Ling Zhang, Hai Wei Wu, Weien Yuan, Jia Wei Zheng

Infantile hemangiomas (IHs) are the most common benign vascular tumor of infancy. They occur more frequently in female infants. The cause of hemangioma is currently unknown; however, current studies suggested the importance of estrogen (E2) signaling in hemangioma proliferation. Hemangioma-derived stem cells (HemSCs) were cultured with estrogen for 48-72 h; the cell viability and proliferation were evaluated with the messenger RNA (mRNA) and protein expression levels of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor-A (VEGF-A) and estrogen receptor-α (ER-α), by application of several in vitro assays, such as methyl thiazolyl tetrazolium (MTT), reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, enzyme-linked immunosorbent assay (ELISA) and Western blotting. Also, the cell population's response to external estrogen was investigated by in vivo experiments. HemSCs and human umbilical vein endothelial cells (HUVECs) were mixed and injected subcutaneously into 20 flank of BALB/c-nu mice, which were randomly divided into 5 groups based on different E2 treatment doses (0, 0.01, 0.1 and 1 mg, respectively), 0.1 mg dimethyl sulfoxide (DMSO) as control. Each group of mice were treated intramuscularly every week, then 2 and 4 weeks later, the subcutaneous implants were harvested and evaluated the tumor tissues with microvessel density (MVD) assay and immunohistochemistry. The study demonstrated that application of E2 increased the expression of FGF2, VEGF-A, and ER-α in HemSCs with the optimal concentration from 10(-9) to 10(-5) M. Two-week treatment of E2 promoted expression of VEGF-A and FGF2 in HemSCs culture. Morphological, histological and immunohistological improvements were observed in vivo using murine IH model in which HemSCs and HUVECs were implanted into BALB/c-nu mice that were post-injected with E2. In the grafts, mean MVD was markedly increased. The results suggested that E2 promotes angiogenesis via combination with ER-α to up-regulate the expression of VEGF-A in HemSCs, promoting proliferation of IHs. These findings provide critical insight into the potential mechanisms of E2 action on IHs.