Non-Coding RNAs: Novel Regulators of Macrophage Homeostasis in Ocular Vascular Diseases.

Ocular neovascularization can impair vision and threaten patients' quality of life. However, the underlying mechanism is far from transparent. In all mammals, macrophages are a population of cells playing pivotal roles in the innate immune system and the first line of defense against pathogens. Therefore, it has been speculated that the disfunction of macrophage homeostasis is involved in the development of ocular vascular diseases. Moreover, various studies have found that non-coding RNAs (ncRNAs) regulate macrophage homeostasis. This study reviewed past studies of the regulatory roles of ncRNAs in macrophage homeostasis in ocular vascular diseases.

Single-cell profiling transcriptomic reveals cellular heterogeneity and cellular crosstalk in choroidal neovascularization model.

Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (nAMD) and a major contributor to vision loss in nAMD cases. However, the identification of specific cell types associated with nAMD remains challenging. Herein, we performed single-cell sequencing to comprehensively explore the cellular diversity and understand the foundational components of the retinal pigment epithelium (RPE)/choroid complex. We unveiled 10 distinct cell types within the RPE/choroid complex. Notably, we observed significant heterogeneity within endothelial cells (ECs), fibroblasts, and macrophages, underscoring the intricate nature of the cellular composition in the RPE/choroid complex. Within the EC category, four distinct clusters were identified and EC cluster 0 was tightly associated with choroidal neovascularization. We identified five clusters of fibroblasts actively involved in the pathogenesis of nAMD, influencing fibrotic responses, angiogenic effects, and photoreceptor function. Additionally, three clusters of macrophages were identified, suggesting their potential roles in regulating the progression of nAMD through immunomodulation and inflammation regulation. Through CellChat analysis, we constructed a complex cell-cell communication network, revealing the role of EC clusters in interacting with fibroblasts and macrophages in the context of nAMD. These interactions were found to govern angiogenic effects, fibrotic responses, and inflammatory processes. In summary, this study reveals noteworthy cellular heterogeneity in the RPE/choroid complex and provides valuable insights into the pathogenesis of CNV. These findings will open up potential avenues for deep understanding and targeted therapeutic interventions in nAMD.

Indirubin alleviates retinal neurodegeneration through the regulation of PI3K/AKT signaling.

Retinal neurodegenerative disease is a leading cause of blindness among the elderly in developed countries, including glaucoma, diabetic retinopathy, traumatic optic neuropathy and optic neuritis, etc. The current clinical treatment is not very effective. We investigated indirubin, one of the main bioactive components of the traditional Chinese medicine Danggui Longhui Pill, in the present study for its role in retinal neurodegeneration. Indirubin exhibited no detectable tissue toxicity in vivo or cytotoxicity in vitro. Moreover, indirubin improved visual function and ameliorated retinal neurodegeneration in mice after optic nerve crush injury in vivo. Furthermore, indirubin reduced the apoptosis of retinal ganglion cells induced by oxidative stress in vitro. In addition, indirubin significantly suppressed the increased production of intracellular reactive oxygen species and the decreased activity of superoxide dismutase induced by oxidative stress. Mechanically, indirubin played a neuroprotective role by regulating the PI3K/AKT/BAD/BCL-2 signaling. In conclusion, indirubin protected retinal ganglion cells from oxidative damage and alleviated retinal neurodegeneration induced by optic nerve crush injury. The present study provides a potential therapeutic medicine for retinal neurodegenerative diseases.

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.

Hyperglycemia-regulated tRNA-derived fragment tRF-3001a propels neurovascular dysfunction in diabetic mice.

Neurovascular dysfunction is a preclinical manifestation of diabetic complications, including diabetic retinopathy (DR). Herein, we report that a transfer RNA-derived RNA fragment, tRF-3001a, is significantly upregulated under diabetic conditions. tRF-3001a downregulation inhibits Müller cell activation, suppresses endothelial angiogenic effects, and protects against high-glucose-induced retinal ganglion cell injury in vitro. Furthermore, tRF-3001a downregulation alleviates retinal vascular dysfunction, inhibits retinal reactive gliosis, facilitates retinal ganglion cell survival, and preserves visual function and visually guided behaviors in STZ-induced diabetic mice and db/db diabetic mice. Mechanistically, tRF-3001a regulates neurovascular dysfunction in a microRNA-like mechanism by targeting GSK3B. Clinically, tRF-3001a is upregulated in aqueous humor (AH) samples of DR patients. tRF-3001a downregulation inhibits DR-induced human retinal vascular endothelial cell and Müller cell dysfunction in vitro and DR-induced retinal neurovascular dysfunction in C57BL/6J mice. Thus, targeting tRF-3001a-mediated signaling is a promising strategy for the concurrent treatment of vasculopathy and neuropathy in diabetes mellitus.

EYE-503: A Novel Retinoic Acid Drug for Treating Retinal Neurodegeneration.

Retinal neurodegeneration is a major cause of vision loss. Retinoic acid signaling is critical for the maintenance of retinal function, and its dysfunction can cause retinal neurodegeneration. However, the therapeutic effects of retinoic acid drugs on retinal neurodegeneration remain unclear. In this study, we designed a novel retinoic acid drug called EYE-503 and investigated its therapeutic effects of EYE-503 on retinal neurodegeneration. The optic nerve crush (ONC) model was selected for the retinal neurodegeneration study. H&E staining, TUNEL staining, immunofluorescence staining, and visual electrophysiology assays were performed to determine the role of EYE-503 in retinal neurodegeneration in vivo. The CCK-8 assay, EdU incorporation assay, PI staining, and flow cytometry assays were performed to investigate the effects of EYE-503 administration on retinal neurodegeneration in vitro. The potential mechanism of EYE-503 in retinal neurodegeneration was investigated by network pharmacology and Western blots. The results showed that EYE-503 administration had no detectable cytotoxicity and tissue toxicity. EYE-503 administration alleviated ONC-induced retinal injury and optic nerve injury in vivo. EYE-503 administration attenuated retinal ganglion cell apoptosis, inhibited reactive gliosis, and retarded the progression of retinal neurodegeneration. Mechanistically, EYE-503 regulated retinal neurodegeneration by targeting the JNK/p38 signaling pathway. This study suggests that EYE-503 is a promising therapeutic agent for retinal neurodegenerative diseases.

A novel retinoic acid drug, EYE-502, inhibits choroidal neovascularization by targeting endothelial cells and pericytes.

Choroidal neovascularization (CNV) occurs in neovascular age-related macular degeneration (AMD) and often leads to permanent visual impairment. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is the gold standard for the treatment of CNV. However, anti-VEGF treatment did not always cause vision improvement and sometimes had detrimental effects on normal retinal tissues. Herein, we identified a novel retinoic acid drug, EYE-502, which had great therapeutic effects on CNV. Administration of EYE-502 could inhibit VEGF-induced dysfunction of endothelial cells (ECs) and reduce platelet-derived growth factor (PDGF)-induced recruitment of pericytes to ECs in vitro. Administration of EYE-502 could reduce the area of choroidal sprouting and laser-induced CNV, exhibiting similar anti-angiogenic effects as aflibercept. Moreover, administration of EYE-502 could reduce pericyte coverage in the sprouting vessels and choroidal neovascularization. Mechanistically, EYE-502 primarily bound to retinoic acid receptors (RARs) and exerted the anti-angiogenic effects by targeting ECs and pericytes via affecting the activation of Wnt/ß-catenin and PDGF/PDGFR/PI3K/Akt signaling. Taken together, this study reports a novel retinoic acid drug, EYE-502, which can exert the anti-angiogenic effects by simultaneous targeting of ECs and pericytes.

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.

A Novel PAX6 Frameshift Mutation Identified in a Large Chinese Family with Congenital Aniridia.

Congenital aniridia is a rare autosomal dominant congenital ocular disorder. Genetic studies suggest that heterozygous mutations in the developmental regulator PAX6 gene or the related regulatory regions leading to haploinsufficiency are the main cause of congenital aniridia. In this study, the clinical characteristics and pathogenic mutation of a four-generation Chinese family with congenital aniridia were investigated. All members recruited in this study underwent comprehensive ophthalmic examinations. Targeted gene capture sequencing and Sanger sequencing were performed to screen and confirm the candidate pathogenicity gene and its mutation. A multiple alignment of homologous sequences covering the identified mutation from different species was investigated, and the mutant protein structure was predicted using Swiss-Model. Additionally, the prediction of pathogenicity was analyzed using the ACMG Guidelines. Thirteen patients in this pedigree were diagnosed with congenital aniridia. A novel heterozygous frameshift mutation (c.391_398dupATACCAAG, p.Ser133Argfs*8) in exon 7 of the PAX6 gene was identified in all affected individuals in the family. This study demonstrates that this frameshift mutation of the PAX6 gene might be the causative genetic defect of congenital aniridia in this family. This mutation is predicted to cause the premature truncation of the PAX6 protein, leading to the functional haploinsufficiency of PAX6, which may be the major molecular mechanism underlying the aniridia phenotype. To the best of our knowledge, this is the first report of a novel pathogenic PAX6 gene variant c.391_398dupATACCAAG(p.Ser133Argfs*8) identified in a Chinese family with congenital aniridia.

Regulatory effect of long-stranded non-coding RNA-CRNDE on neurodegeneration during retinal ischemia-reperfusion.

Ischemia/reperfusion (I/R) injury is a common pathological mechanism involved in many ocular diseases. I/R is characterized by microvascular dysfunction and neurodegeneration. However, the mechanisms of neurodegeneration induced by I/R remain largely unknown. This study showed that the expression of long non-coding RNA-CRNDE was significantly upregulated after retinal ischemia-reperfusion (RIR). LncRNA-CRNDE knockdown alleviated retinal neurodegeneration induced by RIR injury, as shown by decreased reactive gliosis and reduced retinal cells loss. Furthermore, lncRNA-CRNDE knockdown directly regulated Müller cell function and indirectly affected RGC function in vitro. In addition, lncRNA-CRNDE knockdown led to a significant reduction in the release of several cytokines after RIR. This study suggests that lncRNA-CRNDE is a promising therapeutic target for RIR.

Long Non-Coding RNA PNKY Modulates the Development of Choroidal Neovascularization.

Long non-coding RNAs (lncRNAs) have been widely implicated in human diseases. Our aim was to explore the regulatory role of changes in the expression levels of PNKY and its linked signaling networks in mediating stress-induced choroidal neovascularization. PNKY expression levels were reduced in mice by laser and exposure of endothelial cell to hypoxic stress. PNKY silencing exacerbated the formation of CNV in a laser-induced CNV model and an ex vivo model, while overexpression inhibited CNV development. Silencing or overexpression of PNKY altered the viability, proliferation, migration, and tube-forming capacity of endothelial cells in vitro. Mechanistically, through the lncRNA-RNA binding protein-miRNA interaction analysis involving loss of function and gain-of-function experiments, we found that lncRNA PNKY inhibited the binding of miR124 to PTBP1 and maintained the homeostasis of choroidal vascular function by promoting Bcl-2 like protein 11 (BIM), and its dysfunction led to exacerbation of CNV lesion. Therefore, this study suggests that the lncPNKY/PTBP1-miR-124 axis is involved in regulating the development of CNV, providing a potential therapeutic target for the treatment of CNV.

Targeting long noncoding RNA-AQP4-AS1 for the treatment of retinal neurovascular dysfunction in diabetes mellitus.

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in the working-age population, which is characterized by retinal neurodegeneration and vascular dysfunction. Long non-coding RNAs (LncRNAs) have emerged as critical regulators in several biological processes and disease progression. Here we investigated the role of lncRNA AQP4-AS1 in retinal neurovascular dysfunction induced by diabetes. METHODS: Quantitative RT-PCR was used to detect the AQP4-AS1 expression pattern upon diabetes mellitus-related stresses. Visual electrophysiology examination, TUNEL staining, Evans blue staining, retinal trypsin digestion and immunofluorescent staining were conducted to detect the role of AQP4-AS1 in retinal neurovascular dysfunction in vivo. MTT assays, TUNEL staining, PI/Calcein-AM staining, EdU incorporation assay transwell assay and tube formation were conducted to detect the role of AQP4-AS1 in retinal cells function in vitro. qRT-PCR, western blot and in vivo studies were conducted to reveal the mechanism of AQP4-AS1-mediated retinal neurovascular dysfunction. FINDINGS: AQP4-AS1 was significantly increased in the clinical samples of diabetic retinopathy patients, high glucose-treated Müller cells, and diabetic retinas of a murine model. AQP4-AS1 silencing in vivo alleviated retinal neurodegeneration and vascular dysfunction as shown by improved retinal capillary degeneration, decreased reactive gliosis, and reduced RGC loss. AQP4-AS1 directly regulated Müller cell function and indirectly affected endothelial cell and RGC function in vitro. Mechanistically, AQP4-AS1 regulated retinal neurovascular dysfunction through affecting AQP4 levels. INTERPRETATION: This study reveals AQP4-AS1 is involved in retinal neurovascular dysfunction and expected to become a promising target for the treatment of neurovascular dysfunction in DR. FUNDING: This work was generously supported by the grants from the National Natural Science Foundation of China (Grant No. 81800858, 82070983, 81870679 and 81970823), grants from the Medical Science and Technology Development Project Fund of Nanjing (Grant No ZKX17053 and YKK19158), grants from Innovation Team Project Fund of Jiangsu Province (No. CXTDB2017010), and the Science and Technology Development Plan Project Fund of Nanjing (Grant No 201716007, 201805007 and 201803058).

Suppression of choroidal neovascularization by silencing of long non-coding RNA IPW.

Long noncoding RNAs (lncRNAs) have emerged as the key regulators in the pathogenesis of human disorders. This study aimed to investigate the role of lncRNA-IPW in the progression of choroidal neovascularization (CNV) and the underlying molecular mechanism. IPW was significantly up-regulated in the choroidal tissues of laser-induced CNV mice and in the endothelial cells in response to hypoxic stress. IPW silencing led to reduced formation of CNV in laser-induced CNV model and ex vivo choroidal sprouting model, which could achieve similar therapeutic effects of anti-VEGF on CNV formation. Silencing or transgenic overexpression of IPW could alter endothelial cell viability, proliferation, migration, and tube formation ability in vitro. Mechanistically, IPW silencing led to increased expression of miR-370. Increased miR-370 could mimic the effects of IPW silencing on CNV formation and endothelial angiogenic phenotypes in vivo and in vitro. This study suggests that IPW silencing is a promising strategy for the treatment of neovascular ocular diseases.

A small molecular multi-targeting tyrosine kinase inhibitor, anlotinib, inhibits pathological ocular neovascularization.

Ocular neovascularization is a devastating pathology observed in numerous ocular diseases and is a major cause of blindness. However, all current treatments have their limitations. Hence, it is important to explore new therapeutic strategies. This study aimed to investigate the role of anlotinib, a small molecular multi-targeting tyrosine kinase inhibitor, in ocular neovascularization. Anlotinib administration did not induce any cytotoxicity and tissue toxicity at the tested concentrations. Cellular functional experiments demonstrated that anlotinib inhibited the viability, proliferation, migration, and tube formation ability of endothelial cells (ECs) and pericytes. Western blot analysis demonstrated that anlotinib significantly inhibited the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and platelet-derived growth factor receptor ß (PDGFR-ß), as well as their downstream signaling pathways stimulated by VEGF or PDGF-BB, in a concentration-dependent manner in ECs and pericytes. Using an oxygen-induced retinopathy (OIR) model, our results demonstrated that injection of anlotinib reduced avascular areas and pathological neovascular tufts. Furthermore, using a laser-induced choroidal neovascularization (CNV) model, we observed that the combined treatment of anlotinib and Lucentis reduced the size and thickness of CNV lesions compared to Lucentis monotherapy alone. Taken together, our results suggest that anlotinib could be a promising drug candidate for ocular neovascularization.

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.

A Joint Model for Macular Edema Analysis in Optical Coherence Tomography Images Based on Image Enhancement and Segmentation.

Optical coherence tomography (OCT) provides the visualization of macular edema which can assist ophthalmologists in the diagnosis of ocular diseases. Macular edema is a major cause of vision loss in patients with retinal vein occlusion (RVO). However, manual delineation of macular edema is a laborious and time-consuming task. This study proposes a joint model for automatic delineation of macular edema in OCT images. This model consists of two steps: image enhancement using a bioinspired algorithm and macular edema segmentation using a Gaussian-filtering regularized level set (SBGFRLS) algorithm. We then evaluated the delineation efficiency using the following parameters: accuracy, precision, sensitivity, specificity, Dice's similarity coefficient, IOU, and kappa coefficient. Compared with the traditional level set algorithms, including C-V and GAC, the proposed model had higher efficiency in macular edema delineation as shown by reduced processing time and iteration times. Moreover, the accuracy, precision, sensitivity, specificity, Dice's similarity coefficient, IOU, and kappa coefficient for macular edema delineation could reach 99.7%, 97.8%, 96.0%, 99.0%, 96.9%, 94.0%, and 96.8%, respectively. More importantly, the proposed model had comparable precision but shorter processing time compared with manual delineation. Collectively, this study provides a novel model for the delineation of macular edema in OCT images, which can assist the ophthalmologists for the screening and diagnosis of retinal diseases.

Retina as a window to cerebral dysfunction following studies with circRNA signature during neurodegeneration.

Ischemia-induced cerebral injury is a major cause of dementia or death worldwide. The pre-diagnosis is still challenging due to the retarded symptoms. The retina is regarded as the extension of cerebral tissue. Circular RNAs have emerged as the crucial regulators in gene regulatory network and disease progression. However, it is still unknown whether circRNAs can be used as the common regulators and diagnostic markers for cerebral neurodegeneration and retinal neurodegeneration. Methods: C57BL/6J mice were subjected to transient middle cerebral artery occlusion and circRNA microarray profiling was performed to identify neurodegeneration-related circRNAs. Quantitative reverse-transcription PCR (qRT-PCR) assays were performed to verify circRNA expression pattern. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to determine the biologic modules and signaling pathway. TTC staining, Nissl's staining, and immunofluorescence staining assays were performed to investigate the role of circRNA in cerebral neurodegeneration and retinal neurodegeneration in vivo. MTT assay, Propidium iodide (PI)/Calcein-AM staining, and Rhodamine 123 assays were performed to investigate the role of circRNA in neuronal injury in vitro. Bioinformatics, RIP, and luciferase activity assays were performed to determine the regulatory mechanism of circRNA in neurodegeneration. Results: 217 differentially expressed circRNAs were identified between ischemic cerebral tissues and normal controls. Among them, cGLIS3 was shown as the common regulator of cerebral neurodegeneration and retinal neurodegeneration. cGLIS3 silencing alleviated ischemia-induced retinal neurodegeneration and MCAO-induced cerebral neurodegeneration in vivo. cGLIS3 silencing protected against OGD/R-induced RGC injury in vitro. The circulating levels of cGLIS3 were significantly increased in the patients with ischemic stroke compared to healthy subjects. cGLIS3 levels were also increased in the aqueous humor of the patients with retinal vein occlusion. cGLIS3 regulated neuronal cell injury by acting as miR-203 sponge and its level was controlled by EIF4A3. Conclusions: This study provides molecular evidence that the retina is window of the brain from circRNA perspective. cGLIS3 is a common regulator and diagnostic marker of cerebral neurodegeneration and retinal neurodegeneration.