Cyclic Mechanical Stretching Induces Autophagic Cell Death in Tenofibroblasts Through Activation of Prostaglandin E2 Production

Abstract

Background/Aims: Autophagic cell death has recently been implicated in the pathophysiology of tendinopathy. Prostaglandin E2 (PGE2), a known inflammatory mediator of tendinitis, inhibits tenofibroblast proliferation in vitro; however, the underlying mechanism is unclear. The present study investigated the relationship between PGE2 production and autophagic cell death in mechanically loaded human patellar tendon fibroblasts (HPTFs) in vitro. Methods: Cultured HPTFs were subjected to exogenous PGE2 treatment or repetitive cyclic mechanical stretching. Cell death was determined by flow cytometry with acridine orange/ethidium bromide staining. Induction of autophagy was assessed by autophagy markers including the formation of autophagosomes and autolysosomes (by electron microscopy, AO staining, and formation of GPF-LC3-labeled vacuoles) and the expression of LC3-II and BECN1 (by western blot). Stretching-induced PGE2 release was determined by ELISA. Results: Exogenous PGE2 significantly induced cell death and autophagy in HPTFs in a dose-dependent manner. Blocking autophagy using inhibitors 3-methyladenine and chloroquine, or small interfering RNAs against autophagy genes Becn-1 and Atg-5 prevented PGE2-induced cell death. Cyclic mechanical stretching at 8% and 12% magnitudes for 24 h significantly stimulated PGE2 release by HPTFs in a magnitude-dependent manner. In addition, mechanical stretching induced autophagy and cell death. Blocking PGE2 production using COX inhibitors indomethacin and celecoxib significantly reduced stretching-induced autophagy and cell death. Conclusion: Taken together, cyclic mechanical stretching induces autophagic cell death in tenofibroblasts through activation of PGE2 production.