Non-platelet P2Y Receptor in Vascular Inflammation and Thrombogenesis

非血小板 P2Y 受体在血管炎症和血栓形成中的作用

• 批准号: 9237082
• 负责人: Jianzhong Shen
• 金额: $ 37万
• 依托单位: AUBURN UNIVERSITY AT AUBURN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9237082
• 关键词: 3' Untranslated Regions; ATF2 gene; Acute; Address; Affect; Apyrase; Arterial Fatty Streak; Aspirin; Binding; Binding Proteins; Binding Sites; Biological Assay; Blood Platelets; Blood Vessels; Blood coagulation; Blood flow; CYP2C19 gene; Cardiovascular Diseases; Cause of Death; Cell Line; Cells; Cessation of life; Chronic; Clinical; Complication; Coronary; Coronary artery; Disease; Disseminated Intravascular Coagulation; Drug Targeting; EMSA; Elements; Endothelial Cells; Endothelium; Expenditure; Family; Fos-Related Antigens; Gene Expression; Genes; Genetic Polymorphism; Genetic Transcription; Goals; Growth Factor; Healthcare; Hemorrhage; Hormones; Human; Infiltration; Inflammation; Inflammatory; Knockout Mice; Knowledge; LDL-Receptor Related Protein 1; Leukocytes; Link; Luciferases; MAPK14 gene; MAPK8 gene; MK-2; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Modeling; Molecular Target; Monitor; Mus; Myocardial Infarction; Necrosis; Northern Blotting; Nucleotidases; Nucleotides; P2Y2 receptor; PF4 Gene; Pathologic; Pathway interactions; Performance; Pharmaceutical Preparations; Phosphotransferases; Plavix; Population; Post-Transcriptional Regulation; Prevention; Process; Prophylactic treatment; Receptor Activation; Receptor Signaling; Regulation; Reporting; Research; Resistance; Role; Sepsis; Series; Site; Solid; Source; Stents; Stimulus; Stroke; Testing; Therapeutic Agents; Thromboplastin; Thrombosis; Time; Time Factors; Transcription Factor AP-1; United States; Up-Regulation; Vascular Endothelial Cell; Western Blotting; Wild Type Mouse; atherothrombosis; base; cell type; clopidogrel; combat; cytokine; extracellular; ferric chloride; in vivo; liver injury; mRNA Stability; mRNA Transcript Degradation; member; monocyte; mouse model; new therapeutic target; novel; nucleotide receptor; prevent; receptor; response; rho; transcription factor; vascular inflammation

Thrombosis is a fatal complication of major diseases, such as stroke, sepsis, and heart attack. In addition to aspirin, current medication for thrombosis prevention/treatment mainly focus on platelet P2Y12 receptor with plavix as an antagonist in routine clinical use. However, plavix has showed unstable performance, either failing to avoid thrombosis or causing bleeding. The long-term goal of this project is to establish non-platelet P2Y receptors, e.g. P2Y2 receptor (P2Y2R), as a new molecular target that controls inflammation-induced vascular thrombogenesis. Although it is well established that tissue factor (TF) is the initiator of thrombosis, very little is known on the contribution of P2Y receptors in regulation of various inflammatory stimuli-induced TF expressions. This is an important question because many pro-inflammatory stimuli promote cellular nucleotide release and inactivate ectonucleotidases, leading to accumulation of extracellular nucleotides, which in turn amplify original receptors’ signaling via P2Y receptor co-activation. The PI reported previously that P2Y2R is up-regulated in stented coronary arteries and it is the predominant subtype of all P2Y receptors in human coronary artery endothelial cells (HCAEC). They also reported that activation of P2Y2R dramatically induces TF expression and activity in HCAEC. The PIs’ preliminary studies showed that TF induction by P2Y2R is also applicable to human primary blood monocytes and mouse coronary endothelium in vivo, and it involves both transcriptional and post-transcriptional mechanisms. Further, LPS-induced TF induction is significantly decreased when extracellular nucleotides are removed by apyrase, and importantly, P2Y2R-null mice are protected from endotoxic death. Based on these original findings, the PI proposes the hypothesis: The non-platelet P2Y2R is a previously unrecognized key mediator in inflammation-induced thrombosis via TF induction. This hypothesis will be tested by the pursuit of three specific aims: 1) Determine the transcriptional mechanism underlying P2Y2R activation of the TF gene in vascular endothelial cells and blood monocytes; 2) Define the post-transcriptional mechanism by which P2Y2R activation leads to increased TF mRNA stability in vascular endothelial cells and blood monocytes; 3) Assess the role of P2Y2R/TF axis in mouse models of inflammation-induced thrombosis. Aim 1 will determine the role of a new AP-1 binding site with new AP-1 components Fra-1/ATF2 in TF mRNA induction by P2Y2R. Aim 2 will determine the roles of the AU-rich elements in TF 3’UTR along with their binding proteins and the miRNA mechanisms contribute to P2Y2R-mediated TF mRNA stabilization. Aim 3 will verify if deletion of P2Y2R prevents LPS-induced disseminated intravascular coagulation and reduces atherothrombosis. The proposed study will be performed in primary cultured human cells and cells isolated from P2Y2R-null mice, followed by a series of in vivo studies. The proposed research is significant, because it is expected to advance and expand understanding of how inflammation leads to increased thrombogenicity of blood vessels via the new P2Y2R-TF axis. Ultimately, such knowledge has the potential to produce new strategies in combating thrombogenic diseases.

血栓形成是中风、败血症和心脏病等重大疾病的致命并发症。 阿司匹林，目前预防/治疗血栓的药物主要集中在血小板P2Y12受体上 波立维作为拮抗剂在临床常规使用中，但波立维的表现不稳定，要么未能发挥作用。 避免血栓形成或引起出血。该项目的长期目标是建立非血小板P2Y受体， 例如 P2Y2 受体 (P2Y2R)，作为控制炎症诱导的血管的新分子靶点 尽管组织因子 (TF) 是血栓形成的引发剂这一点已得到公认，但这一点却很少。 已知 P2Y 受体在调节各种炎症刺激诱导的 TF 表达中的贡献。 这是一个重要的问题，因为许多促炎刺激物会促进细胞核苷酸释放和 使核酸外切酶失活，导致细胞外核苷酸积累，进而放大原始核苷酸 PI 先前报道，P2Y2R 在 支架冠状动脉，它是人类冠状动脉中所有 P2Y 受体的主要亚型 他们还报道，P2Y2R 的激活可显着诱导 TF 表达和 PI 的初步研究表明 P2Y2R 诱导 TF 也适用于人类。 原代血液单核细胞和小鼠体内冠状动脉内皮细胞，它涉及转录和转录后 此外，当细胞外时，LPS 诱导的 TF 诱导显着降低。 核苷酸被腺苷三磷酸双磷酸酶去除，重要的是，P2Y2R 缺失小鼠免受内毒素死亡。 基于这些原始发现，PI 提出假设：非血小板 P2Y2R 是先前的 通过 TF 诱导炎症诱导血栓形成的关键介质未被识别。 通过追求三个具体目标：1）确定 P2Y2R 激活的转录机制 血管内皮细胞和血液单核细胞中的TF基因2)定义转录后机制 P2Y2R 激活导致血管内皮细胞和血液单核细胞中 TF mRNA 稳定性增加； 3) 评估 P2Y2R/TF 轴在炎症诱导血栓形成小鼠模型中的作用，目标 1 将确定。 新的 AP-1 结合位点与新的 AP-1 成分 Fra-1/ATF2 在 P2Y2R Aim 诱导 TF mRNA 中的作用。 2 将确定 TF 3'UTR 中富含 AU 的元件及其结合蛋白和 miRNA 的作用 有助于 P2Y2R 介导的 TF mRNA 稳定的机制 目标 3 将验证 P2Y2R 的删除是否会阻止。 LPS诱导弥散性血管内凝血并减少动脉粥样硬化血栓形成。 在原代培养的人类细胞和从 P2Y2R 缺失小鼠中分离的细胞中进行，然后进行一系列体内研究 拟议的研究意义重大，因为它有望推进和扩大对这一问题的理解。 炎症如何通过新的 P2Y2R-TF 轴导致血管血栓形成增加。 这些知识有可能产生对抗血栓性疾病的新策略。