Department of Veterinary Medicine, Zhejiang University, Hangzhou, 310058, People's Republic of China.
Center for Veterinary Medicine, Zhejiang University, Hangzhou, 310058, People's Republic of China.
Respir Res. 2022 Oct 23;23(1):290. doi: 10.1186/s12931-022-02210-7.
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.
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.
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.
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.
丛状病变在结构和细胞组成上具有动态表现，是人类严重肺动脉高压的组织学标志。尽管病变发展的发病机制在很大程度上尚不清楚，但它可能与早期祖内皮细胞（eEPC）的局部炎症和功能障碍有关。我们通过检测慢性炎症环境中 eEPC 分化为巨噬细胞来验证假说，即 eEPC 通过分化为巨噬细胞促进丛状病变的发展。
通过免疫组化检测肉鸡模型中丛状病变中的 eEPC 标志物 CD133 和 VEGFR-2、巨噬细胞谱系标志物甘露糖受体 C 型 1（MRC1）、TNFα 和核因子红细胞 2 相关因子 2（Nrf2）。从外周血单核细胞中分离 eEPC，分别通过吞噬作用和 Matrigel 塞试验评估细胞的巨噬细胞分化和血管生成能力。还评估了 Nrf2 在 eEPC 向巨噬细胞转化以及 MRC1 表达中的作用。通过气管内安装 TNFα 来确定局部炎症对丛状病变形成的影响。
早期病变的细胞具有典型的 eEPC 表型，而更成熟病变的细胞则表现出巨噬细胞的分子和形态特征。随着病变的进展，观察到丛状病变中 TNFα 产生增加。体外研究表明，慢性 TNFα 刺激导致 eEPC 向巨噬细胞分化，同时 Nrf2 过度激活，Nrf2 是一种应激反应转录因子。Nrf2 激活（Keap1 敲低）导致 CD133 的明显下调和 MRC1 mRNA 的上调。双荧光素酶报告基因检测表明，Nrf2 结合到 MRC1 的启动子上，触发其表达。与体外观察结果一致，TNFα 暴露诱导 eEPC 在 Matrigel 塞中分化为巨噬细胞，导致塞内新生血管减少。气管内安装 TNFα 导致丛状病变密度显著增加。
这项工作提供了证据表明，慢性炎症刺激导致的 eEPC 向巨噬细胞分化有助于丛状病变的发展。鉴于 Nrf2 在 eEPC 向巨噬细胞表型转换中的关键作用，针对该分子可能有益于丛状病变的干预。
