factor in the pathogenesis of proliferative cardiovascular diseases such as atherosclerosis. Phospholipase A2s (PLA2s) play an important role in inflammation. Arachidonic acid (AA), which is produced primarily by PLA2s, metabolizes via the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 monooxygenase (CYP) pathways producing prostaglandins (PGs), hydroperoxyeicosatetraenoic acids (HPETEs) and epoxyeicosatrienoic acids (EETs), respectively. While some of the COX and LOX products of AA are proinflammatory, CYP, particularly CYP2C8/9 products of AA, are anti-inflammatory. Atherosclerotic arteries produce 15(S)-HETE as a major 15- LOX product of AA. Although the involvement of 15-LOX1 in the oxidation of low-density lipoprotein has been extensively studied in understanding its role in the pathobiology of atherosclerosis, very little is known in regard to its AA product, 15(S)-HETE, in atherosclerosis. In this regard, during the previous funding cycle of this grant application we have shown that 15(S)-HETE via inducing the expression of fibroblast growth factor-2, vascular endothelial growth factor and matrix metalloproteinase-2 modulates angiogenesis, an important factor in vascular wall diseases. During the course of these studies, we have discovered that 15(S)-HETE disrupts endothelial cell (EC) barrier function. Since one of the crucial functions of ECs is to maintain vessel wall integrity, perturbation in EC barrier function could be an initiation point for EC dysfunction and inflammation, two fundamental events in the pathogenesis of atherosclerosis. Based on these novel observations, we predict that eicosanoids, particularly the 15-LOX product of AA, namely, 15(S)-HETE via its capacity to perturb EC barrier function promotes paracellular movement of inflammatory cells into sub-endothelial space and sets the soil for the development of atherosclerosis. To address this hypothesis, we have proposed to test the following specific aims: 1. 15-LOX1-15(S)-HETE axis via tyrosine phosphorylation of tight junction (TJ) proteins disrupts TJs and thereby perturbs EC barrier function; 2. Non-receptor tyrosine kinases, Src and Pyk2 mediate 15- LOX1-15(S)-HETE-induced tyrosine phosphorylation of TJ proteins and their disassembly from TJs resulting in EC barrier dysfunction; 3. Mitogen-activated protein kinases (MAPKs) mediate 15-LOX1-15(S)-HETE-induced TJ protein serine/threonine phosphorylation and their disassembly from TJs resulting in EC barrier dysfunction and 4. 15-LOX1-15(S)-HETE axis via disrupting endothelial barrier function facilitates paracellular movement of inflammatory cells into the subendothelial space and promotes inflammation and atherosclerosis in response to feeding mice with high-fat diet. Thus, the experiments proposed in this grant application will provide novel information on the potential role of 15-LOX1-15(S)-HETE axis in endothelial barrier dysfunction leading to inflammation and atherosclerosis, which could be useful in the development of therapeutic drugs.