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

Abstract

Background Adipose-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. Methods The 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. Results Our 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. Conclusion ADSCs 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.

TBX3 regulates the transcription of VEGFA to promote osteoblasts proliferation and microvascular regeneration

“…VEGFC, another form of the VEGF family protein, has a specific homologous region with VEGFA. It can be induced by TNF-α (Li et al, 2018) and improve diabetic foot by improving microvascular regeneration in diabetes ( Zhou, Wei & He, 2021 ). These studies showed that VEGFA, VEGFB, and VEGFC could all be involved in vascular regeneration, including microvascular regeneration.…”

Adipose-Derived Stem Cells for the Treatment of Diabetic Wound: From Basic Study to Clinical Application

“…Some growth factors that are related to angiogenesis were evaluated in some studies. As the most important and relevant growth factor in angiogenesis, the level of VEGF is reported to be upregulated in rat ASCs studies (34,37,41,42,45,53,56). It is reported that the level of VEGF, as well as TGF-b, is upregulated in granulations, while it can be further increased by combining the use of bio-modified gel (41).…”

“…This may attribute to the enhancement of endothelial cell secretion function by ASCs. The molecular mechanism of this process is explored by Zhou et al in 2021, indicating endothelial cell is promoted by ASCs to migration and forming tubule via VEGF-C-mediated METTL3/IGF2BP2−m6A pathway in diabetic mice (53). Studies using human ASCs also found a significantly higher level of VEGF in the tissue around the wound, no matter ASCs are harvested from diabetic or healthy individuals (32,39,42).…”

The Role of Podoplanin in Skin Diseases

“…Since lymphangiogenesis plays important roles in tissue regeneration and angiogenesis, lymphatic dysfunction can prevent wound healing due to impaired tissue fluid homeostasis [60]. Several studies have indicated that the induction of lymphangiogenesis could be a therapeutic target for impaired wound healing in patients with diabetic ulcers [56,61]. Maruyama et al demonstrated that podoplanin neutralization by anti-podoplanin antibodies inhibited lymphangiogenesis in a model of ear skin wound healing and inhibited tube formation by human lymphatic endothelial cells in vitro [59].…”