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. 2023 Sep;56(9):1092-1107.
doi: 10.1111/iej.13943. Epub 2023 Jun 20.

Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion

Yuchen Zhang  1 , Jialin Zhong  1 , Shulan Lin  1 , Mingxin Hu  1 , Junqing Liu  1 , Jun Kang  1 , Yubingqing Qi  1 , Mohammed S Basabrain  1 , Ting Zou  1 , Chengfei Zhang  1
Affiliations

Direct contact with endothelial cells drives dental pulp stem cells toward smooth muscle cells differentiation via TGF-β1 secretion

Yuchen Zhang et al. Int Endod J. 2023 Sep.

Abstract

Aim: Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vein ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model.

Methodology: Human umbilical vascular ECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-β1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM) and HUVEC+DPSC cocultures (E+D-CM) were analysed by enzyme-linked immunosorbent assay. TGF-β RI kinase inhibitor VI, SB431542, was used to block TGF-β1/ALK5 signalling in DPSCs.

Results: The expression of SMC-specific markers, α-SMA, SM22α and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-β1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-β1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-β1/ALK5 signalling inhibited the expression of α-SMA, SM22α and Calponin in DPSCs.

Conclusions: TGF-β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-β1/ALK5 signalling pathway played a vital role in this process.

Keywords: TGF-β1; dental pulp stem cells; differentiation; smooth muscle cells.

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References

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