The Interplay of M1 Macrophages and Dental Pulp Stem Cells Promotes Angiogenesis Through IL-8-Dependent VEGF...

Dental pulp inflammation triggers immune responses involving macrophages and dental pulp stem cells (DPSCs), which interact to regulate angiogenesis essential for tissue repair. M1 pro-inflammatory macrophages predominate early in pulpitis, and clarifying their angiogenic role is vital in identifying inflammatory regenerative mechanisms.

THP-1 cells and peripheral blood monocyte (PBM)-derived macrophages were polarized to M1 or M2 phenotypes, characterized by qRT-PCR, ELISA, and angiogenesis arrays. A vasculature-on-a-chip comprising DPSCs, human umbilical vein endothelial cells (HUVECs) and THP-1-derived macrophages was imaged, and the vascular segments/sprouts were quantified using ImageJ. Density effects used 5 × 10⁴ versus 7.5 × 10⁴ M1 macrophages/device, with propidium iodide staining for cytotoxicity. IL-8 effects on DPSC VEGF secretion were assessed by ELISA (with/without Reparaxin 1 μM), Matrigel tube formation assays, and exogenous IL-8 (0.5 ng/mL). Transwell co-cultures underwent RNA sequencing and bioinformatics analysis, which identified candidate hub genes and signalling pathways; the results were validated by Western blotting (p-ERK, HIF-1α; ERK inhibitor SCH772984, 25 nM). Statistical testing was performed using ANOVA with Tukey's post hoc test (p < 0.05).

M1 macrophages at low density (5 × 10⁴ cells/device) significantly enhanced vascularization in the vasculature-on-a-chip, increasing vascular segments (p < 0.0001) and free sprouts (p < 0.05-0.01) compared to M0 or no-macrophage controls, with effects comparable to M2. High-density M1 seeding (7.5 × 10⁴ cells/device) reduced sprouts (p < 0.0001 day 4, p < 0.01 day 5) due to increased cytotoxicity (p < 0.0001). Both THP-1- and PBM-derived M1-conditioned media (CM) showed significantly elevated IL-8 levels. M1 CM (THP-1/PBM) induced DPSC VEGF secretion, blocked by Reparaxin (p < 0.01-0.0001), confirming IL-8 mediation via CXCR1/2. Exogenous IL-8 (0.5 ng/mL) upregulated DPSC VEGF protein/mRNA (p < 0.05) and Matrigel tube formation (segments/junctions p < 0.05). M1-DPSC CM enhanced vascular meshes/segments on Matrigel (p < 0.05), reduced by Reparaxin. RNA-seq of M1 co-cultured DPSCs identified 17 angiogenic genes (logFC > 1.2), with HIF-1α as a hub gene and an enriched MAPK/ERK pathway. Western blot analysis confirmed MAPK/ERK-HIF-1α as a contributory pathway in IL-8-induced upregulation of VEGF in DPSCs.

M1 macrophages promote angiogenesis via IL-8-induced DPSC VEGF secretion through CXCR1/2-MAPK/ERK-HIF-1α signalling density-dependently, suggesting that therapeutic modulation rather than total suppression of M1 activity could improve outcomes in vital pulp therapy.