不同表型巨噬细胞对先心病肺动脉高压中肺血管重塑的作用机制及干预研究

Pulmonary arterial hypertension (PAH) is one of most common complications of congenital heart disease and causes a incurable progressive to right ventricular failure and death. Pulmonary vascular smooth muscle cell (PVSMC) proliferation plays a key role in the development of pulmonary vascular remodeling (PVR), which ultimately leads to elevated pulmonary artery pressure and the failure of drug treatment. Recent studies demonstrated the recruitment and activation of macrophages is involved in triggering and maintaining PVR, however, the exact role of macrophages in PAH pathobiology is still controversial, due to its efficiently responding to environmental signals with remarkable plasticity and undergoing different forms of polarized activation. Macrophages can be roughly categorized as classically activated (M1), alternatively activated (M2), and anti-inflammatory (regulatory) macrophages. . In our previous studies, we have already induced every phenotype of macrophages and made their major functions clear. M1 macrophages produce inducible nitric oxide synthase and IL-12 and exhibit enhanced microbicidal or tumoricidal capacity. M2 macrophages play the role of promoting fibrosis and angiogenesis. The major characteristic of the third population, regulatory macrophages, is the production of high IL-10 and low IL-12 levels and the promotion of immunosuppression. . We found that MCT-induced PAH can be effectively alleviated by reducing inflammation. And our previous studies indicated that early recruitment and activation of M2 macrophages is critical for high blood flow-induced PAH, but the activation state of the recruited macrophages and their contribution to disease has remained unclear. . This research project plans to continue the work above, sought to find out if macrophages really initiates vascular remodeling(“initial hit”), participates in its progression (“secondary hits”) or represents a reactive response to remodeling only (“bystander phenomenon”) in the development of PAH. First, we characterize the response of the macrophages in rat PAH model and congenital heart disease-derived PAH patients. Next, we explore how macrophages affect PVSMCs proliferation in vitro, and in vivo, elucidate its role in PAH and PVR by intervening of macrophage polarization and transferring specific phenotype to macrophage-free rat model. Further, we will explore the role of Ym1 (an important molecular in M2 macrophage) in process and pathogenesis of PAH with human samples and rat models in vivo and in vitro. In short, the present study aims to provide new insight in the pathophysiologic process of PAH, and provide potential implications in designing future therapeutic strategies.

肺动脉高压是先天性心脏病常见的并发症，严重影响患者的生存及预后。以肺血管平滑肌细胞的表型转换为核心环节的肺血管重塑，是肺高压持续发展、恶化的主要原因。研究表明，巨噬细胞的浸润可能是肺高压和肺血管重塑发生的关键，但其表型的多样性（M1、M2和调节型）决定了其功能的不同，其在肺高压中的具体机制尚不明确。在前期研究中，我们发现通过减轻炎症反应可有效缓解肺动脉高压，并证实M2型巨噬细胞与高肺血流性肺高压的发展有密切关系；同时，我们已在体外诱导出三种表型的巨噬细胞并应用于动物模型开展研究。本课题拟延续以上研究，从细胞、动物和临床三个层面探讨不同表型巨噬细胞在肺高压发生发展中的变化规律；通过体外定向诱导并回输的方法，干预巨噬细胞分化并探讨其对疾病发展的影响；并首次就M2型巨噬细胞分化中重要分子Ym1和肺高压的关系进行探讨。本研究旨在揭示肺高压发展的新机制，从而为该病的治疗提供重要的理论基础及干预靶点。

肺动脉高压严重影响心血管、呼吸系统疾病患者的生存、预后。肺血管重构是肺高压持续恶化、药物治疗失败的重要原因。巨噬细胞是肺动脉周围炎性细胞浸润的主要细胞之一，也是调控肺血管重构局部因子的重要来源。巨噬细胞具有异质性和可塑性，M1型巨噬细胞被认为参与了肺高压早期损伤过程，释放大量炎症因子损伤组织。M2型巨噬细胞（M2a/M2c）被证实具有促纤维化、促平滑肌细胞增殖的作用。然而，调节型巨噬细胞（M2b）不同于M1和M2，它不参与纤维化并能维持抗炎和促炎功能之间的平衡。于是我们推测M2b型巨噬细胞对肺高压肺血管重塑具有改善作用。.本研究从野百合碱诱导的肺高压大鼠中分离出肺动脉平滑肌细胞，同时我们在体外成功诱导极化各亚型巨噬细胞。将各型巨噬细胞的上清与平滑肌细胞共培养，发现M2b型巨噬细胞较其他组更能抑制平滑肌细胞的增殖、迁移。于是聚焦至M2b型巨噬细胞，发现它确实促进了平滑肌细胞的凋亡，并与Bcl-2家族蛋白介导的线粒体凋亡密切相关。虽然自噬和凋亡的发生关系密切，但我们发现在这一过程中自噬流不通畅，并未起真正作用。进一步筛选信号通路，我们发现M2b型巨噬细胞抑制了PI3K/Akt/FoxO3a通路。于是利用PI3K的抑制剂LY294002，发现它同样可以抑制细胞增殖、迁移，也促进了Bcl-2家族蛋白相关的凋亡。当两者同时处理细胞时，上述的趋势更为显著。综上所述，M2b型巨噬细胞可能通过抑制PI3K/Akt/FoxO3a通路发挥抑制平滑肌细胞增殖、迁移和凋亡抵抗的作用，具有改善肺高压肺血管重塑的潜力。为了探索其发挥作用的通讯分子，我们提取并发现M2b型巨噬细胞分泌大量的外泌体，其内还含有丰富的miRNA。. 在体实验中利用改良颈动静脉分流模型成功诱导肺高压大鼠，并发现了M2型巨噬细胞的浸润。我们还发现该模型中Akt/mTOR通路的激活，于是以其为靶点利用雷帕霉素成功缓解了肺高压，其作用与抑制平滑肌细胞的增殖相关。在缺氧诱导的平滑肌细胞增殖模型中，NIX介导的线粒体自噬可以清理损伤的线粒体从而抑制细胞增殖。在体模型中，我们发现ABT737可以激活NIX介导的线粒体自噬从而缓解缺氧诱导的肺动脉高压。