Abstract
Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to age-related injury. Tendons have poor healing capacity and a lack of effective treatments can lead to ongoing pain, reduced function and re-injury. It is therefore important to identify the mechanisms underpinning age-related tendinous changes in order to develop more effective treatments. Our recent single cell sequencing data has shown that tendon cell populations have extensive heterogeneity and cells housed in the tendon interfascicular matrix (IFM) are preferentially affected by ageing. There is, however, a lack of established surface markers for cell populations in tendon, limiting the capacity to isolate distinct cell populations and study their contribution to age-related tendon degeneration. Here, we investigate the presence of the cell surface proteins MET proto-oncogene (MET), integrin subunit alpha 10 (ITGA10), fibroblast activation protein alpha (FAP) and platelet derived growth factor receptor alpha (PDGFRA) in the equine SDFT cell populations and their co-localisation with known markers.
Using Western blot we validated the specificity of selected antibodies in equine tissue before performing immunohistochemistry to establish the location of the respective proteins in the SDFT. We subsequently used double labelling immunofluorescence with the established mural cell marker desmin (DES) to distinguish between tenocyte and mural cell populations.
In situ, MET, ITGA10, and FAP presence was found in cells throughout the tendon whereas PDGFRA was present in cells within the IFM. Double labelling immunofluorescence with the mural cell marker DES showed lack of co-localisation between PDGFRA and DES suggesting PDGFRA is labelling an IFM cell population distinct from those associated with blood vessels.
PDGFRA is a promising target for the specific cell sorting of IFM-localised tenocytes, enabling their isolation and subsequent characterisation.
Acknowledgments: The authors acknowledge the Biotechnology and Biological Sciences Research Council (BB/W007282/1) for funding this work.
