背景:丛状病变,在结构和细胞组成上具有动态外观,是人类严重肺动脉高压的组织学标志。病变发展的发病机制仍然很大程度上未知,尽管它可能与早期内皮祖细胞(eEPCs)的局部炎症和功能障碍有关。我们测试了以下假设:在慢性炎症的情况下,eEPCs通过分化为巨噬细胞来促进丛状病变的发展。
方法:eEPC标记CD133和VEGFR-2,巨噬细胞谱系标记甘露糖受体C型1(MRC1),通过免疫组织化学测定了肉鸡模型丛状病变中的TNFα和核因子类2相关因子2(Nrf2)。来自外周血单核细胞的eEPCs暴露于TNFα,和巨噬细胞的分化和血管生成能力的细胞通过吞噬和基质胶塞试验进行评估,分别。还评估了Nrf2在eEPC至巨噬细胞转化以及MRC1表达中的作用。进行TNFα的气管内安装以确定局部炎症对丛状病变形成的影响。
结果:由早期病变组成的细胞具有典型的eEPC表型,而在更成熟的病变中的细胞具有巨噬细胞的分子和形态特征。随着病变进展,观察到丛状病变中TNFα的产生增加。体外研究表明,慢性TNFα攻击使eEPCs向巨噬细胞分化,并伴随着应激反应转录因子Nrf2的过度激活。Nrf2激活(Keap1敲低)导致CD133明显下调,但MRC1mRNA上调。双荧光素酶报告基因测定表明Nrf2与MRC1的启动子结合以触发其表达。与体外观察结果吻合良好,TNFα暴露诱导基质胶塞中eEPCs的巨噬细胞分化,导致栓塞的新血管形成减少。气管内安装TNFα导致丛状病变密度显着增加。
结论:这项工作提供的证据表明,由慢性炎症刺激引起的eEPCs的巨噬细胞分化有助于丛状病变的发展。鉴于Nrf2在eEPCs向巨噬细胞的表型转换中的关键作用,靶向该分子可能有利于干预丛状病变。
BACKGROUND: Plexiform lesions, which have a dynamic appearance in structure and cellular composition, are the histological hallmark of severe pulmonary arterial hypertension in humans. The pathogenesis of the lesion development remains largely unknown, although it may be related to local inflammation and dysfunction in early progenitor endothelial cells (eEPCs). We tested the hypothesis that eEPCs contribute to the development of plexiform lesions by differentiating into macrophages in the setting of chronic inflammation.
METHODS: The eEPC markers CD133 and VEGFR-2, macrophage lineage marker mannose receptor C-type 1 (MRC1), TNFα and nuclear factor erythroid 2-related factor 2 (Nrf2) in plexiform lesions in a broiler model were determined by immunohistochemistry. eEPCs derived from peripheral blood mononuclear cells were exposed to TNFα, and macrophage differentiation and angiogenic capacity of the cells were evaluated by phagocytotic and Matrigel plug assays, respectively. The role of Nrf2 in eEPC-to-macrophage transition as well as in MRC1 expression was also evaluated. Intratracheal installation of TNFα was conducted to determine the effect of local inflammation on the formation of plexiform lesions.
RESULTS: Cells composed of the early lesions have a typical eEPC phenotype whereas those in more mature lesions display molecular and morphological characteristics of macrophages. Increased TNFα production in plexiform lesions was observed with lesion progression. In vitro studies showed that chronic TNFα challenge directed eEPCs to macrophage differentiation accompanied by hyperactivation of Nrf2, a stress-responsive transcription factor. Nrf2 activation (Keap1 knockdown) caused a marked downregulation in CD133 but upregulation in MRC1 mRNA. Dual luciferase reporter assay demonstrated that Nrf2 binds to the promoter of MRC1 to trigger its expression. In good agreement with the in vitro observation, TNFα exposure induced macrophage differentiation of eEPCs in Matrigel plugs, resulting in reduced neovascularization of the plugs. Intratracheal installation of TNFα resulted in a significant increase in plexiform lesion density.
CONCLUSIONS: This work provides evidence suggesting that macrophage differentiation of eEPCs resulting from chronic inflammatory stimulation contributes to the development of plexiform lesions. Given the key role of Nrf2 in the phenotypic switching of eEPCs to macrophages, targeting this molecular might be beneficial for intervention of plexiform lesions.