中性粒细胞NETosis对脑缺血后血管新生与功能重塑的作用和机制研究

Angiogenic response is essential for ischemic brain repair. However, currently there are still no effective therapies available for promoting post-stroke neovascularization. Neutrophil extracellular traps (NETs) arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Recently, increased release of NETs has been found to play important roles in the growth of tumor blood vessels and atherosclerosis. Our preliminary data show that neutrophils migrated from blood vessels into the brain and produced NETs after ischemic stroke. Disruption of NETs by administration of DNaseI markedly increased vascularization and capillary perfusion in the peri-infarct cortex at 14 days after stroke. In this project, we will employ mouse stroke model and OGD-challenged human brain microvessel endothelial cells, using PAD4 knockout mice, PAD4 inhibitor, neutrophil-depleted antibody, DNaseI, multiphoton microscopy, and molecular biological approaches to determine the effects and mechanism of inhibition of NETosis and disruption of NETs on formation and remodeling of the newly formed vessels after ischemic stroke. We will also determine whether the increased neovascularization by inhibiting NETosis and cleaving NETs is accompanied by improved neurological functions. Furthermore, we will assess whether inhibition of NETosis and disruption of NETs promote neovascularization and vascular remodeling after stroke via regulation of STING-TBK1-IRF3 and TLR9/MyD88 pathways in brain microvessels. These proposed studies may provide new theoretical and experimental basis for improving post-stroke vascular remodeling.

血管新生是缺血性脑卒中后脑功能修复的重要环节，但是缺乏有效的调控手段。NETosis是近年发现的一种新的细胞死亡方式，通过形成中性粒细胞胞外诱捕网(NETs)，对肿瘤血管发生和动脉硬化发挥重要的作用。我们近期预实验发现，脑缺血导致中性粒细胞由血管内渗出到脑组织产生大量的NETs，而使用DNaseI降解NETs可增加小鼠脑血管长度和血管灌注，相关研究至今尚无报道。本项目拟利用小鼠脑缺血模型和体外脑内皮细胞OGD模型，结合多光子显微镜活体成像、中性粒细胞清除、PAD4基因敲除和抑制剂、药物干预等技术和方法，研究NETosis对脑缺血后血管新生和功能重塑的调控作用，阐明抑制NETs产生和促进NETs降解能否通过调控STING-TBK1-IRF3和TLR9/MyD88信号通路，促进脑缺血后的血管新生，进而改善受损的神经功能。这些研究将为揭示缺血性脑卒中后血管新生的机制提供新的视点和理论基础。

血管新生对缺血性脑卒中后神经功能损伤的修复具有重要的意义。我们研究发现脑内中性粒细胞在脑缺血后第1天到第14天明显增加，并在第3天达到峰值。活体多光子显微镜和激光共聚焦成像进一步表明，中性粒细胞可以通过血脑屏障进入脑损伤周边皮层脑组织。中性粒细胞捕获外网NETs标记物H3Cit在脑缺血后第1天到第5天表达明显增加，并在第3天达到峰值。多光子活体检测NETs标记物Sytox green发现，脑缺血第3天小鼠脑组织中的线性或网状NETs产生明显增多。在清除小鼠体内的中性粒细胞后，脑缺血小鼠第3天的NETs产生减少，脑缺血恢复期损伤周边皮层的血管新生明显增加，血脑屏障破坏减轻。给予小鼠核酸酶DNaseⅠ促进NETs降解，可增加脑缺血恢复期损伤周边皮层新生血管的长度和分支及灌注血管的长度，减轻血脑屏障损伤，促进血管成熟。随后，我们研究发现脑损伤周边皮层中PAD4的表达明显增加。给予小鼠过表达PAD4，可增多NETs产生，明显减少脑缺血后损伤周边皮层的血管新生，并加剧血脑屏障破坏。而敲除PAD4或给予PAD4抑制剂降低NETs产生，引起脑缺血后损伤周边皮层的血管新生增加，血脑屏障破坏减轻，并且小鼠的神经功能明显得到改善。最后，我们发现PAD4抑制可逆转脑缺血引起的STING介导的TBK1-pIRF3蛋白通路的激活和IFN-βI型干扰素产生，并且沉默STING和IFN受体的中和抗体均可增加脑缺血后血管重塑，减轻血脑屏障损伤。以上结果提示，中性粒细胞NETsis可能通过STING介导的I型干扰素损害脑缺血后血管新生和重塑。