Although the molecular mechanisms behind tendon disease remain obscure, aberrant stromal matrix turnover and tissue hypervascularity are known hallmarks of advanced tendinopathy. We harness a tendon explant model to unwind complex cross-talk between the stromal and vascular tissue compartments. We identify the hypervascular tendon niche as a state-switch that gates degenerative matrix remodeling within the tissue stroma. Here pathological conditions resembling hypervascular tendon disease provoke rapid cell-mediated tissue breakdown upon mechanical unloading, in contrast to unloaded tendons that remain functionally stable in physiological low-oxygen/-temperature niches. Analyses of the stromal tissue transcriptome and secretome reveal that a stromal niche with elevated tissue oxygenation and temperature drives a ROS mediated cellular stress response that leads to adoption of an immune-modulatory phenotype within the degrading stromal tissue. Degradomic analysis further reveals a surprisingly rich set of active matrix proteases behind the progressive loss of tissue mechanics. We conclude that the tendon stromal compartment responds to aberrant mechanical unloading in a manner that is highly dependent on the vascular niche, with ROS gating a complex proteolytic breakdown of the functional collagen backbone.

Stem cells have great potential for tissue regeneration, but before stem cell populations can be used in the clinic, it is crucial that the stem cells have been definitely characterized by a set of specific markers. Although there have been attempts to identify a set of immunophenotypic markers to characterize equine mesenchymal stromal cells (MSCs), immunophenotyping of equine MSCs is still challenging due to the limited availability of suitable antibodies of high quality and consistent performance across different laboratories. The aim of this study was to evaluate a strategy for mapping the equine MSCs surface proteome by use of biotin-enrichment and mass spectrometry (MS) analysis and mine the proteome for potential equine MSCs surface markers belonging to the cluster of differentiation protein group. Gene expression analysis was used for verification. Equine MSCs derived from bone marrow (BM) (n = 3) and adipose tissue (AT) (n = 3) were expanded to P3 and either used for (1) cell differentiation into mesodermal lineages (chondrogenic and osteogenic), (2) enrichment of the MSCs surface proteins by biotinylation followed by in-gel digest of the isolated proteins and nanoLC-MS/MS analysis to unravel the enriched cell surface proteome, and (3) RNA isolation and quantitative real-time reverse transcriptase PCR analysis of the CD29, CD44, CD90, CD105, CD166, CD34, CD45, and CD79a gene expression. A total of 1239 proteins at 1% FDR were identified by MS analysis of the enriched MSCs surface protein samples. Of these proteins, 939 were identified in all six biological samples. The identified proteins included 19 proteins appointed to the cluster of differentiation classification system as potential cell surface targets. The protein and gene expression pattern was positive for the commonly used positive MSCs markers CD29, CD44, CD90, CD105, and CD166, and lacked the negative MSCs markers CD34, CD45, and CD79a. The findings of this study show that enrichment of the MSCs surface proteome by biotinylation followed by MS analysis is a valuable alternative to immunophenotyping of surface markers, when suitable antibodies are not available. Further, they support gene expression analysis as a valuable control analysis to verify the data.