Zhu Hao, Li Cheng, Hu Fang, Wu Lifu, Wu Ling, Zhou Meihua, Liu Wei, Dai Aiguo
Department of Respiratory and Critical Care Medicine, Hunan Provincial People's Hospital/The First Affiliated Hospital of Hunan Normal University, Changsha, 410016, Hunan, China.
Department of Respiratory Diseases, Medical School, Hunan University of Chinese Medicine, Changsha, 410208, Hunan, China.
In Vitro Cell Dev Biol Anim. 2025 Jan;61(1):107-116. doi: 10.1007/s11626-024-00976-2. Epub 2024 Dec 2.
Pulmonary hypertension (PH) is a condition in which the smooth muscle cells (SMCs) in the pulmonary arteries multiply excessively, causing the arteries to narrow. This can ultimately result in right heart failure and premature death. Notch3 is an important factor involved in pulmonary vascular remodeling in PH. RO4929097, as a γ-secretase inhibitor that inhibits Notch3 signaling pathway, may be a potential drug for the treatment of PH, but its feasibility and related mechanism of action need to be further investigated. In vitro modeling by hypoxic incubation of human pulmonary artery SMCs (HPASMCs). RO4929097 and plasmids including overexpression-NICD3 (oe-NICD3) and NICD3 small interfering RNA (siRNA) were used to alter the expression of NICD3, and HIF-2α inhibitor PT-2385 was used to alter the expression of HIF-2α. Western blot, EdU incorporation assay was used to investigate the alteration of NICD3, HIF-2α, FoxM1 protein expression, and cell proliferation. The severity of PH in rats was assessed by measuring the weight ratio of right ventricle (RV) to left ventricle (LV) and septum (S) (RV/[LV + S]) and hematoxylin-eosin (H&E) staining of lung tissues in a hypoxia-induced PH rat model. We first determined that hypoxia induction for 48 h had the strongest induction of NICD3 and Notch3 in HPASMCs, and the strongest inhibition by 10 μM RO4929097. Treatment of HPASMCs under hypoxic conditions with RO4929097 inhibited hypoxia-induced expression of NICD3, HIF-2α, FoxM1, and proliferation of HPASMCs. The inhibitory effect of RO4929097 was reversed after overexpression of NICD3 in HPASMCs. Further, we found that PT-2385 reversed the promotional effect of overexpression of NICD3 on the proliferation of HPASMCs. In vivo experiments, hypoxia-induced PH rats treated with RO4929097 showed a reduction in right ventricular hypertrophy index (RVHI) and a return to normal pulmonary artery morphology, indicating a reduction in the severity of PH. Our data suggest that RO4929097 regulates the Notch3/HIF-2α/FoxM1 signaling pathway by inhibiting the expression of NICD3, thereby inhibiting hypoxia-induced proliferation of HPASMCs. In vivo experiments also confirmed that RO4929097 could alleviate PH as a potential therapeutic strategy.
肺动脉高压（PH）是一种肺动脉平滑肌细胞（SMC）过度增殖导致动脉狭窄的病症。这最终可能导致右心衰竭和过早死亡。Notch3是参与PH肺血管重塑的一个重要因素。RO4929097作为一种抑制Notch3信号通路的γ-分泌酶抑制剂，可能是治疗PH的一种潜在药物，但其可行性和相关作用机制需要进一步研究。通过对人肺动脉平滑肌细胞（HPASMCs）进行缺氧孵育建立体外模型。使用RO4929097以及包括过表达NICD3（oe-NICD3）和NICD3小干扰RNA（siRNA）的质粒来改变NICD3的表达，并使用HIF-2α抑制剂PT-2385来改变HIF-2α的表达。采用蛋白质免疫印迹法、EdU掺入试验来研究NICD3、HIF-2α、FoxM1蛋白表达的变化以及细胞增殖情况。通过测量缺氧诱导的PH大鼠模型中右心室（RV）与左心室（LV）及室间隔（S）的重量比（RV/[LV + S]）以及肺组织苏木精-伊红（H&E）染色来评估大鼠PH的严重程度。我们首先确定缺氧诱导48小时对HPASMCs中NICD3和Notch3的诱导作用最强，且10μM RO4929097的抑制作用最强。在缺氧条件下用RO4929097处理HPASMCs可抑制缺氧诱导的NICD3、HIF-2α、FoxM1的表达以及HPASMCs的增殖。在HPASMCs中过表达NICD3后，RO4929097的抑制作用被逆转。此外，我们发现PT-2385可逆转NICD3过表达对HPASMCs增殖的促进作用。在体内实验中，用RO4929097处理缺氧诱导的PH大鼠，其右心室肥厚指数（RVHI）降低，肺动脉形态恢复正常，表明PH严重程度减轻。我们的数据表明，RO4929097通过抑制NICD3的表达来调节Notch3/HIF-2α/FoxM1信号通路，从而抑制缺氧诱导的HPASMCs增殖。体内实验也证实，RO4929097作为一种潜在的治疗策略可以缓解PH。
