Differential effects of vesicular urokinase receptor uPAR on vascular cell migration and proliferation

Angiogenesis is essential for organ and tissue regeneration, as blood vessels provide necessary nutrients and oxygen for cellular restoration and growth. The present study explores a novel mechanism of uPAR involvement in angiogenesis through its incorporation into extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs). MSC-derived vesicles were isolated using ultrafiltration and characterized by flow cytometry, western blotting (WB) and nanoparticle tracking analysis. Model of aortic ring in Matrigel was used to assess capillary-like structure formation and vascular cells migration. To analyze cell migration from explants immunofluorescent staining, phase-contrast and confocal microscopy were used. Endothelial  cells (ECs) and smooth muscle cells (SMCs) images of growing cell populations were captured with microscope and then counted with deep learning object detection model. The activation of Akt signaling in these cells was evaluated by WB. uPA and uPAR content in MSCs and vesicles samples was analyzed using WB and ELISA. uPA and uPAR are incorporated into vesicles secreted by MSCs and regulate the proliferation and migration of ECs and SMCs. Vesicles from MSCs isolated from adipose tissue of mice lacking uPAR stimulate SMCs migration but lose their ability to promote ECs migration and capillary-like structure formation. Moreover, in MSCs lacking uPAR, uPA expression is dysregulated, leading to its increased presence in vesicles. These data suggest that ECs are more sensitive to the absence of uPAR, while uPA preferentially stimulates SMCs. These findings offer new insights into the regulatory roles of extracellular vesicles in angiogenesis, while broadening our understanding of the urokinase system's functional diversity.