RESUMEN
We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Adolescente , Adulto , Western Blotting , Recuento de Células , Ensayos de Migración Celular , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Células Cultivadas , Colágeno , Pulpa Dental/fisiología , Combinación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Laminina , Neovascularización Fisiológica/fisiología , Proteoglicanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Reproducibilidad de los Resultados , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Adulto JovenRESUMEN
Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.
Asunto(s)
Humanos , Adolescente , Adulto , Adulto Joven , Factor de Necrosis Tumoral alfa/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Pulpa Dental/citología , Pulpa Dental/efectos de los fármacos , Inductores de la Angiogénesis/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Proteoglicanos , Valores de Referencia , Factores de Tiempo , Recuento de Células , Células Cultivadas , Western Blotting , Reproducibilidad de los Resultados , Colágeno , Laminina , Neovascularización Fisiológica/fisiología , Pulpa Dental/fisiología , Factor A de Crecimiento Endotelial Vascular/análisis , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Combinación de Medicamentos , Ensayos de Migración Celular , Células Endoteliales de la Vena Umbilical Humana/fisiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
OBJECTIVE: The dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration. DESIGN: hDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50ng/mL) on hDPSCs' migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated. RESULTS: Our results showed significant increase of hDPSCs' migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs. CONCLUSION: In this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs.