中性粒细胞胞外诱捕网调控C3-FH补体系统损伤内皮细胞的分子机制及其在肺动脉高压中的作用

Pulmonary hypertension (PH) is a common but devastating disease lacking curative treatment, characterized by pulmonary vascular remodeling with endothelial cell injury and dysfunction. Inflammation and immune responses are emerging as important contributing factors in the disease process. It was showed in our pilot experiments that neutrophil extracellular traps (NETs), released by neutrophils via NETosis, and related proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), correlated with PH progression. However, the underlying mechanism of NETs injuring endothelium is still unknown. Recently, NETs was found to be capable of activating complement system in Systemic Lupus Erythematosus, mainly by affecting C3 pathway and its key regulator complement Factor H (FH). Whereas, we know that complement system could induce endothelial injury. Furthermore, it was reported that complement C3 deficiency attenuated chronic hypoxia-induced PH in mice. Base on the above pilot data and evidence, we hypothesize that increasing neutrophil extracellular traps (NETs) could damage endothelium in pulmonary hypertension via regulating C3-FH complement system. To test this hypothesis, effectiveness of NETs intervention on PH progression will be further assessed in PAD4-/- mice or in PAD4-inhibited rats after confirming the correlation between PH progression and NETs level in humanized mice with chronic hypoxia-induced PH. Secondly, the direct interaction between NETs and endothelial cells will be evaluated, followed by exploration the underlying mechanism with NETs modification, e.g. DNase-I-degradation, or neutralization with monoclonal anti-MPO/NE/MMP-9 antibodies. Furthermore, impact of NETs on activation of complement system and on expression of complement-related receptors on endothelial surface will be analyzed. Then, the role of C3-FH complement system in NETs damaging endothelium will be investigated in NETs-endothelial cells-complement co-culture environment, by C3 clearance or blocking FH. The research would unveil a novel mechanism of inflammation/immune response regulating PH progression from the point view of NETs and complement system, and provide theoretical basis of new method of diagnosis and treatment of PH.

肺高压（pulmonary hypertension, PH）是一类潜在致死性疾病，缺乏有效治疗手段。内皮细胞损伤和功能异常是PH肺血管重塑的重要病理变化。文献及预实验提示中性粒细胞胞外诱捕网（neutrophil extracellular traps, NETs）及其相关蛋白影响PH进展，但NETs损伤肺动脉内皮细胞的机制不清楚。NETs参与C3-FH补体系统活化过程；而C3-FH补体系统可调节内皮细胞损伤及功能。据此我们提出：PH中NETs很可能通过调控C3-FH补体系统损伤肺动脉内皮细胞损伤，调控肺血管重塑，促进PH发展。项目通过基因敲除动物工具，观察PH进程中NETs变化规律及干预NETs控制PH的效果；构建不同组分NETs或去除NETs中不同成分，明确NETs作用内皮细胞直接机制；进而研究NETs调控C3-FH补体系统损伤内皮细胞的分子机制，为PH诊断和治疗提供理论依据。

肺动脉高压是一类潜在致死性疾病，缺乏有效治疗手段。内皮细胞损伤和功能异常是肺动脉高压肺血管重塑的重要病理变化。文献及预实验提示中性粒细胞胞外诱捕网（neutrophil extracellular traps, NETs）及其相关蛋白影响肺动脉高压进展，但NETs生成及其损伤肺动脉内皮细胞的机制不清楚。我们在肺动脉高压患者中发现，合并与不合并肺动脉高压患者的中性粒细胞/淋巴细胞的比值间存在差异，且该比值和肺动脉收缩压呈正相关；中性粒细胞的NETs生成能力与肺动脉高压患者的肺动脉压力具有相关性，且显著高于正常对照组。在离体细胞实验中，我们发现肺动脉高压的低剪切力是NETs生成的重要刺激因素。通过平行板流动培养舱模拟不同剪切力刺激，我们证实低剪切力可促进外周血来源中性粒细胞或dHL-60类中性粒细胞NETs生成，其机制可能与中性粒细胞机械敏感离子通道Piezo1相关信号通路有关。与机械应力感知相关的中性粒细胞Piezo1表达下调，细胞内钙离子浓度下降，促进NETs生成的细胞内ROS和HDAC2表达水平上调；从而促进NETs生成。siRNA下调Piezo1，模拟低剪切力可促进NETs生成效应；而Piezo1的激动剂Yodal干预，可抑制上述过程。肺动脉高压条件下生成的NETs能促进内皮细胞的凋亡，而重塑肺血管。上述研究结果，一方面证实了重要的免疫细胞——中性粒细胞及其胞外诱捕网（NETs）在肺动脉高压进程中的作用，另一方面也发现了血流动力学相关的机械刺激调控中性粒细胞NETs生成，损伤肺血管内皮细胞，从而影响肺动脉高压肺血管重塑；为未来探索肺动脉高压诊治新靶点提供了有力依据。