Roles of thrombospondin-1 and nuclear factor-kappa B signal pathways in propranolol inhibiting hemangioma-derived endothelial cells

Abstract

Objective To explore the roles of thrombospondin-1 (TSP-1) and nuclear factor-kappa B (NF-κB) signal pathways in propranolol inhibiting hemangioma-derived endothelial cells (HemECs) to improve the therapeutic mechanism of propranolol and provide new targets for its clinical treatment. Methods The specimens of proliferative hemangioma were obtained operatively. Cell suspension was prepared after incubation and digestion. Then CD31-positive HemECs were sorted out by flow cytometry and sub-cultured. Propranolol in different concentrations (0, 25, 50, 100, 150, 200, 250 μmol/L) was added into HemECs solution and cultured for 24, 48 and 72h respectively. And CCK-8 culture and BrdU labeling solutions were added. Activity and proliferation of HemECs before and after intervention of propranolol were respectively tested by enzyme-linked imunosorbent assay (ELISA) and protein expressions of TSP-1 and nf-kappa B pathways detected by Western blot. After immunofluorescent double-labeling, activation of TSP-1 pathway and inactivation of nf-kappa B pathway were simultaneously observed by laser confocal microscope. All data were analyzed by SPSS 13.0 statistical software. Results The rate of CD31-positive cells was 98.0%. The longer incubation time, the more marked inhibitions of cell activity and proliferation after propranolol dosing. The inhibition of cell activity and proliferation were significant at a propranolol concentration of 100-150 μmol/L. And significant differences existed between them and those of control group (P<0.05), especially at 72 h. Expressions of TSP-1 and its receptor CD36 increased with rising propranolol concentrations while the expressions of nf-kappa Bp65, p-IκBα and p-IKKβ gradually decreased (P<0.05). Two pathways were subject to negative regulations, that is, TSP-1 was gradually activated while nf-kappaBp65 became gradually inactivated. Conclusions The effective treatment of propranolol for infantile hemangioma is probably correlated with promoting TSP-1 induced anti-angiogenesis and/or block nf-kappa B-mediated angiogenesis. Key words: Hemangioma; Propranolol; In vitro