Targeting endothelial glycolytic reprogramming by tsRNA-1599 for ocular anti-angiogenesis therapy

Rationale: Current treatments for ocular angiogenesis primarily focus on blocking the activity of vascular endothelial growth factor (VEGF), but unfavorable side effects and unsatisfactory efficacy remain issues. The identification of novel targets for anti-angiogenic treatment is still needed. Methods: We investigated the role of tsRNA-1599 in ocular angiogenesis using endothelial cells, a streptozotocin (STZ)-induced diabetic model, a laser-induced choroidal neovascularization model, and an oxygen-induced retinopathy model. CCK-8 assays, EdU assays, transwell assays, and matrigel assays were performed to assess the role of tsRNA-1599 in endothelial cells. Retinal digestion assays, Isolectin B4 (IB4) staining, and choroidal sprouting assays were conducted to evaluate the role of tsRNA-1599 in ocular angiogenesis. Transcriptomic analysis, metabolic analysis, RNA pull-down assays, and mass spectrometry were utilized to elucidate the mechanism underlying angiogenic effects mediated by tsRNA-1599. Results: tsRNA-1599 expression was up-regulated in experimental ocular angiogenesis models and endothelial cells in response to angiogenic stress. Silencing of tsRNA-1599 suppressed angiogenic effects in endothelial cells in vitro and inhibited pathological ocular angiogenesis in vivo. Mechanistically, tsRNA-1599 exhibited little effect on VEGF signaling but could cause reduced glycolysis and NAD+/NADH production in endothelial cells by regulating the expression of HK2 gene through interacting with YBX1, thus affecting endothelial effects. Conclusions: Targeting glycolytic reprogramming of endothelial cells by a tRNA-derived small RNA represents an exploitable therapeutic approach for ocular neovascular diseases.


Clinical sample collection
The clinical samples were obtained from 2021 to 2023 at the Affiliated Eye Hospital of Nanjing Medical University, in accordance with the Declaration of Helsinki.Informed consents were obtained from all patients before inclusion.Aqueous humor (AH) was collected prior to intravitreal anti-VEGF therapy (for nAMD and PDR group) or prior to phacoemulsification surgery (for control group).

Immunofluorescent staining
The eyeballs were placed in the Fekete's solution at 4 ℃ for 2 h.The cornea, lens, and vitreous humor was removed.The remaining tissues were fixed in 4% paraformaldehyde (PFA; BL539A, Biosharp Biotechnology, China) overnight at 4℃.Then, they were dehydrated in 30% sucrose solution, embedded in OCT compound, and stored at -80 °C until sectioning.The frozen samples were sectioned to 5-μm thickness on a cryostat (Thermo Scientific, USA) and placed on the adhesion microscope slides (Citotestt, China).The frozen sections were washed with PBS for 3 times, permeabilized, and blocked with 5% bovine serum albumin (BSA) and 1% Triton X-100 for 1 h at 37 ℃.Then, the sectioned tissues were stained with the primary antibodies, including rabbit anti-NeuN

TUNEL assay
The apoptosis of ocular tissues was assessed using TUNEL assays with the In Situ Cell Detection Kit (C1086, Beyotime, China).In brief, paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated through a series of alcohol gradients followed by distilled water.The sections were then treated with permeabilization solution (containing 20 μg/ml proteinase K in 10 mM Tris/HCl) at room temperature for 15 min and rinsed with distilled water.Subsequently, the sections were incubated with the TUNEL reaction mixture for 1 h at 37°C.The nuclei were counterstained with DAPI solution for 5 min.Finally, the sections were visualized under a fluorescence microscope (Olympus, Tokyo, Japan).

Quantification of avascular area and neovascular tuft area in OIR retinas
Retinal images were imported into Adobe Photoshop 2021.The entire retina, avascular area, and retinal neovascularization (NV) area were selected using the polygonal lasso tool, quick selection tool, and magic wand tool, respectively.Finally, pixel measurements were conducted and recorded for the subsequent analysis.

Quantification of RGCs stained with RBPMS
To assess RGC number, the images of whole-mounted retinas were captured using a fluorescence microscope at 40 ╳ magnification.Six sample areas were chosen from each flat-mounted retina stained with anti-RBPMS: two from the central, mid-peripheral, and peripheral retina regions (approximately 1/6, 3/6, and 5/6 of the retinal radius from the optic nerve head, respectively).The number of RBPMS-positive cells was counted in each area and cell count was derived from the average across the three retinal regions.

Quantification of vascular length and branch point number
Retinal images were imported into AngioTool 64.0 (https://ccrod.cancer.gov/confluence/display/ROB2/Home).The identified vessels are demarcated with an outline on the displayed image.Then, the parameter of "vessel diameter and intensity" was set as 8-20.Meantime, the image dynamically updated its shape in response to the adjustments done using the controls included in the analysis tab.
Once the outline overlay matched the vessels in the displayed image, the analysis was conducted.Finally, AngioTool was used to calculate vascular length and branch points.

Cell Counting Kit-8 assay
Cell viability was evaluated by Cell Counting Kit-8 (CCK-8, Beyotime, C0071S) assays according to manufacturer's instruction.ECs were re-suspended and seeded in a 96well plate.Following the required treatment, 100 μl of CCK-8 solution diluted by ECM (1:10) was added to each well of 96-well plate.These cells were incubated for 2 h.Finally, the absorbance at 450 nm was determined by a microplate reader (FilterMax F5, Molecular Devices).

Cell proliferation assay
Cell proliferation was evaluated by an EdU kit (Beyotime, China, C0071S).Briefly, ECs were cultured into 24-well plates.EdU staining reagent was added to each well and incubated at 37 ℃ for 2 h.Then, these cells were fixed with 4% PFA and permeabilized with 0.3% Triton X-100 for 15 min.After washing, these cells were stained with the click reaction solution for 30 min to label the proliferating cells.Cell nuclei was stained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, C1002) for 10 min at room temperature.
The stained cells were observed by a fluorescence microscope (Olympus IX70, Japan).

Calcein-AM/propidium iodide (PI) double staining
Cell apoptosis was determined by Calcein-AM/propidium iodide (PI) double staining (Beyotime, C2015S).Briefly, ECs were seeded into 24-well plates followed the required treatment.An equal volume of PI (10 μmol/L) and Calcein (10 μmol/L) was added to each well of plate and incubated with the cells for 20 min at room temperature.The images were observed by a fluorescence microscope (Olympus IX70, Japan).

Transwell assay
Cell migration was determined by Transwell assays.ECs were seeded into the transwell chamber (8 μm pore size; Costar, USA) in 24-well plates.After the required treatment, these cells were re-suspended in serum-free ECM medium and 100 μl of cell suspension (3.5 × 10 4 cells) was added to the upper chamber.500 μl of complete medium was added to the lower chamber.These cells were cultured for 20 h at 37 ℃ and the nonmigrated cells in the upper chamber were removed.The cells attached the lower surface were fixed with methanol for 15 min and stained with the crystal violet (C805211, Macklin) for 10 min.The images were observed by a phase-contrast microscope (Olympus, Tokyo, Japan).

Tube formation assay
The 96-well plates were pre-cooled and pre-coated with Matrigel (Corning, NY, USA, CAT#356230) and returned to CO2 incubator for gel solidification at 37 ℃ for 30 min.
After the required treatment, ECs were digested with trypsin and seeded onto Matrigel (3 × 10 5 cells per well) in ECM medium containing 10% FBS.After 8 h-culture at 37 ℃ in 5% CO2, the total tube lengths in each well were measured by Image J software.

Quantitative reverse transcription PCR assay
Total RNAs were extracted from the cell lysates or the retinas with an RNA isoplus kit (Takara, Kyoto, Japan).The extracted RNAs were reversely transcribed into cDNAs using a microRNA Reverse Transcription Kit (EZB-miRT4, EZBioscience, China) with the specific stem-loop RT-primer according to the manufacturer's instruction.PCR products were amplified using the EZ-probe qPCR Master Mix (EZB-miProbe-R2, EZBioscience, China) on an Applied Biosystems Step One Real-time system (Applied Biosystems).For other genes, RNA samples were reversely transcribed into cDNAs using the HiScript III RT SuperMix (R323, Vazyme Biotech Co.,Ltd, China).qPCR assays were conducted using the ChamQ SYBR qPCR Master Mix (Q321, Vazyme Biotech Co.,Ltd, China).Relative tsRNA expression was normalized to U6 and other genes were normalized β-actin.Each reaction was performed at least in triplicate and calculated by the 2 − ΔΔCt method.The primer sequences were shown in Table S3 and S4.

Western blot
Total proteins were extracted from ECs using RIPA lysis buffer (Beyotime Biotech, Shanghai, China) supplemented with protease inhibitor cocktail and phosphatase inhibitors.
Protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit

Subcellular fractionation assay
The Cytoplasmic & Nuclear RNA Purification Kit (NGB-21000, Norgen Biotek) was used to extract nucleus and cytoplasmic RNAs following the manufacturer's instructions.
Subsequently, qPCR assays were conducted to assess the levels of tsRNA-1599 in the nucleus and cytoplasmic fractions.β-actin served as the cytoplasmic endogenous control, while U6 was used as the nucleus endogenous control.

Fluorescence in situ hybridization assay (FISH)
FISH assays were carried out to detect the subcellular distribution of tsRNA-1599 in ECs.Briefly, ECs were seeded in 24-well plate and fixed with 4% PFA.Then, they were treated with 0.5% Triton X-100 followed by pre-hybridization.Overnight hybridization was performed with 20 µM probe.RNA FISH kit was purchased from RiboBio (Guangzhou, China).FISH experiment was conducted according to the manufacturer's instruction.Cy3-labeled 18S rRNA, U6, and tsRNA-1599 probes were synthesized by RiboBio (Guangzhou, China).The nuclei were labeled with DAPI reagent.Finally, the cells were visualized by a fluorescence microscope (Olympus, Tokyo, Japan).

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photographed by a fluorescence microscope (Olympus IX70, Japan) and analyzed by