Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing

Abstract

Background: Diminished wound healing is a major complication of diabetes mellitus and can lead to foot ulcers. However, there are limited therapeutic methods to treat this condition. Exendin-4 (Ex-4), a glucagon-like peptide-1 receptor agonist, is known to have many beneficial effects on diabetes. In addition, mesenchymal stem cells are known to have wound healing effects. We investigated the effects of Ex-4 in combination with human adipose tissue-derived stem cells (ADSCs) on diabetic wound healing in a diabetic animal model.

Methods: Diabetic db/db (blood glucose levels, >500 mg/dl) or C57BL/6 mice were subjected to wounding on the skin of the back. One day after wounding, each wound received ADSCs (2.5 × 10⁵ cells) injected intradermally around the wound and/or Ex-4 (50 μl of 100 nM Ex-4) topically applied on the wound with a fine brush daily. Wound size was monitored and wound histology was examined. Human endothelial cells and keratinocyte cells were used to assess angiogenesis and vascular endothelial growth factor expression in vitro.

Results: Topical administration of Ex-4 or injection of ADSCs resulted in a rapid reduction of wound size in both diabetic and normoglycemic animals compared with vehicle treatment. Histological analysis also showed rapid skin reconstruction in Ex-4-treated or ADSC-injected wounds. A combination of Ex-4 and ADSCs showed a significantly better therapeutic effect over either treatment alone. In vitro angiogenesis assays showed that both Ex-4 and ADSC-conditioned media (CM) treatment improved migration, invasion and proliferation of human endothelial cells. ADSC-CM also increased migration and proliferation of human keratinocytes. In addition, both Ex-4 and ADSC-CM increased the expression of vascular endothelial growth factor. Co-culture with ADSCs increased migration and proliferation of these cells similar to that found after ADSC-CM treatment.

Conclusions: We suggest that Ex-4 itself is effective for the treatment of diabetic skin wounds, and a combination of topical treatment of Ex-4 and injection of ADSCs has a better therapeutic effect. Thus, a combination of Ex-4 and ADSCs might be an effective therapeutic option for the treatment of diabetic wounds, such as foot ulcers.

Keywords: Adipose-derived stem cells; Angiogenesis; Diabetic wound; Exendin-4; GLP-1.

Fig. 1

Effect of Ex-4, ADSCs, or a combination of Ex-4 and ADSCs on diabetic wound healing. Diabetic db/db mice (a, b) or normal C57BL/6 mice (c, d) were subjected to wounding (6 mm in diameter) by punch biopsy on the skin of the back. One day later, each wound received 2.5 × 10⁵ ADSCs injected intradermally around the wound and/or 50 μl of 100 nM Ex-4 was applied to the wound with a fine brush, daily. a Changes of wound area at each of the time points compared with the original wound area. Values represent mean ± SEM. *p < 0.05 vs vehicle, ^$ p < 0.05 vs ADSC, ^# p < 0.05 vs EX-4. b Wound shapes were recorded over 14 days. c Wound area up to 4 days after artificial wound creation. *p < 0.05 vs vehicle, ^$ p < 0.05 vs ADSC, ^# p < 0.05 vs EX-4. d Wound shapes 2 and 4 days after artificial wound creation

Fig. 2

Histological analysis of skin morphology of wounds on day 14 after treatment with Ex-4, ADSCs, or combination of Ex-4 and ADSCs. Diabetic db/db mice were subjected to wounding (6 mm in diameter) by punch biopsy on the skin of the back. One day later, each wound received 2.5 × 10⁵ ADSCs injected intradermally around the wound and/or 100 nM Ex-4 was applied to the wound with a fine brush, daily. a H&E staining of wound area (×400). b The thickness of the dermis observed through the H & E staining results. *p < 0.05 vs wound-(-), ^$ p < 0.05 vs wound-ADSC, ^# p < 0.05 vs wound-EX-4. c Masson’s trichrome staining of wound area (×400). c Masson’s trichrome staining of wound area (×400). d The collagen density observed through Masson’s trichrome staining results. *p < 0.05 vs wound-(−), ^$ p < 0.05 vs wound-ADSC, ^# p < 0.05 vs wound-EX-4. e Immunofluorescence staining of VEGF. (×800). f VEGF expression levels observed through VEGF staining results. *p < 0.05 vs vehicle, ^# p < 0.05 vs EX-4

Fig. 3

The effect of treatment with Ex-4, ADSCs, or combination of Ex-4 and ADSCs on diabetes. a, b Diabetic db/db mice were subjected to wounding (6 mm in diameter) by punch biopsy on the skin of the back. One day later, each wound received 2.5 × 10⁵ ADSCs injected intradermally around the wound and/or 100 nM Ex-4 was applied on the wound with a fine brush, daily. Fourteen days after wounding, a blood glucose and b. HbA1c levels were measured

Fig. 4

The effects of Ex-4 and ADSCs on migration, proliferation and invasion of HUVECs. a, d Migration assay. Plated HUVECs were wounded and the injury line was marked. a Cells were cultured with Ex-4 and/or ADSC-CM. d Cells were co-cultured with or without ADCSs in the upper chamber of a transwell plate with or without Ex-4 treatment. Migration patterns were observed under a phase contrast microscope and photographed (×200). b, e Proliferation assay. b HUVECs were plated and incubated with or without ADSC-CM and/or 10 nM Ex-4. e HUVECs were plated on transwell plates and co-cultured with ADSCs (in the upper chamber) and/or 10 nM Ex-4. After 24 h, cell proliferation was determined with a cell counting Kit-8 assay. Results are expressed as a percentage of the control. *p < 0.05 vs CON, ^# p < 0.05 vs ADSC. c Invasion assay. HUVECs (2 × 10⁴ cells) were seeded into the upper compartment of a transwell and incubated with or without ADSC-CM and/or 10 nM Ex-4 for 24 h. Sample images are shown of transmigrated cells stained with crystal violet (×200)

Fig. 5

The effect Ex-4 or ADSC-CM on migration, proliferation and angiogenic factor secretion in keratinocytes. a, d Migration assay. Plated HaCaT cells were wounded and the injury line was marked. a Cells were cultured with ADSC-CM and/or 10 nM Ex-4. d Cells were co-cultured with or without ADCSs in the upper chamber of a transwell plate with or without Ex-4 treatment. Migration patterns were observed under a phase contrast microscope and photographed (×200). b, e Proliferation assay. b HaCaT cells were plated on 96-well plates and incubated with or without ADSC-CM and/or 10 nM Ex-4. e HaCaT cells were plated on transwell plates and co-cultured with ADSCs (in the upper chamber) and/or 10 nM Ex-4. After 24 h, cell proliferation was determined with a cell counting Kit-8 assay. Results are expressed as a percentage of the control. *p < 0.05 vs CON. c Western blotting assay. HaCaT cells were incubated with or without ADSC-CM and/or 10 nM Ex-4. After 24 h, cells were harvested and western blotting assay for VEGF was carried out. The results were analyzed through ImageJ software for Windows. *p < 0.05 vs CON, ^# p < 0.05 vs ADSC + EX-4