Abstract: 【Abstract】 Objective To investigate the effect of transforming growth factor beta 1 （TGF-β1） on the biological activity of infantile hemangioma （IH）-derived endothelial cells （HemECs）. Methods Three proliferating IH tissues and three involuting IH tissues were collected from IH patients receiving surgical resection at the Department of Pediatric Surgery, West China Hospital, Sichuan University from February to August 2021. Primary HemECs were isolated from proliferating IH tissues, and human umbilical vein endothelial cells （HUVECs） served as the control. The TGF-β1 expression levels in tissues and cells were detected by immunohistochemical study and Western blot analysis. Cell counting kit-8（CCK8）assay was performed to assess the effect of 0 （control group） - 100 ng/ml TGF-β1 on HemEC proliferation. HemECs were treated with 5 ng/ml TGF-β1 or without （control group）, and after several hours of treatment, Transwell assay was performed to evaluate cell migration ability, and immunofluorescence assay to assess the changes in the expression of endothelial markers （platelet-endothelial cell adhesion molecule-1 ［CD31］, vascular endothelial cadherin ［VE-cadherin］） and mesenchymal markers （α-smooth muscle actin ［α-SMA］, collagen type Ⅰ α 1 ［COL1A1］）. Comparisons between groups were conducted by t test or one-way analysis of variance. Results Immunohistochemical study showed that proliferating IH tissues were stained positively for TGF-β1, which was expressed relatively abundantly; the percentages of TGF-β1-positive signal area were higher in the proliferating IH tissues （24.68% ± 3.74%） than in the involuting IH tissues （almost no expression）. Western blot analysis revealed that the relative expression level of TGF-β1 was significantly higher in HemECs （1.08 ± 0.13） than in HUVECs （0.30 ± 0.04, t = 9.93, P < 0.001）. CCK8 assay showed increased proliferative activity of HemECs in the 3.125-, 6.25-, 12.5-, 25-, 50- and 75-ng/ml TGF-β1 groups compared with the control group （all P < 0.05）, and no significant difference was found between the 100-ng/ml TGF-β1 group and the control group （P > 0.05）. Transwell assay revealed an increased number of migratory HemECs in the 5-ng/ml TGF-β1 group （127 ± 6） compared with the control group （103 ± 9; t = 5.32, P < 0.01）. Immunofluorescence assay showed significantly decreased fluorescence intensity of endothelial markers CD31 and VE-cadherin in the 5-ng/ml TGF-β1 group （5.441 ± 1.254, 5.073 ± 0.412, respectively） compared with the control group （9.518 ± 1.728,7.671 ± 0.921, t = 3.31, 4.46, P = 0.030, 0.011, respectively）, and significantly increased fluorescence intensity of mesenchymal markers α-SMA and COL1A1 in the 5-ng/ml TGF-β1 group （8.074 ± 0.846, 5.885 ± 0.216, respectively） compared with the control group （0.393 ± 0.342, 0.295 ± 0.125, t = 14.58, 38.76, P < 0.001, < 0.000 1, respectively）. Conclusion TGF-β1 was relatively highly expressed in the proliferating IH tissues and HemECs, and could promote the proliferation, migration and endothelial-to-mesenchymal transition of HemECs.

Key words: Hemangioma, Transforming growth factor beta 1, Cell proliferation, Cell migration assays, Hemangioma-derived endothelial cells, Endothelial-to-mesenchymal transition