Pulmonary veno-occlusive disease (PVOD) is a fatal disease of pulmonary vascular lesions leading to right heart failure. Heritable PVOD (hPVOD) is related to biallelic mutation of EIF2AK4 (encoding GCN2), but its molecular mechanism remains unclear. In this study, we aimed to investigate the pathogenesis of PVOD and to find potential drug targets for PVOD. GCN2 dysfunction led to an enhanced transcription of collagen I gene (col1a1 and col1a2) through decreasing ATF3-dependent p38 phosphorylation inhibition in PVOD, which promotes the collagen I synthesis in pulmonary arterial smooth muscle cells (PASMCs) and eventually leads to increased collagen deposition in pulmonary artery. Four GCN2 knockout (KO) cell lines (exon 15 or 33 mutation) were successfully constructed by epiCRISPR system. Two induced pluripotent stem cells (iPSCs) were generated by reprogramming peripheral blood mononuclear cells (PBMCs) of PVOD patient. It was also comfirmed that GCN2 dysfunction could lead to increased expression of collagen I in lateral plate mesoderm lineage-smooth muscle cells (LM-SMCs) differentiated from both GCN2 KO cell lines and iPSCs. SB203580 (a specific inhibitor of p38) improved hemodynamics and pulmonary vascular remodeling in mitomycin C (MMC)-induced PVOD rats by right ventricle echocardiography. On the whole, we proposed that GCN2 deficiency decreased ATF3-dependent p38 phosphorylation inhibition in PVOD development and suggested a potential therapeutic reagent of SB203580 for the treatment of the disease.

肺静脉闭塞性疾病（PVOD）是一种致命的肺血管病变导致右心衰竭。可遗传的 PVOD (hPVOD) 与EIF2AK4（编码 GCN2）的双等位基因突变有关，但其分子机制仍不清楚。在这项研究中，我们旨在研究 PVOD 的发病机制并寻找 PVOD 的潜在药物靶点。GCN2 功能障碍导致胶原蛋白 I 基因（col1a1和col1a2) 通过降低 PVOD 中 ATF3 依赖性 p38 磷酸化抑制，促进肺动脉平滑肌细胞 (PASMCs) 中胶原蛋白 I 的合成，最终导致肺动脉中胶原蛋白沉积增加。通过epiCRISPR系统成功构建了4个GCN2敲除（KO）细胞系（外显子15或33突变）。通过对 PVOD 患者的外周血单个核细胞 (PBMC) 进行重编程，产生了两种诱导多能干细胞 (iPSC)。还证实 GCN2 功能障碍可导致从 GCN2 KO 细胞系和 iPSC 分化的外侧板中胚层谱系平滑肌细胞 (LM-SMC) 中胶原蛋白 I 的表达增加。SB203580（p38 的特异性抑制剂）通过右心室超声心动图改善丝裂霉素 C (MMC) 诱导的 PVOD 大鼠的血流动力学和肺血管重塑。总体而言，我们提出 GCN2 缺乏降低了 PVOD 发展中 ATF3 依赖性 p38 磷酸化抑制，并提出了治疗该疾病的潜在治疗试剂 SB203580。