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-ß1 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.

Comprehensive metabolic profiling of diabetic retinopathy.

Diabetic retinopathy (DR) is an important complication of diabetes mellitus and a prevalent blind-causing ophthalmic disease. Despite years of efforts, rapid and accurate diagnosis of DR remains a challenging task. Metabolomics has been used as a diagnostic tool for disease progression and therapy monitoring. In this study, retinal tissues were collected from diabetic mice and age-matched non-diabetic mice. An unbiased metabolic profiling was performed to identify the altered metabolites and metabolic pathways in DR. 311 differential metabolites were identified between diabetic retinas and non-diabetic retinas under the criteria of variable importance in projection (VIP) > 1 and P < 0.05. These differential metabolites were highly enriched in purine metabolism, amino acid metabolism, glycerophospholipid metabolism, and pantaothenate and CoA biosynthesis. We then evaluated the sensitivity and specificity of purine metabolites as the candidate biomarkers for DR through the area under the receiver-operating characteristic curves (AUC-ROCs). Compared with other purine metabolites, adenosine, guanine, and inosine had higher sensitivity, specificity, and accuracy for DR prediction. In conclusion, this study sheds new light on the metabolic mechanism of DR, which can facilitate clinical diagnosis, therapy, and prognosis of DR in the future.

Targeting pericyte-endothelial cell crosstalk by circular RNA-cPWWP2A inhibition aggravates diabetes-induced microvascular dysfunction.

The crosstalk between vascular pericytes and endothelial cells (ECs) is critical for microvascular stabilization and remodeling; however, the crosstalk is often disrupted by diabetes, leading to severe and even lethal vascular damage. Circular RNAs are a class of endogenous RNAs that regulate several important physiological and pathological processes. Here we show that diabetes-related stress up-regulates cPWWP2A expression in pericytes but not in ECs. In vitro studies show that cPWWP2A directly regulates pericyte biology but indirectly regulates EC biology via exosomes carrying cPWWP2A. cPWWP2A acts as an endogenous miR-579 sponge to sequester and inhibit miR-579 activity, leading to increased expression of angiopoietin 1, occludin, and SIRT1. In vivo studies show that cPWWP2A overexpression or miR-579 inhibition alleviates diabetes mellitus-induced retinal vascular dysfunction. By contrast, inhibition of cPWWP2A-mediated signaling by silencing cPWWP2A or overexpressing miR-579 aggravates retinal vascular dysfunction. Collectively, this study unveils a mechanism by which pericytes and ECs communicate. Intervention of cPWWP2A or miR-579 expression may offer opportunities for treating diabetic microvascular complications.

CircRNA expression profile and functional analysis in retinal ischemia-reperfusion injury.

Retinal ischemia-reperfusion (I/R) is involved in the pathogenesis of many vision-threatening diseases. circRNAs act as key players in gene regulation and human diseases. However, the global circRNA expression profile in retinal I/R injury has not been fully uncovered. Herein, we established a murine model of retinal I/R injury and performed circRNA microarrays to identify I/R-related circRNAs. 1265 differentially expressed circRNAs were identified between I/R retinas and normal retinas. Notably, the detection of cWDR37 level in aqueous humor could discriminate glaucoma patients from cataract patients (AUC = 0.9367). cWdr37 silencing protected against hypoxic stress- or oxidative stress-induced retinal ganglion cell (RGC) injury. cWdr37 silencing alleviated IR-induced retinal neurodegeneration as shown by increased NeuN staining, reduced retinal reactive gliosis, and decreased retinal apoptosis. Collectively, this study provides a novel insight into the pathogenesis of retinal I/R injury. cWdr37 is a promising target for the diagnosis or treatment of I/R-related ocular diseases.

Identification of aberrantly expressed circular RNAs in hyperlipidemia-induced retinal vascular dysfunction in mice.

Hyperlipidemia-induced retinal vascular dysfunction is a complex pathological process. circRNAs are important regulators of biological processes and disease progression. However, the expression pattern of circRNAs in hyperlipidemia-induced retinal vascular dysfunction remains unclear. Herein, we used a murine model of hyperlipidemia and identified 317 differentially expressed circRNAs between hyperlipidemic retinas and normolipidemic retinas by circRNA microarrays. GO analysis indicated that the host genes of dysregulated circRNAs were targeted to cell differentiation (ontology: biological process), cytoplasm (ontology: cellular component), and protein binding (ontology: molecular function). Pathway analysis revealed that circRNAs-mediated network was mostly enriched in focal adhesion signaling. Notably, circLDB1 was significantly up-regulated in the serum of coronary artery disease patients and aqueous humor of age-related macular degeneration patients. circLDB1 regulated endothelial cell viability, proliferation, and apoptosis in vitro. Thus, circRNAs are the promising targets for the prediction and diagnosis of hyperlipidemia-induced vascular diseases.

Role of METTL3-Dependent N6-Methyladenosine mRNA Modification in the Promotion of Angiogenesis.

Epigenetic alterations occur in many physiological and pathological processes. N6-methyladenosine (m6A) modification is the most prevalent modification in eukaryotic mRNAs. However, the role of m6A modification in pathological angiogenesis remains elusive. In this study, we showed that the level of m6A modification was significantly upregulated in endothelial cells and mouse retinas following hypoxic stress, which was caused by increased METTL3 levels. METTL3 silencing or METTL3 overexpression altered endothelial cell viability, proliferation, migration, and tube formation in vitro. METTL3 knockout in vivo decreased avascular area and pathological neovascular tufts in an oxygen-induced retinopathy model and inhibited alkali burn-induced corneal neovascularization. Mechanistically, METTL3 exerted its angiogenic role by regulating Wnt signaling through the m6A modification of target genes (e.g., LRP6 and dishevelled 1 [DVL1]). METTL3 enhanced the translation of LRP6 and DVL1 in an YTH m6A RNA-binding protein 1 (YTHDF1)-dependent manner. Collectively, this study suggests that METTL3-mediated m6A modification is an important hypoxic stress-response mechanism. The targeting of m6A through its writer enzyme METTL3 is a promising strategy for the treatment of angiogenic diseases.

Osthole: A Traditional Chinese Medicine for Ocular Anti-Angiogenic Therapy.

PURPOSE: Osthole is an agent isolated from Cnidium monnieri (L.) Cusson and has been used to treat several disorders. Corneal neovascularization is a sight-threatening condition associated with several inflammatory or infectious ocular disorders. In this study, we investigated the anti-angiogenic effects of osthole on corneal neovascularization and the underlying mechanism. METHODS: MTT assay, HE staining, and calcein-AM/propidium iodide staining was conducted to detect the toxicity of osthole in vitro and in vivo. Corneal neovascularization of ICR mice was induced by alkali burn and observed by a slit lamp microscopy on day 7 after alkali injury. EdU assay, Ki67 immunofluorescence assay, Transwell migration assay, and Matrigel assay were conducted to investigate the role of osthole in endothelial angiogenic effects in vitro. Western blots were conducted to investigate the anti-angiogenic mechanism of osthole in corneal neovascularization. RESULTS: Administration of osthole ranging from 0.05 to 25 µM had no detectable cytotoxicity or tissue toxicity in vivo and in vitro. Topical administration of osthole inhibited corneal neovascularization induced by alkali burn. Osthole decreased the proliferation, migration, and tube-formation of endothelial cells induced by VEGF. Osthole inhibited endothelial angiogenic functions through blocking the phosphorylation of ERK1/2, JNK, and p38. CONCLUSION: Our study provides evidence that osthole is a promising drug for the treatment of corneal neovascularization.

Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

VEGF-induced neovascularization plays a pivotal role in corneal neovascularization (CoNV). The current study investigated the potential effect of ginsenoside Rh2 (GRh2) on neovascularization. In HUVECs, pretreatment with GRh2 largely attenuated VEGF-induced cell proliferation, migration, and vessel-like tube formation in vitro. At the molecular level, GRh2 disrupted VEGF-induced VEGF receptor 2 (VEGFR2)-Grb-2-associated binder 1 (Gab1) association in HUVECs, causing inactivation of downstream AKT and ERK signaling. Gab1 knockdown (by targeted short hairpin RNA) similarly inhibited HUVEC proliferation and migration. Notably, GRh2 was ineffective against VEGF in Gab1-silenced HUVECs. In a mouse cornea alkali burn model, GRh2 eyedrops inhibited alkali-induced neovascularization and inflammatory cell infiltrations in the cornea. Furthermore, alkali-induced corneal expression of mRNAs/long noncoding RNAs in cornea were largely attenuated by GRh2. Overall, GRh2 inhibits VEGF-induced angiogenic effect via inhibiting VEGFR2-Gab1 signaling in vitro. It also alleviates angiogenic and inflammatory responses in alkali burn-treated mouse corneas.-Zhang, X.-P., Li, K.-R., Yu, Q., Yao, M.-D., Ge, H.-M., Li, X.-M., Jiang, Q., Yao, J., Cao, C. Ginsenoside Rh2 inhibits vascular endothelial growth factor-induced corneal neovascularization.

Genome-Wide Identification of Circular RNAs as a Novel Class of Putative Biomarkers for an Ocular Surface Disease.

BACKGROUND/AIMS: Pterygium is a common ocular surface disease with an unknown etiology and threatens vision as it invades into the cornea. Circular RNAs (circRNAs) are a novel class of RNA transcripts that participate in several physiological and pathological processes. However, the role of circRNAs in pathogenesis of pterygium remains largely unknown. METHODS: Genome-wide circRNA expression profiling was performed to identify pterygium -related circRNAs. GO analysis, pathway analysis, and miRNA response elements analysis was performed to predict the function of differentially expressed circRNAs in pterygium. MTT assays, Ki67 staining, Transwell assay, Hoechst 33342 staining, and Calcein-AM/PI staining were performed to determine the effect of circRNA silencing on pterygium fibroblast and epithelial cell function. RESULTS: Approximately 669 circRNAs were identified to be abnormally expressed in pterygium tissues. GO analysis demonstrated that the host genes of differentially expressed circRNAs were targeted to extracellular matrix organization (ontology: biological process), cytoplasm (ontology: cellular component), and protein binding (ontology: molecular function). Pathway analysis showed that dysregulated circRNAs-mediated regulatory networks were mostly enriched in focal adhesion signaling pathway. Notably, circ_0085020 (circ-LAPTM4B) was shown as a potential biomarker for pterygium. circ_0085020 (circ-LAPTM4B) silencing affected the viability, proliferation, migration, and apoptosis of pterygium fibroblast and epithelial cells in vitro. CONCLUSIONS: This study provides evidence that circRNAs are involved in the pathogenesis of pterygium and might constitute promising targets for the therapeutic intervention of pterygium.

Neuroligin-3 protects retinal cells from H2O2-induced cell death via activation of Nrf2 signaling.

Intensified oxidative stress can cause severe damage to human retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs). The potential effect of neuroligin-3 (NLGN3) against the process is studied here. Our results show that NLGN3 efficiently inhibited hydrogen peroxide (H2O2)-induced death and apoptosis in human RPE cells and RGCs. H2O2-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage in retinal cells were alleviated by NLGN3. NLGN3 activated nuclear-factor-E2-related factor 2 (Nrf2) signaling, enabling Nrf2 protein stabilization, nuclear translocation and expression of key anti-oxidant enzymes (HO1, NOQ1 and GCLC) in RPE cells and RGCs. Further results demonstrate that NLGN3 activated Akt-mTORC1 signaling in retinal cells. Conversely, Akt-mTORC1 inhibitors (RAD001 and LY294002) reduced NLGN3-induced HO1, NOQ1 and GCLC mRNA expression. Significantly, Nrf2 silencing by targeted shRNAs reversed NLGN3-induced retinal cytoprotection against H2O2. We conclude that NLGN3 activates Nrf2 signaling to protect human retinal cells from H2O2. NLGN3 could be further tested as a valuable retinal protection agent.

Long non-coding RNA MEG3 silencing protects against light-induced retinal degeneration.

Excessive light exposure leads to retinal degeneration and accelerates the progression and severity of several ocular diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa. Long non-coding RNAs (LncRNAs) have emerged as important regulators of photoreceptor development and ocular diseases. In this study, we investigated the role of lncRNA-MEG3 in light-induced retinal degeneration. MEG3 expression was significantly up-regulated after light insult in vivo and in vitro. MEG3 silencing protected against light-induced retinal degeneration in vivo and light-induced photoreceptor cell apoptosis in vitro. Mechanistically, MEG3 regulated retinal photoreceptor cell function by acting as p53 decoy. MEG3 silencing decreased caspase 3/7 activity, up-regulated anti-apoptotic protein (Bcl-2) expression, and down-regulated pro-apoptotic protein (Bax) expression. Taken together, this study provides a promising method of MEG3 silencing for treating light-induced retinal degeneration.

lncRNA-MIAT regulates microvascular dysfunction by functioning as a competing endogenous RNA.

RATIONALE: Pathological angiogenesis is a critical component of diseases, such as ocular disorders, cancers, and atherosclerosis. It is usually caused by the abnormal activity of biological processes, such as cell proliferation, cell motility, immune, or inflammation response. Long noncoding RNAs (lncRNAs) have emerged as critical regulators of these biological processes. However, the role of lncRNA in diabetes mellitus-induced microvascular dysfunction is largely unknown. OBJECTIVE: To elucidate whether lncRNA-myocardial infarction-associated transcript (MIAT) is involved in diabetes mellitus-induced microvascular dysfunction. METHODS AND RESULTS: Using quantitative polymerase chain reaction, we demonstrated increased expression of lncRNA-MIAT in diabetic retinas and endothelial cells cultured in high glucose medium. Visual electrophysiology examination, TUNEL staining, retinal trypsin digestion, vascular permeability assay, and in vitro studies revealed that MIAT knockdown obviously ameliorated diabetes mellitus-induced retinal microvascular dysfunction in vivo, and inhibited endothelial cell proliferation, migration, and tube formation in vitro. Bioinformatics analysis, luciferase assay, RNA immunoprecipitation, and in vitro studies revealed that MIAT functioned as a competing endogenous RNA, and formed a feedback loop with vascular endothelial growth factor and miR-150-5p to regulate endothelial cell function. CONCLUSIONS: This study highlights the involvement of lncRNA-MIAT in pathological angiogenesis and facilitates the development of lncRNA-directed diagnostics and therapeutics against neovascular diseases.

Nmnat 1: a Security Guard of Retinal Ganglion Cells (RGCs) in Response to High Glucose Stress.

BACKGROUND/AIMS: Retinal neurodegeneration is an early event in the pathological process of diabetic retinopathy (DR). Retinal ganglion cell (RGC) injury is an important pathological feature during neurodegenerative process. Protecting RGCs from high glucose-induced injury is a promising strategy for delaying or hindering diabetes mellitus-related retinal neuropathy. This study aims to investigate the role of Nmnat1, an enzyme which catalyzes a key step in the biosynthesis of nicotinamide adenine dinucleotide (NAD), in high glucose-induced RGC injury. METHODS: Western blot and immunofluorescence analysis was conducted to detect Nmnat1 expression pattern in the retina and RGC-5 cell. MTT assay, Hoechst staining, trypan blue staining, and calcein-AM/ propidium iodide (PI) staining was conducted to determine the effect of Nmnat1 knockdown on RGC-5 cell function. Microarray and bioinformatics analysis was conducted to identify potential signaling pathways affected by Nmnat1 knockdown. Pharmacological intervention, molecular intervention, and in vitro experiments were conducted to reveal molecular mechanism of Nmnat1-mediated protective effect on RGC-5 cell function. RESULTS: Nmnat1 is constitutively expressed in retina and RGC-5 cells. Nmnat1 knockdown aggravates RGC injury, and accelerates the development of RGC-5 cell apoptosis upon high glucose stress. MAPK signaling is the primary signaling pathway affected by Nmnat1 knockdown. Under high glucose stress, Nmnat1 knockdown leads to p38-MAPK signaling inactivation. p38-MAPK pathway inhibitor strongly blocks Nmnat1-mediated protective effect on RGC-5 cell function. CONCLUSION: Nmnat1 protects RGC against high glucose-induced injury via p38-MAPK signaling pathway. Nmnat1 may serve as a neuroprotective target for diabetes mellitus-related retinal neuropathy.

Regulation of autophagy by high glucose in human retinal pigment epithelium.

BACKGROUND: Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Retinal pigment epithelium (RPE) works as the outer blood retina barrier and is vulnerable to energy stress-induced injury. However, the effect of high glucose treatment on autophagy is still unclear in RPE. METHODS: Transmission electron microscopy was used to detect the generation of autophagosome. Small interfering RNA (siRNA) and MTT was used to determine the effect of autophagy on cell viability. Western blots and immunohistochemistry were used to detect the expression pattern of autophagic markers, including LC3 and p62. RESULTS: High glucose treatment results in a significant increase in the generation of autophagosome and altered expression of LC3 and p62. High glucose-induced autophagy is independent of mTOR signaling, but is mainly regulated via ROS-mediated ER stress signaling. CONCLUSION: In the scenario of high glucose-induced oxidative stress, autophagy may be required for the removal of damaged proteins, and provide a default mechanism to prevent high glucose-induced injury in RPE.

Long non-coding RNAs: new players in ocular neovascularization.

Pathological neovascularization are the most prevalent causes of moderate or severe vision loss. Long non-coding RNAs (lncRNAs) have emerged as a novel class of regulatory molecules involved in numerous biological processes and complicated diseases. However, the role of lncRNAs in ocular neovascularization is still unclear. Here, we constructed a murine model of ocular neovascularization, and determined lncRNA expression profiles using microarray analysis. We identified 326 or 51 lncRNAs that were significantly either up-regulated or down-regulated in the vaso-obliteration or neovascularization phase, respectively. Based on Pearson correlation analysis, lncRNAs/mRNAs co-expression networks were constructed. GO enrichment analysis of lncRNAs-co-expressed mRNAs indicated that the biological modules were correlated with chromosome organization, extracellular region and guanylate cyclase activator activity in the vaso-obliteration phase, and correlated with cell proliferation, extracellular region and guanylate cyclase regulator activity in the neovascularization phase. KEGG pathway analysis indicated that MAPK signaling was the most significantly enriched pathway in both phases. Importantly, Vax2os1 and Vax2os2 were not only dynamically expressed in the vaso-obliteration and neovascularization phases, but also significantly altered in the aqueous humor of patients with neovascular age-related macular degeneration (AMD), suggesting a potential role of lncRNAs in the regulation of ocular neovascularization. Taken together, this study provided novel insights into the molecular pathogenesis of ocular neovascularization. The intervention of dysregulated lncRNA could become a potential target for the prevention and treatment of ocular vascular diseases.

Abnormal glutamate metabolism in the retina of aquaporin 4 (AQP4) knockout mice upon light damage.

Glutamate is a major excitatory neurotransmitter in the retina. Glutamate neurotoxicity has been implicated in the pathogenesis of several ocular diseases. Aquaporin 4 (AQP4) is a water-selective membrane transport protein, and its knockout could alter retinal neuron excitability. However, the effect of AQP4 knockout on glutamate metabolism is still unclear in the retina. Here, we reported that the retinas in AQP4 knockout mice showed higher glutamate levels than that in wild-type mice upon light damage. AQP4 knockout could result in accelerated apoptosis of retinal cells, increased reactive gliosis, and attenuated survival of RGCs in response to light damage. Moreover, AQP4 knockout could affect the expression pattern of glutamate metabolism-related proteins such as GLAST and GS. Taken together, this study revealed a novel role of AQP4 in regulating glutamate metabolism. Pharmacological manipulation of AQP4 function may represent as a potent therapeutic target in the treatment of neurological ocular disorders.

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.

Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: a potential role for reducing UVB light-induced retinal damage.

Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

Targeting circular RNA-Glra2 alleviates retinal neurodegeneration induced by ocular hypertension.

Glaucoma is a leading cause of irreversible vision loss characterized by retinal neurodegeneration. Circular RNAs (circRNAs) have emerged as the potential biomarkers and therapeutic targets for neurodegenerative diseases. However, the expression profiling of circRNAs in glaucomatous neurodegeneration has not been fully understood. In this study, we built a glaucomatous neurodegeneration model via the injection of microbeads into anterior chamber. circRNA expression profile and bioinformatics analysis revealed that compared with normal retinas, 171 circRNAs were dysregulated in the glaucomatous retinas, including 101 up-regulated circRNAs and 70 down-regulated circRNAs. Detecting the level of circular RNA-glycine receptor α2 subunit gene (cGlra2) in aqueous humor made it possible to distinguish glaucoma patients from cataract patients. Silencing of cGlra2 protected against oxidative stress- or hydrostatic pressure-induced retinal ganglion cell (RGC) injury in vitro. Moreover, silencing of cGlra2 retarded ocular hypertension-induced retinal neurodegeneration in vivo as shown by increased TUJ1 staining, reduced reactive gliosis, decreased retinal cell apoptosis, enhanced visual acuity, and improved retinal function. cGlra2 acted as a miRNA sponge to regulate RGC function through cGlra2/miR-144/BCL2L11 signaling axis. Collectively, this study provides novel insights into the underlying mechanism of retinal neurodegeneration and highlights the potential of cGlra2 as a target for the diagnosis and treatment of glaucoma.

Single-cell RNA sequencing reveals a unique pericyte type associated with capillary dysfunction.

Background: Capillary dysfunction has been implicated in a series of life- threatening vascular diseases characterized by pericyte and endothelial cell (EC) degeneration. However, the molecular profiles that govern the heterogeneity of pericytes have not been fully elucidated. Methods: Single-cell RNA sequencing was conducted on oxygen-induced proliferative retinopathy (OIR) model. Bioinformatics analysis was conducted to identify specific pericytes involved in capillary dysfunction. qRT-PCRs and western blots were conducted to detect Col1a1 expression pattern during capillary dysfunction. Matrigel co-culture assays, PI staining, and JC-1 staining was conducted to determine the role of Col1a1 in pericyte biology. IB4 and NG2 staining was conducted to determine the role of Col1a1 in capillary dysfunction. Results: We constructed an atlas of > 76,000 single-cell transcriptomes from 4 mouse retinas, which could be annotated to 10 distinct retinal cell types. Using the sub-clustering analysis, we further characterized retinal pericytes into 3 different subpopulations. Notably, GO and KEGG pathway analysis demonstrated that pericyte sub-population 2 was identified to be vulnerable to retinal capillary dysfunction. Based on the single-cell sequencing results, Col1a1 was identified as a marker gene of pericyte sub-population 2 and a promising therapeutic target for capillary dysfunction. Col1a1 was abundantly expressed in pericytes and its expression was obviously upregulated in OIR retinas. Col1a1 silencing could retard the recruitment of pericytes toward endothelial cells and aggravated hypoxia-induced pericyte apoptosis in vitro. Col1a1 silencing could reduce the size of neovascular area and avascular area in OIR retinas and suppressed pericyte-myofibroblast transition and endothelial-mesenchymal transition. Moreover, Col1a1 expression was up-regulated in the aqueous humor of the patients with proliferative diabetic retinopathy (PDR) or retinopathy of prematurity (ROP) and up-regulated in the proliferative membranes of PDR patients. Conclusions: These findings enhance the understanding of the complexity and heterogeneity of retinal cells and have important implications for future treatment of capillary dysfunction.