M1 and M2 Macrophages Differentially Modulate DPSC-Supported Angiogenesis

Abstract

<p>Objectives: To investigate the effects of differential macrophage phenotypes (M1 and M2) on dental pulp stem cell (DPSC) supported vascular constructs.<br>Methods: THP-1 cells were treated with 320 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours to differentiate them into M0 macrophages. M0 macrophages were then cultured for 48 hours in media containing IFN-Υ(100ng/ml) and LPS (100ng/ml) for M1 activation or<br>IL-4 (40ng/ml) and IL-13 (20ng/ml) for M2a activation. Macrophages or their conditioned medium (CM) were added into microfluidic devices with cocultures of human umbilical vein endothelial cells (HUVECs) and DPSCs to determine their functional angiogenic effects. The presence of angiogenic growth factors in CM of M0, M1, and M2a macrophages was assessed via the Angiogenesis array kit and ELISA.<br>Results: Enhanced vascularization was observed when M1 macrophages were seeded into the side channel of the microfluidic device where DPSCs and HUVECs encapsulated in fibrin gel were seeded in the center channel. This was manifested by the significantly higher (p〈0.05)<br>number of vascular segments, vascular sprouts, and branching points observed, compared to M0-seeded groups and negative controls. In addition, significantly high (p〈0.05) levels of IL-8 were detected in the CM of M1 macrophages in Angiogenesis array and ELISA. In the<br>presence of M1 Macrophage CM, an increase in vascular endothelial growth factor (VEGF) levels was observed in DPSC cultures, which was mediated via IL-8 secreted by M1 macrophages. Conversely, in microfluidic devices with M2a CM, a significantly lower count of free sprouts (p〈0.05) and a significantly higher percentage of mural cell-covered vessels (p〈0.05) compared to M0, M1, and negative controls, indicative of a more stabilized vascular network were observed.<br>Conclusions: M1 macrophages promote initial vascularization via IL-8-mediated enhanced VEGF secretion from DPSCs, while M2a macrophages improve the stabilization of vessels in DPSC-supported vascular constructs.<br></p>