Alba George A, Samokhin Andriy O, Wang Rui-Sheng, Wertheim Bradley M, Haley Kathleen J, Padera Robert F, Vargas Sara O, Rosas Ivan O, Hariri Lida P, Shih Angela, Thompson Boyd Taylor, Mitchell Richard N, Maron Bradley A
Division of Pulmonary and Critical Care Medicine Massachusetts General Hospital Boston Massachusetts USA.
Division of Cardiovascular Medicine Brigham and Women's Hospital Boston Massachusetts USA.
Pulm Circ. 2022 May 11;12(2):e12071. doi: 10.1002/pul2.12071. eCollection 2022 Apr.
The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia increases neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and induces expression of a prothrombotic NEDD9 peptide (N9) on the extracellular plasma membrane surface. We hypothesized that the SARS-CoV-2-ARDS pathophenotype involves increased pulmonary endothelial N9. Paraffin-embedded autopsy lung specimens were acquired from patients with SARS-CoV-2-ARDS ( = 13), ARDS from other causes ( = 10), and organ donor controls ( = 5). Immunofluorescence characterized the expression of N9, fibrin, and transcription factor 12 (TCF12), a putative binding target of SARS-CoV-2 and known transcriptional regulator of . We performed RNA-sequencing on normal HPAECs treated with normoxia or hypoxia (0.2% O) for 24 h. Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) profiled protein-protein interactions involving N9 relevant to thrombus stabilization. Hypoxia increased TCF12 messenger RNA significantly compared to normoxia in HPAECs in vitro (+1.19-fold, = 0.001; false discovery rate = 0.005), and pulmonary endothelial TCF12 expression was increased threefold in SARS-CoV-2-ARDS versus donor control lungs ( < 0.001). Compared to donor controls, pulmonary endothelial N9-fibrin colocalization was increased in situ in non-SARS-CoV-2-ARDS and SARS-CoV-2-ARDS decedents (3.7 ± 1.2 vs. 10.3 ± 3.2 and 21.8 ± 4.0 arb. units, < 0.001). However, total pulmonary endothelial N9 was increased significantly only in SARS-CoV-2-ARDS versus donor controls (15 ± 4.2 vs. 6.3 ± 0.9 arb. units, < 0.001). In HPAEC plasma membrane isolates, IP-LC-MS identified a novel protein-protein interaction between NEDD9 and the β-subunit of the αβ-integrin, which regulates fibrin anchoring to endothelial cells. In conclusion, lethal SARS-CoV-2-ARDS is associated with increased pulmonary endothelial N9 expression and N9-fibrin colocalization in situ. Further investigation is needed to determine the pathogenetic and potential therapeutic relevance of N9 to the thrombotic pathophenotype of SARS-CoV-2-ARDS.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染所致急性呼吸窘迫综合征（ARDS）中肺原位血栓形成的病理生物学特征尚未完全明确。在人肺动脉内皮细胞（HPAECs）中，缺氧会增加神经前体细胞表达且发育下调的9（NEDD9），并诱导细胞外质膜表面促血栓形成的NEDD9肽（N9）的表达。我们推测SARS-CoV-2-ARDS病理表型涉及肺内皮N9增加。从患有SARS-CoV-2-ARDS的患者（n = 13）、其他原因导致的ARDS患者（n = 10）以及器官捐献者对照（n = 5）获取石蜡包埋的尸检肺组织标本。免疫荧光法鉴定N9、纤维蛋白和转录因子12（TCF12）的表达，TCF12是SARS-CoV-2的假定结合靶点且是已知的转录调节因子。我们对在常氧或缺氧（0.2% O₂）条件下处理24小时的正常HPAECs进行RNA测序。免疫沉淀-液相色谱-质谱（IP-LC-MS）分析与血栓稳定相关的涉及N9的蛋白质-蛋白质相互作用。与常氧相比，体外HPAECs中缺氧显著增加了TCF12信使RNA（+1.19倍，P = 0.001；错误发现率 = 0.005），并且与捐献者对照肺相比，SARS-CoV-2-ARDS患者肺内皮TCF12表达增加了三倍（P < 0.001）。与捐献者对照相比，非SARS-CoV-2-ARDS和SARS-CoV-2-ARDS死者肺原位肺内皮N9-纤维蛋白共定位增加（3.7 ± 1.2对10.3 ± 3.2和21.8 ± 4.0任意单位，P < 0.001）。然而，仅在SARS-CoV-2-ARDS与捐献者对照相比时，肺内皮总N9显著增加（15 ± 4.2对6.3 ± 0.9任意单位，P < 0.001）。在HPAEC质膜分离物中，IP-LC-MS鉴定出NEDD9与αβ整合素的β亚基之间存在一种新的蛋白质-蛋白质相互作用，αβ整合素调节纤维蛋白锚定到内皮细胞。总之，致死性SARS-CoV-2-ARDS与肺内皮N9表达增加和原位N9-纤维蛋白共定位增加有关。需要进一步研究以确定N9对SARS-CoV-2-ARDS血栓形成病理表型的发病机制及潜在治疗相关性。
