Laboratory of Cardiovascular Diseases, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China.
Cardiovascular Medicine Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China.
J Cell Biochem. 2019 Dec;120(12):19567-19577. doi: 10.1002/jcb.29264. Epub 2019 Jul 11.
This study aimed to investigate the transdifferentiation of human pulmonary arterial endothelial cells (HPAECs) into smooth muscle like (SM-like) cells under hypoxic conditions and reveal the role of endogenous small molecular compound 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE) in this process. HPAECs were treated by hypoxia and hypoxia + ITE with different durations. The endothelial markers (CD31 and VE-cad) and smooth muscle markers (α-SMA, SM22α, and OPN) were investigated by immunofluorescence double staining, and their expressions, along with the differentiation regulators transforming growth factor-β (TGF-β) ligands and downstream signals including TGF-β1, bone morphogenetic protein (BMP2), BMP9, Samd2/3, ERK, and p38 MAPK, were determined by Western blot analysis. The viability and proliferation of HPAECs were detected by Cell Counting Kit-8 (CCK-8) method and bromodeoxyuridine (BrdU) assays. As a result, hypoxia induced HPAECs transdifferentiation from paving-stone-like into polygonal or spindle cells, whose number increased greatly after additional ITE stimulation for 7 days. Compared with the normoxic HPAECs, the expression of endothelial markers reduced and smooth muscle markers were enhanced with the extension of hypoxia + ITE treatment, and meanwhile the cell viability increased significantly. Hypoxia could promote expression of TGF-β1 protein rather than BMP2 and BMP9, and regulate phosphorylation levels of Samd2/3, ERK and p38 MAPK in different manners. In conclusion, ITE can promote the hypoxia-induced transdifferentiation of HPAECs into SM-like cells via TGF-β/Smads and MAPK/ERK pathways.
本研究旨在探讨低氧条件下人肺动脉内皮细胞（HPAEC）向平滑肌样（SM-like）细胞的转分化，并揭示内源性小分子化合物 2-(1'H-吲哚-3'-羰基)-噻唑-4-羧酸甲酯（ITE）在这一过程中的作用。用不同时间的缺氧和缺氧+ITE 处理 HPAEC。通过免疫荧光双重染色检测内皮标志物（CD31 和 VE-cad）和平滑肌标志物（α-SMA、SM22α 和 OPN），并通过 Western blot 分析检测其表达以及分化调节剂转化生长因子-β（TGF-β）配体和下游信号，包括 TGF-β1、骨形态发生蛋白（BMP2）、BMP9、Samd2/3、ERK 和 p38 MAPK。通过细胞计数试剂盒-8（CCK-8）法和溴脱氧尿苷（BrdU）检测 HPAEC 的活力和增殖。结果表明，低氧诱导 HPAEC 从铺路石样转分化为多角形或纺锤形细胞，在添加 ITE 刺激 7 天后数量显著增加。与常氧 HPAEC 相比，内皮标志物的表达减少，平滑肌标志物的表达增强，随着缺氧+ITE 处理时间的延长，细胞活力显著增加。低氧可以促进 TGF-β1 蛋白的表达，而不是 BMP2 和 BMP9 的表达，并以不同的方式调节 Samd2/3、ERK 和 p38 MAPK 的磷酸化水平。总之，ITE 可以通过 TGF-β/Smads 和 MAPK/ERK 通路促进低氧诱导的 HPAEC 向 SM-like 细胞的转分化。
