Are Endothelial Cell and Granulocyte Protein Arginine Deiminase 4 on the Pathway of Inflammasome-Activation, Advancing Myocardial Ischemia Reperfusion Injury?

随着经皮冠状动脉介入治疗技术的进步，急性冠状动脉综合征患者的预后在过去的几十年中得到了显著改善。然而，大约6%的入院患者不能存活出院，心脏功能下降仍然是急性心肌梗死的常见后果，尽管进行了积极的药物治疗。越来越多的临床和实验证据表明，血运重建本身触发了称为心肌缺血-再灌注（M/IR）损伤的病理反应。实验和临床数据表明，心肌梗死/再灌注损伤可能是导致最终梗死面积高达50%的原因，自从Jennings等人在20世纪60年代首次描述了M/IR损伤以来，很明显，M/IR损伤是一种炎症驱动的机制，部分取决于骨髓来源细胞的浸润以及经典炎症通路的激活。阐明这一过程的病理生理机制有助于减轻M/IR损伤。2014年，高C。等人发现了NLRP 3-炎症体（一种介导促炎性半胱天冬酶活化的胞质蛋白复合物）的活化，最终导致心脏微血管内皮细胞（CMEC）中白介素-1家族细胞因子的分泌，作为MI/R损伤的新机制。截至2019年8月，Mishra N等人表明蛋白质精氨酸脱亚胺酶4调节巨噬细胞中的NLRP 3炎症体激活。在2019年，瓦格纳等人证明NLRP 3炎症体激活在粒细胞神经细胞胞外陷阱（NET）释放过程中起重要作用，一种防御机制的形式，最初旨在阻止入侵的微生物，由修饰有颗粒衍生蛋白质的DNA支架组成。多年来，NET与越来越多的自身免疫性和炎症性疾病有关，2015年，Ge L.等人提供了在MI/R损伤激发的心肌中存在NET的证据。内皮细胞和粒细胞蛋白精氨酸脱亚胺酶4是否在炎症体激活的途径上，从而促进心肌缺血再灌注损伤尚待建立，该项目的目的是进一步研究在小鼠模型中使用短期结扎左前降支动脉的MI/R损伤的促炎信号通路。将使用离体方法，如组织学、血液和内皮细胞的基因表达分析、蛋白质分析、流式细胞术和免疫荧光显微镜。PAD 4-/--、WT-和NLRP 3-/--小鼠的比较分析将有助于挑选出它们在MI/R损伤中的个体功能，从而潜在地揭示减轻MI/R损伤的新治疗方式。

With the advances in percutaneous coronary intervention technology, the prognosis of patients with acute coronary syndrome has improved significantly over the last decades. However, approximately 6% of admitted patients do not survive hospital discharge and a decrease in cardiac function is still a common consequence of acute myocardial infarction, despite vigorous medical therapy. Increasing clinical and experimental evidence suggests that revascularisation itself triggers a pathological response termed myocardial ischemia-reperfusion (M/IR) injury. Experimental and clinical data suggests that MI/R injury may be responsible for up to 50% of the ultimate infarction area.Since the first description of M/IR injury by Jennings et al. in the 1960s it became evident that M/IR injury is an inflammatory-driven mechanism, in part depending on the infiltration of bone-marrow derived cells as well as the activation of classic inflammatory pathways. To unravel the pathophysiology of this process could help to attenuate M/IR injury. In 2014 Gao C. et al. discovered the activation of NLRP3-Inflammasome, a cytosolic protein complex that mediates the activation of pro-inflammatory caspases, ultimately resulting in the secretion of cytokines of the interleukin-1 family, in cardiac microvascular endothelial cells (CMECs) as a novel mechanism of MI/R injury. As of August 2019 Mishra N et al. showed that the Protein Arginine Deiminase 4 regulates NLRP3 Inflammasome activation in macrophages.In 2019 Wagner et al. demonstrated that NLRP3 Inflammasome activation plays an important role in the process of granulocyte Neutrophil Extracellular Traps (NETs) release, a form of defence mechanism originally intended to immobilize invading microorganisms consisting of a DNA scaffold decorated with granule-derived proteins. Over the years NETs have been implicated in a growing list of autoimmune and inflammatory conditions and in 2015 Ge L. et al. provided evidence for the existence of NETs in MI/R injury challenged myocardium. Whether endothelial cell and granulocyte Protein Arginine Deiminase 4 are on the pathway of Inflammasome activation and thus advancing myocardial ischemia reperfusion injury is yet to be established.The aim of the proposed project is to further investigate pro-inflammatory signalling pathways of MI/R injury in a mouse model using short-term ligation of the left anterior descending artery. Ex-vivo methods such as histology, gen expression analysis of blood and endothelial cells, protein analysis, flow cytometry and Immunofluorescence microscopy will be used. A comparative analysis of PAD4-/--, WT- and NLRP3-/-- mice will help to single out their individual function in MI/R injury potentially unveiling novel therapeutic modalities in attenuating MI/R injury.