Int J Stem Cells.  2021 Nov;14(4):447-454. 10.15283/ijsc21050.

Hypoxic Pretreatment of Adipose-Derived Stem Cells Accelerates Diabetic Wound Healing via circ-Gcap14 and HIF-1α/VEGF Mediated Angiopoiesis

• Wang Z
• Feng C
• Liu H
• Meng T
• Huang W
• Long X
• Wang X

• KMID: 2522566
• DOI: http://doi.org/10.15283/ijsc21050

Abstract

Background and Objectives
Adipose-derived stem cell (ADSC) transplantation improves stem cell paracrine function and can enhance wound healing. However, in diabetic patients, glucose-associated effects on this function and cell survival lead to impaired wound closure, thereby limiting ADSC transplantation efficiency. The hypoxia-inducible factor HIF-1α has an important protective function during wound healing. Here, we aim to clarify the regulatory mechanism of ADSCs.
Methods and Results
ADSCs were isolated from BALB/C mice adipose samples. We then used high-throughput se-quencing to assess abnormal expression of circular RNAs (circRNAs). We also used an in vivo full-thickness skin defect mouse model to assess the effects of transplanted ADSC on diabetic wound closure. Hypoxic pretreatment of ADSCs accelerated diabetic wound closure, which enhanced angiogenic growth factor expression in our mouse model. High-throughput sequencing and RT-qPCR indicated that circ-Gcap14 was upregulated in hypoxic pretreated ADSCs. Similarly, circ-Gcap14 downregulation also decreased the therapeutic effects of ADSCs; however, circ-Gcap14 overexpression increased the effects of ADSC by promoting angiopoiesis. We also used a luciferase reporter assay to confirm that miR-18a-5p and HIF-1α were downstream targets of circ-Gcap14. HIF-1α expression plays an important role in increased VEGF level.
Conclusions
Based on our data, we suggest that circ-Gcap14 plays an important role in accelerating hypoxic ADSC-mediated diabetic wound closure, by enhancing mouse angiogenic growth factor expression and regulating downstream miR-18a-5p/HIF-1α expression.

Keyword

circ-Gcap14; HIF-1α; Angiopoiesis; Diabetic wound healing, ADSCs

Figure

• 

  Fig. 1 Characterization of adipose-derived mesenchymal stem cells (ADSCs). (A) ADSCs show a typical cobblestone-like morphology. (B∼G) Immunofluorescence staining of cell surface markers. Antibodies were labeled with either fluorescein isothiocyanate (FITC, green) or phycoerythrin (PE, red). CD29, CD44, CD90, and CD105 staining was positive, whereas CD31 and von Wille-brand Factor (vWF) staining was negative. (H, I) Differentiation potential of ADSCs by oil red O (H) and alizarin red staining (I). Scale bar, 50 μm.

• 

  Fig. 2 Wound healing effects of hypoxia-pretreated ADSCs in mice. (A) Schematic of in vivo procedures. (B, C) Wound closure rates were quantified at indicated times after wound generation and ADSC transplantation. Data are presented as the mean±SD. *p<0.05, ***p<0.001 vs. NC group. (D, E) CD31 immunohistochemical staining detection the angiogenesis as black arrow show. Data are presented as mean±SD. ***p<0.001 vs. NC group. (F, G) RT-qPCR and western blot detection show the expression HIF-1α and VEGF in mRNA and protein level from tissue surrounding wounds in the diabetic mouse model. Data are presented as the mean±SD. ***p<0.001 vs. NC group.

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  Fig. 3 Circ-Gcap14 is upregulated in hypoxia-pretreated ADSCs (hypoxic-ADSCs). (A) Heat map of upregulated and downregulated circRNAs with a ≥1.5-fold difference between hypoxic- and wild-type ADSCs. (B) RT-qPCR shows circRNA expression in both hypoxic- and wild-type ADSCs. Data are presented as the mean±SD. **p<0.01, ***p<0.001 vs. ADSCs group.

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  Fig. 4 Circ-Gcap14 plays an important role in ADSC mediated diabetic wound closure. (A) RT-qPCR shows circ-Gcap14 expression in ADSCs upon circ-Gcap14 downregulation or overexpression/silencing. Data are presented as the mean±SD. ***p<0.001 vs. NC. ###p<0.001 vs. si-circ-Gcap14. (B, C) The rate of wound closure was quantified at indicated times after ADSC transplantation. Data are presented as the mean±SD. ***p<0.001 vs. hypoxic-ADSC. ###p<0.001 vs. si-circ-Gcap14-hypoxic-ADSC. (D, E) CD31 immunohistochemical staining detection the angiogenesis as black arrow show. Data are presented as the mean±SD. ***p<0.001 vs. hypoxic-ADSC. ###p<0.001 vs. si-circ-Gcap14-hypoxic-ADSC. (F, G) RT-qPCR and western blot detection shows the expression of HIF-1α and VEGF in both mRNA and protein level from tissue surrounding wounds. Data are presented as the mean±SD. ***p< 0.001 vs. hypoxic-ADSC. ###p<0.001 vs. si-circ-Gcap14-hypoxic-ADSC.

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  Fig. 5 miR-18a-5p and HIF-1α are downstream targets of circ-Gcap14. (A) Predicted binding sites for miR-18a-5p in circ-Gcap14. The mutated circ-Gcap14 is also shown. (B) Relative luciferase activity at 48 h post-transfection of HEK293T cells with miR-18a-5p mimics/NC or circ-Gcap14 wild-type/mutant. Data are presented as the mean±SD. ***p<0.001. (C) Binding site prediction of miR-18a-5p in the HIF-1α 3’UTR. The mutant version of the 3’-UTR-HIF-1α is shown. (D) Relative luciferase activity at 48 h post-transfection of HEK293T cells with miR-18a-5p mimic/NC or 3’UTR-HIF-1α wild-type/mutant. Data are presented as the mean±SD. ***p<0.001. (E∼G) RT-qPCR shows circ-Gcap14 (E), miR-18a-5p (F) and HIF-1α expression (G). Data are presented as the mean±SD. ***p<0.001 vs. NC group. ###p<0.001 vs. circ-Gcap14.

Reference

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