Microvesicles isolated from 5-azacytidine- and-resveratrol-treated mesenchymal stem cells for the treatment of suspensory ligament injury in horse-a case report

Background: In athlete horses, suspensory ligament (SL) injuries are the most common cause of lameness. Healing of SL injury is still problematic, and even proper rehabilitation and pharmacological therapy do not guarantee returning to the initial performance level. In our previous studies, we have shown that a combination of 5-azacytidine (AZA) and resveratrol (RES) exerts beneficial, rejuvenating effects on metabolic syndrome derived adipose-derived stem cells (ASCs). Thus, in the presented research, we investigate whether not only rejuvenated ASC but also microvesicles (MVs(AZA/RES)) secreted by them possess enhanced regenerative properties in SL injury. Methods: In the presented study, a 6-year-old Dutch Warmblood gelding, working in jumping, was diagnosed with SL injury using ultrasonography, Doppler, real-time elastography and thermography. As a therapeutic strategy, the affected animal was treated with extracellular microvesicles derived from ASC treated with the combination of 5-azacytydine (AZA) and resveratrol (RES) (MVs(AZA/RES)). Results: First, anti-apoptotic effects of MVs(AZA/RES) were tested in co-culture with metabolic syndrome derived ASC. The proliferation of cells and expression of pro-apoptotic genes were investigated. Then, MVs(AZA/RES) were injected directly into the injured SL of the Dutch Warmblood gelding. In vitro assays revealed that MVs(AZA/RES) enhance the proliferation of ASC and exert an anti-apoptotic effect. In the affected horse, the application of MVs(AZA/RES) resulted in increased lesion filling and improvement of angiogenesis and elasticity in injured tissue. Conclusions: As MVs(AZA/RES) mimic several of the biological actions exerted by ASC, they have become an alternative for stem cell-based therapies and can be effectively applied for the treatment of SL injury in horses.