MITOCHONDRIAL TRANSPLANTATION AS A NOVEL THERAPEUTIC STRATEGY FOR RETINAL ISCHEMIA-REPERFUSION INJURY

Abstract

Abstract Title: Mitochondrial Transplantation as a Novel Therapeutic Strategy for Retinal Ischemia-Reperfusion Injury Authors: Fernando Rubio-Mijangos1,2, Aybuke Çelik1, Maria Loscertales2, Alexander Bigger-Allen3, Sitaram Emani1, Pedro del Nido1, Demetrios Vavvas2*, James D. McCully1* 1Department of Cardiac Surgery, Boston Children’s Hospital, Department of Surgery, Harvard Medical School, Boston, MA, 02215, USA 2Department of Ophthalmology, Retina Service, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA 3Urological Diseases Research Center, Boston Children’s Hospital, Harvard Medical School, Boston, MA 02114, USA *Co-last authors Keywords: Mitochondrial transplantation; Retinal ischemia-reperfusion injury; Oxidative stress; Mitochondrial dysfunction; Cellular bioenergetics; ARPE-19; Inflammation; Apoptosis; Background Mitochondria are central to cellular homeostasis, providing ATP through oxidative phosphorylation while also regulating oxidative stress, apoptosis, and immune responses. Retinal ischemia-reperfusion injury (RIRI) disrupts mitochondrial function, triggering oxidative stress, inflammation, and neurovascular degeneration. Given the critical role of mitochondria in retinal cell survival, mitochondrial transplantation (MT) has emerged as a promising therapeutic strategy to restore mitochondrial integrity and bioenergetics in ischemic tissues. This study evaluates the potential of MT in rescuing oxidative stress-induced mitochondrial dysfunction in an in vitro model of RIRI. Methods ARPE-19 cells were differentiated into a mature retinal pigment epithelium-like phenotype and subjected to oxidative stress using hydrogen peroxide (H₂O₂). Mitochondria were isolated from ARPE-19 cells and characterized for viability and function. Transplantation was performed at optimized doses, and mitochondrial uptake, ATP production, oxidative stress resilience, and cell survival were assessed. Bulk RNA sequencing was conducted to analyze transcriptional responses following MT, identifying differentially expressed genes and enriched biological pathways involved in cellular stress adaptation. Results Transplanted mitochondria were efficiently internalized by ARPE-19 cells and integrated into the host mitochondrial network, restoring ATP levels in a dose-dependent manner. MT significantly improved cell survival and reduced oxidative stress-induced apoptosis at 0.25-0.5 mM H₂O₂ concentrations. RNA sequencing revealed that MT downregulated inflammatory and apoptotic pathways, including IL-17, VEGF, and CASP9 signaling, while upregulating stress-adaptive pathways such as TNF, NF-κB, SGK1, and efferocytosis. Notably, MT reduced IL6 and IL6R expression, potentially mitigating inflammation-driven mitochondrial dysfunction. Conclusion MT demonstrates significant potential in restoring mitochondrial function and modulating stress-responsive transcriptional programs in RIRI models. By mitigating oxidative stress, preserving ATP levels, and reprogramming inflammatory responses, MT presents a promising therapeutic approach for retinal ischemic diseases. Future Directions Further investigations are required to validate these findings in vivo, refine transplantation protocols, and assess the long-term efficacy and safety of MT. Proteomic and metabolomic analyses could provide deeper insights into mitochondrial integration and functional recovery, advancing MT as a viable intervention for retinal ischemia.