METHODS: Lung tissue samples were collected to detect the expression of Notch4 from patients with HPH and matched controls. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic and normoxic conditions. Real-time quantitative PCR and western blotting were used to examine the mRNA and protein levels of Notch4. HPASMCs were transfected with small interference RNA (siRNA) against Notch4 or Notch4 overexpression plasmid, respectively. Cell viability, cell proliferation, apoptosis, and migration were assessed using Cell Counting Kit-8, Edu, Annexin-V/PI, and Transwell assay. The interaction between Notch4 and ERK, JNK, P38 MAPK were analyzed by co-immunoprecipitation. Adeno-associated virus 1-mediated siRNA against Notch4 (AAV1-si-Notch4) was injected into the airways of hypoxic rats. Right ventricular systolic pressure (RVSP), right ventricular hypertrophy and pulmonary vascular remodeling were evaluated.
RESULTS: In this study, we demonstrate that Notch4 is highly expressed in the media of pulmonary vascular and is upregulated in lung tissues from patients with HPH and HPH rats compared with control groups. In vitro, hypoxia induces the high expression of Delta-4 and Notch4 in HPASMCs. The increased expression of Notch4 promotes HPASMCs proliferation and migration and inhibits cells apoptosis via ERK, JNK, P38 signaling pathways. Furthermore, co-immunoprecipitation result elucidates the interaction between Notch4 and ERK/JNK/P38. In vivo, silencing Notch4 partly abolished the increase in RVSP and pulmonary vascular remodeling caused by hypoxia in HPH rats.
CONCLUSIONS: These findings reveal an important role of the Notch4-ERK/JNK/P38 MAPK axis in hypoxic pulmonary remodeling and provide a potential therapeutic target for patients with HPH.
方法:收集肺组织样本以检测HPH患者和匹配的对照组中Notch4的表达。在低氧和常氧条件下培养人肺动脉平滑肌细胞(HPASMC)。实时定量PCR和蛋白质印迹检测Notch4的mRNA和蛋白水平。用针对Notch4或Notch4过表达质粒的小干扰RNA(siRNA)转染HPASMC,分别。细胞活力,细胞增殖,凋亡,和迁移使用细胞计数试剂盒-8,Edu,膜联蛋白-V/PI,和Transwell分析。Notch4和ERK之间的相互作用,JNK,通过共免疫沉淀分析P38MAPK。将腺相关病毒1介导的针对Notch4的siRNA(AAV1-si-Notch4)注射到缺氧大鼠的气道中。右心室收缩压(RVSP),评估右心室肥厚和肺血管重塑。
结果:在这项研究中,我们证明,与对照组相比,Notch4在肺血管介质中高表达,并且在HPH患者和HPH大鼠的肺组织中上调。体外,低氧诱导HPASMC中Delta-4和Notch4的高表达。Notch4的表达增加通过ERK促进HPASMCs的增殖和迁移,抑制细胞凋亡,JNK,P38信号通路。此外,免疫共沉淀结果阐明了Notch4与ERK/JNK/P38之间的相互作用。在体内,沉默Notch4部分消除了HPH大鼠缺氧引起的RVSP和肺血管重塑的增加。
结论:这些发现揭示了Notch4-ERK/JNK/P38MAPK轴在缺氧性肺重塑中的重要作用,并为HPH患者提供了潜在的治疗靶点。