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Abstract Ischemic retinopathies are the major causes of blindness, yet effective early-stage treatments remain limited due to an incomplete understanding of the underlying molecular mechanisms. Significant changes in gene expression often precede structural and functional alterations. Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are emerging as novel gene regulators, involved in various biological processes and human diseases. In this study, tsRNA-Gln-i-0095 was identified as a novel regulator, which was significantly upregulated in retinal ischemia/reperfusion (I/R) injury. Reducing the levels of tsRNA-Gln-i-0095 suppressed reactive gliosis, lowered inflammatory cytokine levels, and protected retinal ganglion cells from I/R injury. These effects led to reduced structural and functional damage, inhibited glial activation and inflammation, and enhanced neuronal function. Mechanistically, tsRNA-Gln-i-0095 downregulated the expression of NFIA and TGFBR2 through a miRNA-like mechanism. Collectively, this study highlights the potential of targeting tsRNA-Gln-i-0095 as a novel therapeutic approach to reduce retinal I/R injury and preserve visual function. Graphical abstract

Abstract Background Intravitreal injections of angiogenesis inhibitors have proved efficacious in the majority of patients with ocular angiogenesis. However, one-fourth of all treated patients fail to derive benefits from intravitreal injections. tRNA-derived small RNA (tsRNA) emerges as a crucial class of non-coding RNA molecules, orchestrating key roles in the progression of human diseases by modulating multiple targets. Through our prior sequencing analyses and bioinformatics predictions, tRNA-Cys-5-0007 has shown as a potential regulator of ocular angiogenesis. This study endeavors to elucidate the precise role of tRNA-Cys-5-0007 in the context of ocular angiogenesis. Methods Quantitative reverse transcription PCR (qRT-PCR) assays were employed to detect tRNA-Cys-5-0007expression. EdU assays, sprouting assays, transwell assays, and Matrigel assays were conducted to elucidate the involvement of tRNA-Cys-5-0007 in endothelial angiogenic effects. STZ-induced diabetic model, OIR model, and laser-induced CNV model were utilized to replicate the pivotal features of ocular vascular diseases and evaluate the influence of tRNA-Cys-5-0007 on ocular angiogenesis and inflammatory responses. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were employed to elucidate the anti-angiogenic mechanism of tRNA-Cys-5-0007. Exosomal formulation was employed to enhance the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. Results tRNA-Cys-5-0007 expression was down-regulated under angiogenic conditions. Conversely, tRNA-Cys-5-0007 overexpression exhibited anti-angiogenic effects in retinal endothelial cells, as evidenced by reduced proliferation, sprouting, migration, and tube formation abilities. In diabetic, laser-induced CNV, and OIR models, tRNA-Cys-5-0007 overexpression led to decreased ocular vessel leakage, inhibited angiogenesis, and reduced ocular inflammation. Mechanistically, these effects were attributed to the targeting of vascular endothelial growth factor A (VEGFA) and TGF-β1 by tRNA-Cys-5-0007. The utilization of an exosomal formulation further potentiated the synergistic anti-angiogenic and anti-inflammatory efficacy of tRNA-Cys-5-0007. Conclusions Concurrent targeting of tRNA-Cys-5-0007 for anti-angiogenic and anti-inflammatory therapy holds promise for enhancing the effectiveness of current anti-angiogenic therapy.