Sahoo Sanghamitra, Li Yao, de Jesus Daniel, Sembrat John, Rojas Mauricio M, Goncharova Elena, Cifuentes-Pagano Eugenia, Straub Adam C, Pagano Patrick J
Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.
Am J Physiol Heart Circ Physiol. 2021 Sep 1;321(3):H542-H557. doi: 10.1152/ajpheart.00125.2021. Epub 2021 Jul 23.
Pulmonary arterial hypertension (PAH) is a fatal cardiopulmonary disease characterized by increased vascular cell proliferation with apoptosis resistance and occlusive remodeling of the small pulmonary arteries. The Notch family of proteins subserves proximal signaling of an evolutionarily conserved pathway that effects cell proliferation, fate determination, and development. In endothelial cells (ECs), Notch receptor 2 (Notch2) was shown to promote endothelial apoptosis. However, a pro- or antiproliferative role for Notch2 in pulmonary endothelial proliferation and ensuing PAH is unknown. We postulated that suppressed Notch2 signaling drives pulmonary endothelial proliferation in the context of PAH. We observed that levels of Notch2 are ablated in lungs from PAH subjects compared with non-PAH controls. Notch2 expression was attenuated in human pulmonary artery endothelial cells (hPAECs) exposed to vasoactive stimuli including hypoxia, TGF-β, ET-1, and IGF-1. Notch2-deficient hPAECs activated Akt, Erk1/2, and antiapoptotic protein Bcl-2 and reduced levels of p21 and Bax associated with increased EC proliferation and reduced apoptosis. In addition, Notch2 suppression elicited a paradoxical activation of Notch1 and canonical Notch target gene Hes1, Hey1, and Hey2 transcription. Furthermore, reduction in Rb and increased E2F1 binding to the Notch1 promoter appear to explain the Notch1 upregulation. Yet, when Notch1 was decreased in Notch2-suppressed cells, the wound injury response was augmented. In aggregate, our results demonstrate that loss of Notch2 in hPAECs derepresses Notch1 and elicits EC hallmarks of PAH. Augmented EC proliferation upon Notch1 knockdown points to a context-dependent role for Notch1 and 2 in endothelial cell homeostasis. This study demonstrates a previously unidentified role for Notch2 in the maintenance of lung vascular endothelial cell quiescence and pulmonary artery hypertension (PAH). A key novel finding is that Notch2 suppression activates Notch1 via Rb-E2F1-mediated signaling and induces proliferation and apoptosis resistance in human pulmonary artery endothelial cells. Notably, PAH patients show reduced levels of endothelial Notch2 in their pulmonary arteries, supporting Notch2 as a fundamental driver of PAH pathogenesis.
肺动脉高压（PAH）是一种致命的心肺疾病，其特征是血管细胞增殖增加、具有抗凋亡能力以及小肺动脉发生闭塞性重塑。Notch蛋白家族参与了一条进化上保守的信号通路的近端信号传导，该通路影响细胞增殖、命运决定和发育。在内皮细胞（ECs）中，Notch受体2（Notch2）被证明可促进内皮细胞凋亡。然而，Notch2在肺内皮细胞增殖及随后的PAH中是起促进还是抑制增殖的作用尚不清楚。我们推测，在PAH情况下，Notch2信号的抑制会驱动肺内皮细胞增殖。我们观察到，与非PAH对照组相比，PAH患者肺组织中Notch2水平降低。在暴露于包括缺氧、转化生长因子-β（TGF-β）、内皮素-1（ET-1）和胰岛素样生长因子-1（IGF-1）等血管活性刺激的人肺动脉内皮细胞（hPAECs）中，Notch2表达减弱。Notch2缺陷的hPAECs激活了Akt、细胞外信号调节激酶1/2（Erk1/2）和抗凋亡蛋白Bcl-2，并降低了与内皮细胞增殖增加和凋亡减少相关的p21和Bax水平。此外，Notch2的抑制引发了Notch1的反常激活以及Notch经典靶基因Hes1、Hey1和Hey2的转录。此外，视网膜母细胞瘤蛋白（Rb）的减少以及E2F1与Notch1启动子结合的增加似乎可以解释Notch1的上调。然而，当在Notch2抑制的细胞中降低Notch1水平时，伤口损伤反应增强。总的来说，我们的结果表明，hPAECs中Notch2的缺失会解除对Notch1的抑制，并引发PAH的内皮细胞特征。Notch1敲低后内皮细胞增殖增强，这表明Notch1和Notch2在内皮细胞稳态中具有依赖于背景的作用。这项研究证明了Notch2在维持肺血管内皮细胞静止和肺动脉高压（PAH）方面以前未被识别的作用。一个关键的新发现是，Notch2的抑制通过Rb-E2F1介导的信号传导激活Notch1，并诱导人肺动脉内皮细胞增殖和抗凋亡。值得注意的是，PAH患者肺动脉中内皮Notch2水平降低，这支持Notch2是PAH发病机制的一个基本驱动因素。
