PV QA 1 - Poster Viewing Q&A 1
Purpose/Objective(s): Proton beam radiotherapy is used for the treatment of ocular diseases such as retinoblastoma and uveal melanoma, but despite its precision, patients can still experience radiation retinopathy. The retinopathic effects of radiation include endothelial cell injury, retinal ischemia, neovascularization, microaneurysms, and retinal detachment. We hypothesize that irradiation of retinal endothelial cells results in chronic inflammation of the endothelium and mitochondrial dysfunction leading to endothelial cell dysfunction and that administration of AP39, a mitochondrial-targeted hydrogen sulfide (H2S) donor will protect against IR-induced endothelial injury.
Materials/Methods: Mouse retinal endothelial cells (mRECs) were irradiated with either protons (72.7 MeV to 131.1 MeV) using the ProteusONETM pencil beam system or photons (6 MV). AP39 was administered 45 minutes prior to IR and was present during irradiation. AP39 was removed 30-60 minutes after irradiation. The clonogenic assay was used to assess cell survival following irradiation +/- AP39 treatment. Mitochondrial damage and function was measured using mitochondrial copy number, mtDNA common deletion, and Seahorse XF24. RT-PCR and Western analysis were used to determine changes in RNA and protein levels.
Results: The relative biological effectiveness (RBE) of the ProteusONETM pencil proton beam was 1.1 compared to photons in mRECs. Intercellular adhesion molecule-1 (ICAM-1) and platelet-endothelial cell adhesion molecule-1 (PECAM-1) RNA levels increased 72 hr after 10 Gy photon. There was also a 50% reduction in 3-mercaptopyruvate sulfurtransferase (3-MST) RNA, a predominantly mitochondrial H2S synthesis enzyme. There was no observed effect on mitochondrial DNA copy number or mtDNA deletion at 72 hr post-IR. However, there was a significant decrease in mitochondrial respiration and a corresponding increase in glycolysis following 10 Gy photons or protons that was rescued by AP39. AP39 also increased acetylation of mitochondrial proteins with no effect on SIRT3, the major mitochondrial deacetylase.
Conclusion: Endothelial cells are critical for the establishment of barrier function in the eye and mitochondrial dysfunction could lead to barrier disruption and gradual development of radiation retinopathy following radiotherapy. We show that IR increases expression of adhesion molecules and decreases 3-MST, a major H2S synthesis enzyme in the mitochondria. In addition, mitochondrial function was markedly disrupted by 72 hours post-IR, but its rescue by AP39 suggests a novel role for H2S as a mitochondrial and endothelial radio-protectant following IR. Future studies will focus on identifying how AP39 and other mitochondria-targeted H2S delivery molecules restore mitochondrial function in irradiated retinal endothelial cells.
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