Fig 1: Gene expression, protein expression, and endothelial tube forming assays. A, Expression of three potent proangiogenic genes in CD34+CD146+, CD34+CD146−, and CD34+ UF ASCs was compared by PCR. CD34+CD146+ ASCs expressed significantly more VEGF (***P < .001) and ANGTP1 (***P < .001) than CD34+CD146− and CD34+ UF ASCs, and significantly greater levels of FGF than CD34+CD146− ASCs (**P < .01). B, Representative immunofluorescence images (top row) and binarized images from ImageJ analysis (bottom row) showing results from endothelial tube forming assays. Human microvascular endothelial cells (green due to staining with Calcein AM) were cocultured for 18 hours in transwells with CD34+CD46+ ASCs (left), CD34+CD146− ASCs (middle), and CD34+ UF ASCs (right). Scale bars = 100 μm. C, Graphs showing results from endothelial tube forming assay analysis demonstrating, C(i), greater total pixel density, C(ii), tube length, and, C(iii), total branching length in HMECs cocultured with CD34+CD146+ compared to CD34+CD146− ASCs (all *P ≤ .05). D, Graphs showing the results from enzyme‐linked immunosorbent assays comparing protein expression between three ASC subpopulations. CD34+CD146+ ASCs expressed significantly more VEGF (****P < .0001) (D[i]) and ANGTP1 (****P < .0001) (D[ii]) than both CD34+CD146− and CD34+ UF ASCs, as well as significantly greater levels of FGF than CD34+CD146− ASCs (***P < .001) and CD34+ UF ASCs (****P < .0001) (D[iii]). ANGPT1, angiopoietin‐1; ASC, adipose‐derived stromal cell; FGF, fibroblast growth factor‐2; HMEC, human microvascular endothelial cell; PCR, polymerase chain reaction; UF, unfractionated; VEFG, vascular endothelial growth factor
Fig 2: Angiogenesis-related differentiation markers of (A) VEGF and (B) Ang-1 expression of hDPSCs cultured on CA0, CA10, and CA20 scaffolds for different time points. * indicates a significant difference (p < 0.05) from CA0. # indicates a significant difference (p < 0.05) from CA10. Data presented as mean ± SEM; n = 6 for each group.
Fig 3: Effects of 5-fluoro ABICA treatment on the protein expression of VEGF, ANG-1, ANG-2, GSK-3β, and p-GSK-3β in HBMECs, assessed with western blotting. (A) Protein extracts were obtained from HBMECs treated with 5-fluoro ABICA at varying concentrations (0.1 μM, 0.01 μM, or 0.0001 μM). These extracts were then used to quantify the protein levels of VEGF, ANG-1, ANG-2, GSK-3β, and p-GSK-3β within the cells; β-actin served as the reference protein. Proteins were isolated with radioimmunoprecipitation assay lysis buffer containing phosphatase-protease inhibitors, and 20 μg of each protein sample was separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred to a polyvinylidene fluoride membrane, which was blocked with 2 % bovine serum albumin and probed with primary antibodies during an overnight incubation. HRP-conjugated secondary antibodies were used to detect chemiluminescence signals. (B) The VEGF expression levels, normalized to those of β-actin, were significantly higher in cells treated with 5-fluoro ABICA than the control. (C) Significantly elevated ANG-1 in treated HBMECs. (D) Increase in ANG-2 expression after treatment with 5-fluoro ABICA compared with the control. (E) Substantial increase in the expression of p-GSK-3β with respect to GSK-3β. The data are presented as SD ± mean (n = 3). (∗∗∗∗) indicates p < 0.0001, (∗∗∗) indicates p < 0.001, (∗∗) indicates p < 0.01.
Fig 4: ELISA quantification of gene expression in HBMECs treated with different concentrations of 5-fluoro ABICA. ELISA was conducted on HBMECs treated with 5-fluoro ABICA at different concentrations, to quantify the levels of secreted proangiogenic factors. (A–C) Significant increase in the release of VEGF, ANG-1, and ANG-2 concentrations in a dose-dependent manner after treatment with 5-fluoro ABICA compared with the control. Gene expression was calculated for each sample and is presented in the graph as units. The data are presented as SD ± mean (n = 3). (∗∗∗∗) indicates p < 0.0001, (∗∗∗) indicates p < 0.001, (∗) indicates p < 0.05.
Fig 5: Intracellular protein expression rates of p-Ser-9-GSK-3β/GSK-3β, ANG-1, ANG-2, and VEGF in hBMECs
Supplier Page from Abcam for Human Angiopoietin 1 ELISA Kit (ANG1)