M1 macrophages enhance angiogenic properties of DPSCs via IL-8

Abstract

<p>Objectives: To investigate the effects of M1 macrophages on dental pulp stem cell (DPSC) supported vascular constructs.<br>Methods: THP-1 cells were differentiated into macrophages(M0) via 320nm <em>Phorbol</em> 12-myristate 13-acetate (PMA) induction for 24 hours. M0 macrophages were then cultured for 48 hours in media supplemented with IFN-γ (100 ng/ml) and LPS (100 ng/ml) for M1 activation or IL-4 (40 ng/ml) and IL-13 (20 ng/ml) for M2a activation. M0, M1, and M2a macrophages were seeded into microfluidic devices with cocultures of human umbilical vein endothelial cells (HUVECs) and DPSCs to determine their effects on vascular network formation. The angiogenic growth factors, IL-8 and vascular endothelial growth factor (VEGF), in conditioned medium (CM) of M0, M1, and M2a macrophages were assessed via the Angiogenesis array kit and ELISA. DPSCs were treated with macrophage CM for 48hrs with and without Reparaxin (IL-8 inhibitor), and ELISA for VEGF was performed on the supernates. In addition, DPSCs were treated with exogenous IL-8 for 48hrs, and ELISA for VEGF was performed on the supernates.<br>Results: When M1 macrophages were seeded onto the side channel of the microfluidic device where DPSCs and HUVECs were encapsulated in fibrin gel and seeded in the center channel, an enhanced vascular network was observed, as shown by the significantly higher (p<0.05) number of vascular segments, sprouts, and branching points compared to M0-seeded groups and negative controls. According to the Angiogenesis array and ELISA results, CM of M1 macrophages contained significantly high (p<0.05) amounts of IL-8. VEGF levels were increased in supernates of M1 CM-treated DPSCs, which was significantly reduced (p<0.05) when IL-8 inhibitor reperaxin was added. Additionally, significantly high levels (p<0.05) of VEGF were detected in supernates of exogenous IL-8-treated DPSCs compared to control groups.<br>Conclusions: Taken together, M1 macrophages enhance the angiogenic properties of DPSCs via IL-8-mediated induction of VEGF secretion.<br></p>