ATP1A1-Driven Intercellular Contact Between Dental Pulp Stem Cell and Endothelial Cell Enhances Vasculogenic Activity

Abstract

Aim: The interaction between dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) is crucial to the speedy establishment of functional blood circulation within the transplanted pulp tissue. It is a complex process involving direct cell contact and paracrine signalling. The transmembrane domains of α1-Na⁺/K⁺-ATPase (ATP1A1) have been shown to influence tumour angiogenesis. Its role in regulating DPSCs/ECs interaction in vascular formation remains unknown. This study aimed to explore ATP1A1 on DPSCs/ECs communication, vascular network formation, and underlying mechanisms.

Methods: The formation of vessel structures within different culture systems was examined. The expression of pericyte-like markers and Na⁺/K⁺-ATPase-related genes and proteins were systematically analysed. Immunofluorescence staining was performed to examine the localisation of ATP1A1. Total and phosphorylated proteins were evaluated to identify and explore the signalling pathways activated under cocultured conditions. Downstream signalling was also investigated after the inhibition of ATP1A1.

Results: Direct coculture accelerated vessel network formation and prolonged its stability compared to indirect systems. ATP1A1 expression and SMC-specific marker (α-SMA) levels significantly increased in direct coculture systems, with nuclear α-SMA localisation and ATP1A1 enrichment at cell-contact sites. Protein assay revealed activated Src/AKT pathways and upregulated FGF-2/activin A secretion in coculture supernatants. ATP1A1 inhibition reduced α-SMA expression, impairing SMC differentiation.

Conclusion: Direct DPSCs-HUVECs contact stabilises vessel networks via ATP1A1-mediated Src/AKT activation, driving FGF-2/activin A secretion and initiating SMC differentiation. This highlights that ATP1A1 may be critical for pericyte-like transition and vascular microenvironment optimisation in pulp angiogenesis.

Clinical significance: This research informed strategies aimed at pulp tissue regeneration. The findings hold significant implications for enabling the biological restoration of tooth vitality and function in the field of clinical regenerative treatment.

Keywords: ATP1A1; Coculture; Dental pulp stem cells; Smooth muscle cells; Vessel stability.

Fig. 1

Different coculture methods of endothelial cells and dental pulp stem cells in vitro. (A) Methods of direct coculture and indirect coculture. (B) Separation of direct coculture cells. The schematic diagram of methods was generated by Figdraw.

Fig. 2

Different coculture methods influence the formation of vessel-like structures in vitro. (A) Tube formation on Matrigel after 4, 6, 12, 24, 48, and 72 hours of coculture medium incubation. (B-C) ELISA analysis of the FGF-2 and activin A level in CM of HUVECs and DPSCs from monoculture and cocultures. Data are presented as box plots showing the median (horizontal line) and minimum and maximum values (box boundaries) for n = 3 replicates, *P < .05, ***P < .001, Scale Bar = 100 μm.

Fig. 3

RNA-sequencing data and the expression of ATP1A1 and α-SMA after direct coculture. (A-B) Differently expressed Genes (DEGs) analysis of cells after direct coculture. (C-D) Gene ontology (GO) analysis revealed the enrichment of biological processes among the upregulated genes of DPSCs. (E-F) RT-qPCR analysis of the relative mRNA expression of ATP1A1, ATP1B1 and α-SMA in cells after coculture for 24 and 48 hours. (G) Representative immunofluorescence images of ATP1A1(green) and DAPI (blue) in HUVECs after coculture for 2 days. Scale bar = 20 μm. (H-I) Western blot analysis of the expression of ATP1A1 and α-SMA in DPSCs after coculture for 48 hours. Data are mean ± SD for n = 3 replicates, *P < .05, ***P < .001.

Fig. 4

DPSCs exhibited differentiation properties toward SMCs after contact with HUVECs. (A) RT-qPCR analysis of the relative mRNA expression of ATP1A1, ATP1B1 and α-SMA in DPSCs after coculture for 24 and 48 hours. (B) Western blot analysis of the expression of ATP1A1, and α-SMA in DPSCs after coculture for 24 and 48 hours. (C-D) Representative immunofluorescence images of α-SMA (green) and DAPI (blue) in HUVECs after coculture for 2 days. Scale bar = 50 μm. (E-F) Microscopy images showed the location of ATP1A1 and α-SMA in direct coculture. Scale bar = 50 μm. Data are mean ± SD for n = 3 replicates, *P < .05, **P < .01, ***P < .001.

Fig. 5

Inhibition of ATP1A1 suppresses the expression of SMC markers in DPSCs. (A-D) RT-qPCR analysis of the relative expression of ATP1A1, α-SMA and activin A in cells after siRNA transfection. Representative fluorescence images of cells after siRNA transfection. Images were captured at 48 hours post-transfection to visualise transfection efficiency and cell morphology. (E-F) Western blot analysis of the relative expression of proteins in cells after siRNA transfection. (G) The impact of ouabain treatment on DPSCs. (H-J) Ouabain inhibited HUVEC migration and tube formation. Data are mean ± SD for n = 3 replicates, *P < .05, ***P < .001, Scale Bar = 100 μm.

Fig. 6

Involvement of Src/AKT pathway in HUVECs/DPSCs direct coculture. (A-B) Effects of ATP1A1 on the phosphorylation status of AKT and Src after coculture. (C-D) Effects of ATP1A1 on the phosphorylation status of AKT and Src in DPSCs. (E-F) Semi-quantitative analysis of the phosphorylation and total protein after coculture. Data are mean ± SD for n = 3 replicates, *P < .05, **P < .01.

Fig. 7

Proposed model of cell interactions through ATP1A1 promotes SMC activation for vascular stability. Cell interactions between DPSCs and HUVECs induce ATP1A1 over-expression, leading to SMC differentiation, Src/AKT activation and activin A, FGF-2 secretion. The schematic diagram of mechanism was generated by Figdraw.