Redox Regulation of Lung Endothelial PERK, Unfolded Protein Response (UPR) and Proliferation via NOX1: Targeted Inhibition as a Potential Therapy for PAH

Aims: Reactive oxygen species (ROS) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH) and NADPH oxidases (NOXs) as sources of ROS are implicated in the development of the disease. We previously showed that NOX isozyme 1 (NOX1)-derived ROS contributes to pulmonary vascular endothelial cell (EC) proliferation in response to PAH triggers in vitro. However, whether and how NOX1 is involved in PAH in vivo have not been explored nor has NOX1 been examined as a viable and effective therapeutic disease target. Methods and Results: Herein, infusion of mice exposed to Sugen/hypoxia (10% O2) with a specific NOX1 inhibitor, NOXA1ds, delivered via osmotic minipumps (i.p.), significantly suppressed pathological changes in hemodynamic parameters characteristic of PAH. Furthermore, lungs of human patients with idiopathic PAH (iPAH) and exploratory RNA-seq analysis of hypoxic human pulmonary ECs, in which NOX1 was suppressed, were probed. The findings showed a clear indication of NOX1 in the promotion of both protein disulfide isomerase (PDI) and the unfolded protein response (UPR; in particular, the PERK arm of the pathway including eIF2α and ATF4) leading to proliferation. In aggregate, these results are consistent with a causal role for NOX1 in the development of mouse and human PAH and reveal a novel and mechanistic pathway by which NOX1 activates the UPR response during EC proliferation. Conclusion: NOX1 promotes phenotypic changes in ECs that are pivotal to proliferation and PAH through activation of the UPR. Taken together, our results are consistent with selective inhibition of NOX1 as a novel modality for attenuating PAH Human pulmonary artery endothelial cells (HPAECs) were transfected with scramble (Scr) or NOX1 (siNOX1) siRNA for 48 h before cells were exposed to Normoxia (Nx, 21% O2) or Hypoxia (Hx, 1% O2) for 24 h. At the end of treatment, cells were lysed for RNA isolation and purification (RNeasy MinElute cleanup kit, QIAGEN). Total RNA samples were sent to NovoGene (Sacramento, CA) for mRNA library preparation and sequencing. Unstranded and paired 150 bp sequencing data was filtered, trimmed, aligned to the human reference genome (GRCh38), and counted on the gene level.

研究目的：活性氧（reactive oxygen species, ROS）在肺动脉高压（pulmonary arterial hypertension, PAH）的发病机制中发挥关键作用，而作为活性氧来源的烟酰胺腺嘌呤二核苷酸磷酸氧化酶（nicotinamide adenine dinucleotide phosphate oxidases, NOXs）与该疾病的发生发展密切相关。本团队既往研究证实，体外实验中，烟酰胺腺嘌呤二核苷酸磷酸氧化酶同工酶1（NOX isozyme 1, NOX1）产生的活性氧可促进肺动脉血管内皮细胞（pulmonary vascular endothelial cell, EC）在肺动脉高压触发因素作用下的增殖。然而，NOX1在体内是否以及如何参与肺动脉高压的病理进程，目前尚未有相关探索；同时，NOX1作为可行且有效的疾病治疗靶点，也未得到验证。

研究方法与结果：本研究中，我们对经苏生素（Sugen）联合低氧（10%氧气）造模的小鼠，通过渗透微型泵给予腹腔注射（intraperitoneal, i.p.）特异性NOX1抑制剂NOXA1ds，结果显示该处理可显著抑制肺动脉高压特征性血流动力学参数的病理改变。此外，我们还对特发性肺动脉高压（idiopathic PAH, iPAH）患者的肺组织样本进行分析，并对沉默NOX1的低氧人肺动脉内皮细胞开展探索性RNA测序（RNA-seq）分析。结果清晰表明，NOX1可促进蛋白质二硫键异构酶（protein disulfide isomerase, PDI）的表达，以及未折叠蛋白反应（unfolded protein response, UPR；尤其是该通路的PERK分支，包括eIF2α和ATF4），进而介导血管内皮细胞增殖。综上，上述结果证实NOX1在小鼠和人类肺动脉高压的发生发展中发挥因果性作用，并揭示了NOX1在血管内皮细胞增殖过程中激活未折叠蛋白反应的全新分子机制。

研究结论：NOX1通过激活未折叠蛋白反应，诱导血管内皮细胞发生与细胞增殖及肺动脉高压密切相关的表型改变。综上，本研究结果支持选择性抑制NOX1可作为一种全新的肺动脉高压治疗策略。

实验方法：将人肺动脉内皮细胞（human pulmonary artery endothelial cells, HPAECs）分别转染阴性对照小干扰RNA（scramble siRNA, Scr）或靶向NOX1的小干扰RNA（siNOX1），培养48小时后，将细胞置于常氧（Normoxia, Nx, 21%氧气）或低氧（Hypoxia, Hx, 1%氧气）环境中处理24小时。处理结束后，裂解细胞以提取并纯化总RNA（使用RNeasy MinElute 纯化试剂盒，QIAGEN公司）。将总RNA样本送至诺禾致源（NovoGene，美国加利福尼亚州萨克拉门托）进行mRNA文库构建及测序。对非链特异性双端150bp测序数据进行过滤、修剪后，将其比对至人类参考基因组（GRCh38），并进行基因水平的计数分析。