Fractional carbon dioxide laser-induced photothermal activation of mesenchymal stem cell-derived exosomes accelerates diabetic wound healing by enhancing angiogenesis

Figure 5 The sphingosine-1-phosphate/sphingosine-1-phosphate receptor signaling axis mediates exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells-induced angiogenesis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. Data are presented as mean ± SD (n = 3). A: Enzyme-linked immunosorbent assay analysis of sphingosine-1-phosphate concentrations in activated supernatant from non-irradiated adipose-derived mesenchymal stem cells (Ad-MSCs), activated supernatant from laser-irradiated Ad-MSCs, exosomes, and exosomes derived from CO₂ laser-preconditioned Ad-MSCs; B: Relative mRNA expression levels of sphingosine-1-phosphate receptor (S1PR1)-3 in human umbilical vein endothelial cells (HUVECs); C and D: Western blot and quantitative analysis of the relative protein expression of S1PR1-3 in HUVECs; E and F: 5-Ethynyl-2’-deoxyuridine incorporation assay to evaluate HUVEC proliferation. Scale bar = 100 μm; G: Cell Counting Kit-8 assay to assess HUVEC viability; H and I: Transwell migration assay to evaluate HUVEC motility. Scale bar = 200 μm; J and K: In vitro wound healing assay to assess migration. Scale bar = 200 μm; L and M: Tube formation assay to examine capillary-like network formation by HUVECs. Scale bar = 200 μm; N and O: Western blot analysis of the expression levels of phosphorylated protein kinase B, protein kinase B, hypoxia-inducible factor-1α, vascular endothelial growth factor-A, and S1PR1. NC: Activated supernatant from non-irradiated adipose-derived mesenchymal stem cells; NG: Normal glucose; HG: High glucose; HP: High permeability; CO₂ laser-AS: Activated supernatant from laser-irradiated adipose-derived mesenchymal stem cells; Exos: Exosomes; CO₂ laser-Exos: Exosomes derived from CO₂ laser-preconditioned adipose-derived mesenchymal stem cells; EdU: 5-Ethynyl-2’-deoxyuridine; S1P: Sphingosine-1-phosphate; S1PR: Sphingosine-1-phosphate receptor; shS1PR: Short hairpin RNA targeting sphingosine-1-phosphate receptor; AKT: Protein kinase B; p-AKT: Phosphorylated protein kinase B; HIF-1α: Hypoxia-inducible factor-1α; VEGF-A: Vascular endothelial growth factor-A.