Tacrolimus improves therapeutic efficacy of mesenchymal stem cells in diabetic retinopathy by suppressing DRP1-mediated mitochondrial fission

Abstract Background: Diabetic retinopathy (DR) is a leading cause of blindness in diabetic patients. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) are emerging as a promising new drug for degenerative disease associated with diabetes. Recent studies have shown that high glucose-increased excessive calcium levels are a major risk factor for mitochondrial reactive oxygen species (mtROS) accumulation and apoptosis. This study aimed to investigate the role of high glucose-induced NFATC1 signaling in mitochondrial oxidative stress-stimulated apoptosis and the effect of tacrolimus on the therapeutic efficacy of subconjunctival transplantation of UCB-MSCs in a DR rat model. Methods: The UCB-MSCs were pretreated with the drugs prior to high glucose treatment. Then, we conducted experiments using western blot, LDH release, mitoSOX staining, qPCR analysis and MitoTacker staining. For animal experiment, tacrolimus or vehicle-pretreated MSCs were injected subconjunctivally in STZ-induced DR rat model. Results: High glucose increased cytotoxicity, mtROS, and cleaved caspase-9 expression in UCB-MSCs, and high glucose-induced mtROS was critical for apoptosis. High glucose conditions increased O-GlcNAcylated protein expression and nuclear translocation of NFATC1. However, nuclear translocation of NFATC1 was reduced by ST045849 pre-treatment, an O-GlcNAc transferase inhibitor. Tacrolimus pretreatment recovered high glucose-induced mtROS levels and apoptosis. In DR rat model, subconjunctival transplantation of tacrolimus-pretreated MSCs improved retinal vessel formation, retinal function, and uveitis. In high glucose conditions, tacrolimus pretreatment reduced protein and mRNA expression levels of DRP1 and inhibited mitochondrial fission. In addition, Mdivi-1 pretreatment, DRP1 inhibitor, reduced mtROS levels and apoptosis of UCB-MSCs under high glucose conditions. Conclusions : In conclusion, we demonstrated that high glucose-induced O-GlcNAcylation activates NFATC1 signaling, important for DRP1-mediated mitochondrial fission and mitochondrial apoptosis. Finally, we proposed NFATC1 suppression by tacrolimus as a promising therapeutic strategy to improve the therapeutic efficacy of UCB-MSC transplantation for DR treatment.