ADSCs enhance VEGFR3-mediated lymphangiogenesis via METTL3-mediated VEGF-C m6A modification to improve wound healing of diabetic foot ulcers

Jie Zhou,Tianhong Wei,Zhiyou He

doi:10.1186/s10020-021-00406-z

Abstract

BackgroundAdipose-derived mesenchymal stem cells (ADSCs) are an important focus in regenerative medicine. However, the biological function of ADSCs in the wound repair of diabetic foot ulcers (DFUs) remains unclear. This study aimed to determine the underlying mechanisms of ADSCs involved in the wound healing of DFUs.MethodsThe cell surface markers cluster of differentiation 34 (CD34), stromal cell antigen 1 (Stro-1), cluster of differentiation 90 (CD90) and cluster of differentiation 105 (CD105) on ADSCs were identified by flow cytometry. Oil Red O staining and Alizarin Red S staining were performed to identify the multipotential differentiation of ADSCs into adipocytes and bone. The levels of Methyltransferase-like 3 (METTL3), vascular endothelial growth factor C (VEGF-C) and insulin-like growth factor 2 binding protein 2 (IGF2BP2) were assessed by RT-qPCR. CCK-8, Transwell and tubule formation assays were conducted to assess lymphatic endothelial cell (LEC) viability, migration and tubule formation ability, respectively. RIP and RNA pulldown assays were conducted to assess the interaction between IGF2BP2 and VEGF-C. The levels of VEGF-C, VEGFR3, LYVE-1 and IGF2BP2 proteins were assessed by Western blotting. The levels of VEGF-C in LECs were measured by ELISA.ResultsOur findings illustrated that ADSCs accelerate LEC proliferation, migration and lymphangiogenesis via the METTL3 pathway and regulate VEGF-C expression via the METTL3/IGF2BP2-m6A pathway VEGF-C-mediated lymphangiogenesis via the METTL3/IGF2BP2-m6A pathway in DFU mice.ConclusionADSCs enhance VEGFR3-mediated lymphangiogenesis via METTL3-mediated VEGF-C m6A modification to improve wound healing in DFUs, indicating that ADSCs may be regarded as a promising therapeutic strategy to promote wound healing in DFUs.

Highlights

Diabetic foot ulcers (DFUs) are one of the primary causes of amputation in diabetic patients with diabetic peripheral neuropathy and foot deformities (Amin 2020).Delayed or nonhealing wounds are major complications of diabetic foot ulcers (DFUs) (Yin 2020)
The Adipose-derived mesenchymal stem cells (ADSCs) were stained with antiCluster of differentiation 34 (CD34), anti-stromal cell antigen 1 (Stro-1), anti-cluster of differentiation 90 (CD90) and anti-cluster of differentiation 105 (CD105) monoclonal antibodies labelled with phycoglobin (PE) or luciferin isothiocyanate (FITC)
The results of flow cytometry analysis showed that the ADSCs were positive for the mesenchymal markers CD90, Stro-1 and CD105 and negative for the haematopoietic marker cluster of differentiation 34 (CD34) (Fig. 1B)

Summary

Introduction

Diabetic foot ulcers (DFUs) are one of the primary causes of amputation in diabetic patients with diabetic peripheral neuropathy and foot deformities (Amin 2020).Delayed or nonhealing wounds are major complications of DFU (Yin 2020). Aurora A kinase (AURKA) promotes diabetic wound repair by enhancing ADSC autophagy (Yin et al 2020). Li et al showed that ADSCs accelerate skin wound healing by promoting vascularization in DFU rats (Li et al 2018). ADSCs have the ability to mediate DFU wound healing through mitochondrial transfer and the secretion of p-hydroxybenzoic factor and exosomes (Li et al 2018). The specific mechanism by which ADSCs promote wound healing in DFUs has not been fully elucidated. The role and underlying mechanisms of ADSCs involved in the wound repair of DFUs remain unclear. The biological function of ADSCs in the wound repair of diabetic foot ulcers (DFUs) remains unclear.

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