Nicotine triggers endothelial to mesenchymal transition (EndMT) in human aortic endothelial cells (HAECs) in vitro. (A) Morphological changes upon nicotine stimulation were observed under the microscope. Scale bar indicates 200 µm. (B) The protein levels of endothelial markers (VE-cadherin and CD31) were downregulated, while mesenchymal markers (α-SMA and FSP1) were upregulated in HAECs after treatment with nicotine, as indicated by western blot. n = 5. (C) The relative mRNA levels of endothelial markers (VE-cadherin and CD31) were downregulated, while mesenchymal markers (α-SMA and FSP1) and stem cell markers (Oct4, Nanog, Sox2, CD44 and Bmi1) were upregulated in HAECs after treatment with nicotine. n = 5. (D) Endothelial permeability was determined using a transwell permeability assay. n = 5. (E) F-actin staining was performed using phalloidin (green) and imaged under the microscope. The nuclei were stained blue with DAPI. Scale bar indicates 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001.