Figure 5.

CM of equine PB-MSCs stimulates angiogenesis in vitro through the stimulation of VEGF-A production via the secreted factors ET1, IL-8, PDGF-AA, and IGFBP2. (A): Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate whether recombinant uPA, ET1, IL-8, PDGF-AA, or IGFBP2 could change the expression levels of VEGF-A mRNA in ECs. (B): An equine-specific VEGF enzyme-linked immunosorbent assay was used to determine the levels of VEGF secreted by ECs when exposed to PB-MSC-derived CM. Control medium and EC-derived CM and NBL6-derived CM were included as controls. Absorbance was measured at 490 nm on an Infinite M200 Pro microplate reader using i-control software (Tecan). (C): ECs were seeded on an extracellular matrix gel in the presence of VEGF-A to confirm the positive effect of this recombinant protein on the tube-like formation capacity of ECs in vitro. After 1 day of culture, ECs were stained with 10 μl of 10× cell-based calcein. Arrows indicate hollow tube-like structure formation. Scale bars = 100 μm. (D): Quantitative RT-PCR was performed to evaluate whether CM of PB-MSCs changed the expression levels of VEGFR mRNA in ECs. (E): Western blotting was performed to evaluate whether CM of PB-MSCs changed the expression levels of VEGFR protein in ECs. Lane 1 represents ECs incubated in MSC expansion medium; lane 2, ECs in CM of ECs; lane 3, ECs in CM of NBL-6; and lanes 4-6, ECs in CM of PB-MSCs obtained from 3 different horses. Abbreviations: CM, conditioned medium; EC, endothelial cell; ET1, endothelin-1; IGFBP2, insulin-like growth factor binding protein 2; IL, interleukin; PB-MSC, peripheral blood-derived equine mesenchymal stromal cell; PDGF-AA, platelet-derived growth factor AA; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; uPA, urokinase plasminogen activator.