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.

血栓形成是重大疾病的致命并发症，如中风、败血症和心脏病发作。除了 阿司匹林，目前用于血栓预防/治疗的药物主要集中在血小板P2 Y12受体， 在常规临床使用中，血小板减少素作为拮抗剂。然而，planetary表现不稳定， 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P2 Y受体， 例如P2 Y2受体（P2 Y2 R）作为一种新的调控炎症诱导的血管生成的分子靶点， 血栓形成尽管已经确定组织因子（TF）是血栓形成的起始因子， 已知P2 Y受体在调节各种炎症刺激诱导的TF表达中的作用。 这是一个重要的问题，因为许多促炎刺激促进细胞核苷酸释放， 细胞外核苷酸酶，导致细胞外核苷酸的积累，这反过来又放大了原始的 通过P2 Y受体共激活的受体信号传导。PI先前报告P2 Y2 R在 P2 Y受体是人冠状动脉中所有P2 Y受体的主要亚型 内皮细胞（HCAEC）。他们还报告说，P2 Y2 R的激活显着诱导TF表达， HCAEC的活动。PI的初步研究表明，P2 Y2 R诱导的TF也适用于人类。 原代血单核细胞和小鼠冠状动脉内皮细胞在体内，它涉及转录和转录后 机制等此外，LPS诱导的TF诱导显著降低，当细胞外 通过腺苷三磷酸双磷酸酶去除核苷酸，并且重要的是，保护P2 Y2 R缺失小鼠免于内毒素死亡。 基于这些原始发现，PI提出假设：非血小板P2 Y2 R是先前的 在炎症诱导的血栓形成中通过TF诱导未被识别的关键介质。这一假设将得到检验 通过追求三个特定的目标：1）确定P2 Y2 R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）通过以下方式定义转录后机制： 其中P2 Y2 R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P2 Y2 R/TF轴在炎症诱导的小鼠血栓形成模型中的作用。目标1将决定 新AP-1结合位点与新AP-1组分Fra-1/ATF 2在P2 Y2 R诱导TF mRNA表达中的作用。目的 2将决定TF 3 'UTR中富含AU的元件沿着结合蛋白和miRNA的作用 机制有助于P2 Y2 R介导的TF mRNA稳定。目标3将验证删除P2 Y2 R是否会阻止 脂多糖诱导的弥散性血管内凝血和减少动脉粥样硬化血栓形成。拟议的研究将 在原代培养的人细胞和分离自P2 Y2 R缺失小鼠的细胞中进行，然后进行一系列体内实验。 问题研究拟议的研究是重要的，因为它有望推进和扩大对 炎症如何通过新的P2 Y2 R-TF轴导致血管血栓形成性增加。最后， 这些知识有可能产生防治血栓形成疾病的新战略。