Dual anti-angiogenic and anti-inflammatory action of tRNA-Cys-5-0007 in ocular vascular disease

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. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-024-05338-w.


Tube formation assay
The 24-well plates were coated with 35 μL of Matrigel (354230, Corning, USA) per well and incubated at 37°C for 1 h.The pre-treated HRVECs cultured on 6-well plates were harvested.After gelation, the matrigel was overlaid with 500 μL of medium containing 4 × 10 4 of HRVECs per well.These cells were cultured in a CO2 incubator (37°C, 5% [v/v] CO2, 95% humidity).A branched endothelial network was visualized by a light microscope (IX73P1F, Olympus, Japan).The quantification of capillary tube formation was conducted using Image J.

Transwell migration assay
After transfection, endothelial cells were harvest and adjusted to a concentration of 1 × 10 5 cells/mL in ECM.The 8 μm cell culture inserts (353097, BD Falcon, USA) were placed in a 24-well plate with tweezers.Then, 600 µL of ECM complete medium was added to the lower chamber, while 200 µL of cell suspension was added to the upper chamber.After 12 h, the cells in the lower chamber were fixed by methanol for 15 min.The transwell chamber was then placed upside down for 10 min for air dry.
The crystal violets were washed away with ddH2O.These non-migrated cells in the upper chamber were gently removed using a cotton swab.The images were obtained under a light microscope.

5-ethynyl-2'-deoxyuridine (EdU) assay
BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071S, Beyotime, China) was used for the detection of cell proliferation following the manufacturer's instruction.Briefly, the cells were plated into 24-well plate and allowed to grow until 80% cell density.EdU solutions were added to incubate for 2 h.Next, the cells were fixed with 4% PFA and permeabilized with a buffer (0.3 % Triton X-100) for 15 min.
Subsequently, the reaction mix was added to label EdU and incubate for 30 min.The nuclei were labeled with DAPI.The images were obtained by a fluorescence microscope and EdU-positive cells were counted.

Endothelial cell spheroid-based sprouting angiogenesis assay
Endothelial cells were harvested and adjusted to the concentration of 2 × 10 4 cells/mL.Cell suspension was mixed with 1.2% methocel stock solution (419273, Sigma, USA) and dripped onto the lid of a petri dish.The dish was incubated at 37 °C for 24 h.Subsequently, the hanging drops containing spheroids were gently washed off using PBS.The spheroids were then centrifuged at 200 g for 3 min to form the pellets to discard the supernatants.2 mL mixture of methocel stock solution containing 20% FBS and collagen I (354236, Corning, USA) was added and mixed to avoid bubbles.
Next, 1 mL of spheroid mixture was added to a 24-well plate and incubated at 37℃ for 30 min to promote solidification.Finally, the spheroids were cultivated at 37℃ for 24 h and imaged.

Evans blue assay
Evans blue assays were used to detect the breakdown of blood-retinal barrier.The first step was to prepare Evans blue solution (30 g/L, K29136, Kehbio, China) using sterile saline and add 400 U/mL of heparin sodium salt (1170GR001, BioFroxx, German) for anti-coagulation.The solution was shaken overnight at room temperature, filtered with a 0.22 μm sterile filter and stored at 4℃ for the preservation.To perform Evans blue assays, the animals were anesthetized and secured in an upward position with the abdomen facing up.The hair from the inner thighs was removed and a longitudinal incision was made in the inner thigh skin.Blunt dissection of the fascia was then performed to expose femoral vein.Then, the animals were slowly injected with Evans blue solution (30 mg/kg) using an insulin syringe.After 30-min circulation, the eyeballs were gently enucleated and fixed with 4% PFA for 30 min.The anterior segment, lens, and vitreous were removed away from the eyecups and the retinas were dissected free from choroid-sclera complexes and cut into four-leaf clover shape under a stereo surgical microscope.EB extravasation from retinal vessels was observed under a fluorescence microscope.Retinal permeability was evaluated by detecting the concentration of EB dye in the plasma from iliac artery.After measuring wet weight, the retinas were thoroughly dried in a desiccator.To extract EB dye, the retina was incubated with 120 μL of formamide at 70 °C for 18 h.The extract was centrifuged at 12, 000 g for 30 min at 4 °C.Absorbance at 620 nm (blue signal) and 740 nm (background subtracted) of the supernatant was detected by the spectrophotometric method.Concentrations of EB dye was calculated based on the corresponding standard curve in formamide.The concentration of EB dye in blood sample was measured in the same manner.Retinal vascular permeability was calculated using the equation as shown below.

Retinal leukocytosis assay
The adhesion of leukocytes to retinal vessels was detected as previously described.Briefly, the mice were anesthetized and positioned with their abdomen.The chest cavity was opened using the scissors to expose the heart.A 20 G perfusion tube was inserted into the aorta and 10 mL of PBS was perfused to remove these nonadherent blood cells.Subsequently, 10 mL of fluorescent isothiocyanate (FITC)-labeled concanavalin A (Con A) lectin (40 μg/mL, FL-1001, Vector Laboratories, USA) was perfused to label the adherent leukocytes and vascular endothelial cells.After that, the mouse was cannulated with 10 mL of PBS to remove the remaining unbound Con A.
Then, the eyeballs were enucleated and fixed with 4% PFA for 30 min.After fixation, the anterior segment, lens, and vitreous were removed from the eyecups.The retinas were dissected free from the choroid-sclera complexes.Then, the retinas were cut into four-leaf clover shape under a stereo surgical microscope.Retinal flat mounts were imaged under a fluorescent stereoscope and total number of leukocytes adhered to the vessel walls per retina was counted.

Immunofluorescent staining of choroidal flat mounts
After anesthesia, the mice were euthanized by cervical dislocation.The eyeballs were removed and fixed in 4% PFA for 30 min.Then, the fascia and connective tissues on the surfaces of eyeballs were carefully peeled off.The anterior segment, lens, and vitreous were removed away from the eyecups.RPE choroid-sclera complex was dissected and cut into four-leaf clover shape under a stereo surgical microscope.RPEchoroid-sclera complex was fixed in 4% PFA for 30 min and washed with PBS for three times.To facilitate permeabilization, the complex was permeabilized with PBS containing 5% BSA and 1% TritonX-100 at 37℃ for 45 min, incubated with the F4/80 primary antibody (1:200, ab6640, Abcam, England) overnight at 4℃, and then incubated with the secondary antibody or Isolectin B4 (1:25; L2895, Sigma-Aldrich, USA) at room temperature for 2 h.RPE choroid-sclera complexes were observed under a fluorescent scope.

Immunofluorescent staining
After the mice were anesthetized and euthanized, their eyeballs were removed and fixed with 4% PFA for 30 min.Following fixation, the fascia, connective tissue, anterior segment, lens, and vitreous were carefully removed from the eyecups.Subsequently, the retinas were dissected free and cut into a four-leaf clover shape under a stereo surgical microscope.The dissected retinas were fixed again with 4% PFA and permeabilized using a solution containing 5% bovine serum albumin (BSA) and 1% Triton X-100 for 45 min at 37°C.For immunostaining, the retinas were incubated overnight at 4°C with claudin-5 antibody (1:200, sc-374221, Santa Cruz, USA).This was followed by the incubation with the secondary antibody and GS-IB4 Alexa Fluor™ 594 (1:50, I21413, Thermo Fisher, USA) at room temperature for 2 h.Finally, the retinas were imaged under a fluorescent microscope to visualize the distribution of claudin-5 and GS-IB4.

Subcellular fractionation assay
The Cytoplasmic & Nuclear RNA Purification Kit (NGB-21000, Norgen Biotek) was employed to extract both the nucleus and cytoplasmic RNAs.This kit allows for the separation of RNA fractions based on cellular localization.After RNA extraction, qPCR assays were conducted to detect the levels of tRNA-Cys-5-0007 in both the nucleus and cytoplasmic fractions.To ensure accurate normalization and comparison of RNA levels between the two fractions, β-actin and U6 were detected as endogenous controls for cytoplasmic and nucleus RNAs, respectively.qPCR analysis provides insights into subcellular distribution of tRNA-Cys-5-0007 and allows for the assessment of its potential role in different cellular compartments.
Subsequently, qPCR was conducted using the EZ-Probe qPCR Master Mix (EZBioscience, EZB-miprobe-R2, USA).Expression levels of all target genes were normalized to the expression of U6.For mRNA detection, total RNAs were reversetranscribed into complementary DNAs (cDNAs) using the PrimeScript RT Master Mix (RR037A, Takara Bio, Japan).The resulting cDNAs were then subjected to qPCRs using the SYBR Premix Ex Taq II (RR820A, Takara Bio, Japan) according to the manufacturer's protocol.

Western blot
The cells were lysed in RIPA lysis buffer (P0013B, Beyotime, China) supplemented with Complete TM Protease Inhibitor Cocktail (04693116001, Roche, Switzerland).Protein concentration in the lysates was determined and normalized using the BCA method (23225, Thermo Fisher, USA).Protein samples were prepared with a five-fold loading buffer and separated by SDS-PAGE, after which they were transferred to PVDF membranes (IPVH00010, Merck Millipore, Germany).The membranes were then blocked and incubated overnight at 4°C with primary antibodies.The primary antibodies used were VEGFA (Dilution, 1:1000) and TGF-β1 (Dilution, 1:1000).

Enzyme-linked immunosorbent assay (ELISA) assay
After incubation at 37 °C for 24 h, the medium was collected and examined for cytokine and chemokine production with TGF-β1 ELISA Kit (E-EL-0162c, eBioscience, USA) and VEGF-A ELISA Kit (E-EL-H0111, eBioscience, USA) according to the manufacturer's instruction.
This incubation allowed for the immunoprecipitation of RNA-protein complexes, specifically those associated with Ago2 or IgG.Following the incubation, RNA was eluted and purified from the immunoprecipitated complexes.The enriched RNA, including tRNA-Cys-5-0007, was then subjected to qRT-PCRs.This approach facilitated the identification and quantification of RNA molecules that interact with Ago2, providing insights into the regulatory roles of tRNA-Cys-5-0007 within the RNA-induced silencing complex.

RNA pull down assay
The cellular proteins were extracted using IP lysis buffer and then incubated for 30 min.The lysates underwent centrifugation at 3, 000 g for 20 min at 4°C to obtain the total protein.The total protein was combined with a biotinylated probe specific to tRNA-Cys-5-0007 or a negative control probe (RiboBio, China).The mixture was left to incubate overnight at 4°C.Following overnight incubation, the protein-probe complexes were exposed to Streptavidin MagPoly Beads (SM017005, Smart-Lifesciences Biotechnology, China) at 26°C for 2 h.After washing to remove the nonspecific bound proteins, the RNA-binding protein complexes were eluted.To prepare the eluted complexes for western blot, 5 ╳ SDS-PAGE loading buffer (P0015, Beyotime, China) was added, and the mixture was boiled for 25 min.The presence of the target protein in the pull-down complex was assessed.A portion of cell lysate was retained as the input control.For immunoprecipitation, the primary antibody employed was Ago2 antibody (1:1000, 67934-1-Ig, Proteintech, China).This approach allowed for the identification and characterization of protein interactions with tRNA-Cys-5-0007.

Luciferase reporter gene assay
The luciferase reporter assay was performed using the Dual Luciferase Reporter Gene Assay Kit (RG027, Beyotime, China).VEGFA 3'UTR-Wt and Mut, as well as TGF-β1 3'UTR-Wt and Mut sequences, were incorporated into the GP-miRGLO vector (GenePharma, China).These luciferase reporter vectors were then transfected with either tRNA-Cys-5-0007 mimics or negative control mimics.After a 48-h posttransfection period, the luciferase activity was quantified relative to Renilla luciferase activity using the Dual Luciferase Reporter Gene Assay Kit.

Detection of ocular drug distribution
To assess delivery efficiency, bioluminescence imaging was conducted on mice using the IVIS Spectrum In Vivo Imaging System (IVIS® Spectrum, PerkinElmer, USA).Cy5-labeled tRNA-Cys-5-0007 or Exos-Cys-5-0007 were intravitreally injected into ICR mice, which are an albino mouse strain suitable for imaging with IVIS.After 24 h, the mice were euthanized and their eyeballs were collected for imaging.To detect the distribution of exosomes in a laser-induced CNV model, Evlink-labeled exosomes were intravitreally injected following laser injury.Choroids were isolated three days after injection for analysis.

Exosome uptake in vitro
HRVECs were seeded into 24-well plates at the density of 4 × 10 4 cells/well and cultured for 24 h.To detect the uptake of Exos by HRVECs, the isolated Exos (200 μg protein/150 μL DPBS) were labeled with CD63 (1:50, sc-5275, Santa Cruz, USA).The labeled Exos were then incubated at room temperature for 30 min and subsequently centrifuged at 100,000 g to remove excess antibody.HRVECs were incubated with 50 μg/mL labeled Exos in FBS-free medium for 24 h.Then, the medium was discarded and the cells were washed with DPBS (14190144, Thermo Fisher, USA) for 3 times.

OCT and FFA assay
OCT and FFA assays were conducted using a Saris Multi-Modal Ophthalmic ImagingSystem for Animal (Robotrak, China).To dilate the pupils, 0.5% tropicamide with 5% phenylephrine was administered.To visualize blood vessels, the mice were intraperitoneally injected with 10% fluorescein sodium at a volume/weight ratio of 50 μL/20 g.Fluorescence accumulation images were captured during the late stationary phase.FFA and OCT images were captured with a 30 ° angle of view.To quantify vascular permeability, Image J software was used for analysis.

TUNEL assay
TUNEL assays were conducted to detect retinal apoptosis.Following cardiac perfusion, the eyeballs were harvested for making frozen sections.These sections were then allowed to re-warm at 25°C for 30 min and washed with PBS for three times.
Retinal apoptosis was detected using the TUNEL Apoptosis Detection Kit (Beyotime, C1098, China) according to the manufacturer's instruction.DNase included positive controls were performed using the Apoptosis Inducers Kit (C0005, Beyotime, China).
The nuclei were labeled with DAPI and the images were captured by a fluorescence microscope.

Hematoxylin-eosin (H&E) staining
H&E staining was used to detect the change of retinal structure.The eyeballs were removed and preserved in paraffin, followed by cutting them into 5 µm sections.These sections underwent sequential alcohol dehydration and were cleared with xylene.
Subsequently, they were stained with hematoxylin for 1 min and eosin for 30 sec.The changes of retinal structures observed under a light microscope.

Flow cytometry and Calcein-AM/PI double staining
Flow cytometry and Calcein-AM/PI double staining was conducted to detect cell apoptosis.After treating HRVECs with Exos for 24 h, Calcein-AM (22003, AAT Bioquest, USA) and PI (17517, AAT Bioquest, USA) were added to label the live and apoptotic cells at 37°C for 15 min in dark.The nuclei were labeled with Hoechst 33258 (Beyotime, C1011, China).The images were captured using a fluorescence microscope.
For flow cytometry analysis, HRVECs were trypsinized and washed with PBS for 3 times.Annexin V Binding Buffer, Annexin V-FITC, and PI were added according to the manufacturer's instruction and incubated in dark for 15 min.The percentage of cell apoptosis was determined by flow cytometry.

ERG examination
The animals were dark adapted overnight and prepared for ERG recording under dim red light.To dilate the pupils, 0.5% tropicamide with 5% phenylephrine was administered.The mice were anesthetized and one drop of oxybuprocaine (0.4%) was applied for corneal anesthesia.The placement of the recording electrode was placed on the central cornea, while the reference needle electrode was placed behind the ears and the ground electrode in the tail.To prevent corneal desiccation, one drop of 1% carboxymethylcellulose was applied to the cornea.Following recording of a scotopic intensity series, the photopic flash responses were measured using increased light stimuli.The b-wave amplitude was measured from the trough of a-wave to the peak of b-wave.ERG recordings were performed using the Roland Consult Color Ganzfeld Q450C recording machine.