Introduction Chronic tendon pathology (tendinopathy) is a common but poorly treated disease, due to limited understanding of its pathogenesis. The resident tenocytes undergo continuous renewal form tendon progenitor/stem cells (TSPCs).1 How TSPCs are maintained and differentiated into tenocytes in healthy individuals and which key molecular events are defective in patients have been largely unknown. Manipulating endogeous stem cells with small molecules has been proposed as a therapeutic strategy, but practical approaches are still unavailable. In this study, our results demonstrate that digoxin represents a potential approach for calcifying tendinopathy therapy through inhibiting HIF-2α mediated erroneous differentiation of TSPCs.
Methods Human model of tendinopathy: Calcific Achilles tendon and supraspinatus tendon were collected from patients undergoing surgical procedures.
Treatment: Digoxin/saline was injected into rat Achilles tendon subcutaneously every 3 days after collagenase injection.
Results We compared the abundance of HIF-2α (encoded by EPAS1) in uncalcified and calcified regions of human and rat Achilles’s tendon. EPAS1 mRNA and protein levels were elevated in calcific tendons compared with normal tendons. Immunoreactive for stro-1, CD44 and HIF-2α demonstrated that HIF-2α was increased in TSPCs which located in the vicinity of calcific sites. These findings demostrate the HIF-2α level is increased in TSPCs of calcific tendons.
Knockdown of HIF-2α by shRNA significantly decreased the activity of alkaline phosphatase (ALP) and Alizarin red staining (ARS) of human TSPCs underwent osteogenesis induction combined with IL1β treatment. In contrast, overexpression of a constitutive active form of HIF-2α lead to a significant reduction in expression levels of tendon specific markers, such as SCX, EYA1/2. These data suggest HIF-2α signalling plays a significant role in the fate specification of tenocytes and osteoblasts from TSPCs.
Digoxin treatment in vitro inhibited IL1β-induced elevation of HIF-2α protein and significantly decreased the activity of ALP and ARS staining of TSPCs (Figure 1 a,b) and increased expression level of SCX, EYA1/2. Morever, we observed successful inhibition of HIF-2α in vivo by subcutaneously delivery of digoxin. X-ray quantification and histological examination showed that compared with vehicle, digoxin administration led to decreased calcium deposition (Figure 1 c,d).
Discussion Our work provides a appicable therapeutic approach for tendon calcification, through manipulation of HIF-2α pathway in stem/progenitor cells by digoxin.
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Bi Y, Ehirchiou D, Kilts TM, et al. Nat Med. 2007;13(10): 1219–27