Equine Endothelial Colony-Forming Cells: Phenotypic Enhancement and Ex vivo Angiogenesis Studies

Abstract

Ischemic diseases in horses are common and often life-threatening, thus cell-based therapies to improve angiogenesis are gaining interest. Endothelial colony-forming cells (ECFCs) can form de novo blood vessels in areas of ischemia and have been isolated from peripheral blood of horses; however, there is the potential of having a mixed cell population rather than pure ECFCs. Selection of ECFCs with the endothelial cell (EC) phenotype and enhancing the endothelial pathway in culture may improve angiogenesis. Angiogenesis and the use of alternative sources of growth factors can be investigated ex vivo to better mimic the processes seen in vivo. The study objectives were to isolate ECFCs with an EC phenotype using cell sorting and to enhance expression of the EC pathway in culture by the addition of the nitric oxide (NO) donor S-nitroso cysteine (CysNO). To optimize media for EC phenotype, the effects of equine platelet lysate (ePL) were evaluated through the arterial ring assay. Equine ECFCs were sorted by fluorescence- (FACS) or magnetic-activated cell sorting (MACS) based on acetylated low-density lipoprotein uptake or expression of cluster of differentiation (CD) 31. Sorted cells were characterized based on phenotype and in vitro angiogenesis. To evaluate the effects of NO, CysNO was added to the endothelial growth media (EGM) of cultured ECFCs, and cells were evaluated for CD31 expression, viability, growth and tubule formation in vitro. The angiogenic effect of ePL was evaluated by supplementing equine arterial rings with EGM-containing ePL. Equine ECFCs were successfully sorted by FACS and MACS, but once expanded, their phenotype and function were comparable to non-sorted ECFCs. The addition of CysNO had no effect in ECFC function in vitro, viability, or phenotype. Equine arterial rings served as an ex vivo model of angiogenesis, and although ePL was not superior to horse serum, it was successful in supporting angiogenesis. Using appropriate protocols allows for the sorting of equine ECFCs, and both ePL and PPP, similarly to HS, have the potential to be used for the culture of ECFCs. However, future research studying the use of alternative EC surface markers or refined colony selection is needed to confirm identity and reduce the mixed population of cells isolated with current methods.