FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Exosomal lncRNA-MIAT promotes neovascularization via the miR-133a-3p/MMP-X1 axis in diabetic retinopathy.

Diabetic retinopathy (DR), a most common microangiopathy of diabetes, causes vision loss and even blindness. The mechanisms of exosomal lncRNA remain unclear in the development of DR. Here, we first identifed the pro-angiogenic effect of exosomes derived from vitreous humor of proliferative diabetic retinopathy patients, where lncRNA-MIAT was enriched inside. Secondly, lncRNA-MIAT was demonstrated significantly increased in exosomes from high glucose induced human retinal vascular endothelial cell, and can regulate tube formation, migration and proliferation ability to promote angiogenesis in vitro and in vivo. Mechanistically, the pro-angiogenic effect of lncRNA-MIAT was via the lncRNA-MIAT/miR-133a-3p/MMP-X1 axis. The reduced level of lncRNA-MIAT in this axis mitigated the generation of retinal neovascular in mouse model of oxygen-induced retinopathy (OIR), providing crucial evidence for lncRNA-MIAT as a potential clinical target. These findings enhance our understanding of the role of exosomal lncRNA-MIAT in retinal angiogenesis, and propose a promising therapeutic strategy against diabetic retinopathy.

SOCS3 regulates pathological retinal angiogenesis through modulating SPP1 expression in microglia and macrophages.

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.

Role of mRNA-binding proteins in retinal neovascularization.

Retinal neovascularization (RNV) is a pathological process that primarily occurs in diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. It is a common yet debilitating clinical condition that culminates in blindness. Urgent efforts are required to explore more efficient and less limiting therapeutic strategies. Key RNA-binding proteins (RBPs), crucial for post-transcriptional regulation of gene expression by binding to RNAs, are closely correlated with RNV development. RBP-RNA interactions are altered during RNV. Here, we briefly review the characteristics and functions of RBPs, and the mechanism of RNV. Then, we present insights into the role of the regulatory network of RBPs in RNV. HuR, eIF4E, LIN28B, SRSF1, METTL3, YTHDF1, Gal-1, HIWI1, and ZFR accelerate RNV progression, whereas YTHDF2 and hnRNPA2B1 hinder it. The mechanisms elucidated in this review provide a reference to guide the design of therapeutic strategies to reverse abnormal processes.

Differences in Oxygen-Induced Retinopathy Susceptibility Between Two Sprague Dawley Rat Vendors: A Comparison of Retinal Transcriptomes.

PURPOSE: To determine the retinal transcriptomic differences underlying the oxygen-induced retinopathy phenotypes between Sprague Dawley rat pups from two commonly used commercial vendors. This will allow us to discover genes and pathways that may be related to differences in disease severity in similarly aged premature babies and suggest possible new treatment approaches. METHODS: We analyzed retinal vascular morphometry and transcriptomes from Sprague Dawley rat pups from Charles River Laboratories and Envigo (previously Harlan). Room air control and oxygen-induced retinopathy groups were compared. Oxygen-induced retinopathy was induced with the rat 50/10 model. RESULTS: Pups from Charles River Laboratories developed a more severe oxygen-induced retinopathy phenotype, with 3.6-fold larger percent avascular area at P15 and twofold larger % neovascular area at P20 than pups from Envigo. Changes in retinal transcriptomes of rat pups from both vendors were substantial at baseline and in response to oxygen-induced retinopathy. Baseline differences centered on activated pathways of neuronal development in Charles River Laboratories pups. In response to oxygen-induced retinopathy, during the neovascular phase, retinas from Charles River Laboratories pups exhibited activation of pathways regulating necrosis, neuroinflammation, and interferon signaling, supporting the observed increase of neovascularization. Conversely, retinas from Envigo pups showed decreased necrosis and increased focal adhesion kinase signaling, supporting more normal vascular development. Comparing oxygen-induced retinopathy transcriptomes at P15 to those at P20, canonical pathways such as phosphate and tensin homolog, interferon, and coordinated lysosomal expression and regulation element signaling were identified, highlighting potential novel mechanistic targets for future research. CONCLUSION: Transcriptomic profiles differ substantially between rat pup retinas from Charles River Laboratories and Envigo at baseline and in response to oxygen-induced retinopathy, providing insight into vascular morphologic differences. Comparing transcriptomes identified new pathways for further research in oxygen-induced retinopathy pathogenesis and increased scientific rigor of this model.

Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage.

BACKGROUND: Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. METHODS: A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. RESULTS: We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. CONCLUSIONS: The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.

Transcriptional Comparison of Human and Murine Retinal Neovascularization.

Purpose: Retinal neovascularization (RNV) is the leading cause of vision loss in diseases like proliferative diabetic retinopathy (PDR). A significant failure rate of current treatments indicates the need for novel treatment targets. Animal models are crucial in this process, but current diabetic retinopathy models do not develop RNV. Although the nondiabetic oxygen-induced retinopathy (OIR) mouse model is used to study RNV development, it is largely unknown how closely it resembles human PDR. Methods: We therefore performed RNA sequencing on murine (C57BL/6J) OIR retinas (n = 14) and human PDR RNV membranes (n = 7) extracted during vitrectomy, each with reference to control tissue (n=13/10). Differentially expressed genes (DEG) and associated biological processes were analyzed and compared between human and murine RNV to assess molecular overlap and identify phylogenetically conserved factors. Results: In total, 213 murine- and 1223 human-specific factors were upregulated with a small overlap of 94 DEG (7% of human DEG), although similar biological processes such as angiogenesis, regulation of immune response, and extracellular matrix organization were activated in both species. Phylogenetically conserved mediators included ANGPT2, S100A8, MCAM, EDNRA, and CCR7. Conclusions: Even though few individual genes were upregulated simultaneously in both species, similar biological processes appeared to be activated. These findings demonstrate the potential and limitations of the OIR model to study human PDR and identify phylogenetically conserved potential treatment targets for PDR.

RhoA/ROCK Signaling Is Involved in Pathological Retinal Neovascularization.

OBJECTIVE: The aim of the study was to evaluate the effect of the RhoA/ROCK inhibitor Fasudil on retinal neovascularization (NV) in vivo and angiogenesis in vitro. METHODS: C57BL/6 was used to establish an OIR model. First, RhoA/ROCK expression was first examined and compared between OIR and healthy controls. Then, we evaluated the effect of Fasudil on pathological retinal NV. Whole-mount retinal staining was performed. The percentage of NV area, the number of neovascular tufts (NVT), and branch points (BP) were quantified. Finally, human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of Fasudil on angiogenesis. RESULTS: Real-time PCR and Western blotting showed that ROCK expression in retinal tissue was statistically upregulated in OIR. Furthermore, we found that Fasudil attenuated the percentage of NV area, the number of NVT, and BP significantly. In addition, Fasudil could suppress the proliferation and migration of HUVECs induced by VEGF. CONCLUSIONS: RhoA/ROCK might be involved in the pathogenesis of OIR. And its inhibitor Fasudil could suppress retinal NV in vivo and angiogenesis in vitro. Fasudil may be a potential treatment strategy for retinal vascular diseases.

Retinal Angiomatous Proliferation in a Patient with Retinitis Pigmentosa.

Retinal angiomatous proliferation (RAP) and other types of choroidal neovascularization (CNV) are very rarely reported in patients with retinitis pigmentosa (RP). We present a case report of a 91-year-old patient with an obvious RP phenotype, who presented with a sudden onset of vision worsening and metamorphopsia in the left eye. Genetic testing on the UK inherited retinal disease panel did not identify a pathogenic variant. Multimodal imaging comprising optical coherence tomography (OCT), OCT angiography, and fluorescein and indocyanine green angiography showed a RAP lesion in the left macula. The patient received three treatments of monthly injections of aflibercept, with excellent morphological and functional outcomes. Taking into account the patient's age at presentation of the RAP lesion, it is not clear whether the RAP was associated or coincidental with RP. This case report highlights the importance of possessing an awareness that RAP lesions can occur in RP. Moreover, due to a good response and potential safety concerns with continuous anti-VEGF injections in RP patients, a pro re nata (PRN) regimen might be the safest option.

Bioinformatics Tools for Bulk Gene Expression Deconvolution in Diabetic Retinopathy.

Retinal neovascularization is one of the leading causes of vision loss and a hallmark of proliferative diabetic retinopathy (PDR). The immune system is observed to be involved in the pathogenesis of diabetic retinopathy (DR). The specific immune cell type that contributes to retinal neovascularization can be identified via a bioinformatics analysis of RNA sequencing (RNA-seq) data, known as deconvolution analysis. Previous study has identified the infiltration of macrophages in the retina of rats with hypoxia-induced retinal neovascularization and patients with PDR through a deconvolution algorithm, known as CIBERSORTx. Here, we describe the protocols of using CIBERSORTx to perform the deconvolution analysis and downstream analysis of RNA-seq data.

Galectin-1-dependent ceRNA network in HRMECs revealed its association with retinal neovascularization.

BACKGROUND: Retinal neovascularization (RNV) is a leading cause of blindness worldwide. Long non-coding RNA (lncRNA) and competing endogenous RNA (ceRNA) regulatory networks play vital roles in angiogenesis. The RNA-binding protein galectin-1 (Gal-1) participates in pathological RNV in oxygen-induced retinopathy mouse models. However, the molecular associations between Gal-1 and lncRNAs remain unclear. Herein, we aimed to explore the potential mechanism of action of Gal-1 as an RNA-binding protein. RESULTS: A comprehensive network of Gal-1, ceRNAs, and neovascularization-related genes was constructed based on transcriptome chip data and bioinformatics analysis of human retinal microvascular endothelial cells (HRMECs). We also conducted functional enrichment and pathway enrichment analyses. Fourteen lncRNAs, twenty-nine miRNAs, and eleven differentially expressed angiogenic genes were included in the Gal-1/ceRNA network. Additionally, the expression of six lncRNAs and eleven differentially expressed angiogenic genes were validated by qPCR in HRMECs with or without siLGALS1. Several hub genes, such as NRIR, ZFPM2-AS1, LINC0121, apelin, claudin-5, and C-X-C motif chemokine ligand 10, were found to potentially interact with Gal-1 via the ceRNA axis. Furthermore, Gal-1 may be involved in regulating biological processes related to chemotaxis, chemokine-mediated signaling, the immune response, and the inflammatory response. CONCLUSIONS: The Gal-1/ceRNA axis identified in this study may play a vital role in RNV. This study provides a foundation for the continued exploration of therapeutic targets and biomarkers associated with RNV.

Vascular cell-adhesion molecule 1 (VCAM-1) regulates JunB-mediated IL-8/CXCL1 expression and pathological neovascularization.

Vascular adhesion molecules play an important role in various immunological disorders, particularly in cancers. However, little is known regarding the role of these adhesion molecules in proliferative retinopathies. We observed that IL-33 regulates VCAM-1 expression in human retinal endothelial cells and that genetic deletion of IL-33 reduces hypoxia-induced VCAM-1 expression and retinal neovascularization in C57BL/6 mice. We found that VCAM-1 via JunB regulates IL-8 promoter activity and expression in human retinal endothelial cells. In addition, our study outlines the regulatory role of VCAM-1-JunB-IL-8 signaling on retinal endothelial cell sprouting and angiogenesis. Our RNA sequencing results show an induced expression of CXCL1 (a murine functional homolog of IL-8) in the hypoxic retina, and intravitreal injection of VCAM-1 siRNA not only decreases hypoxia-induced VCAM-1-JunB-CXCL1 signaling but also reduces OIR-induced sprouting and retinal neovascularization. These findings suggest that VCAM-1-JunB-IL-8 signaling plays a crucial role in retinal neovascularization, and its antagonism might provide an advanced treatment option for proliferative retinopathies.

Long Noncoding RNA PPT2-EGFL8 Regulates Pathological Retinal Neovascularization in PDR by Functioning as a Competing Endogenous RNA.

Diabetic retinopathy (DR) is a common complication in patients with diabetes, and proliferative DR (PDR) has become an important cause of blindness; however, the mechanisms involved have not been fully elucidated. miRNAs and long noncoding RNAs can play an important role in DR, and they can accurately regulate the expression of target genes through a new regulatory model: competing endogenous RNAs. We isolated total RNA of extracellular vesicles (EVs) in the serum of healthy individuals and individuals with diabetes without DR, non-PDR, or PDR, and performed deep sequencing. We found aberrantly low expression of PPT2-EGFL8 and significantly increased level of miR-423-5p. PPT2-EGFL8 adsorbs miR-423-5p as a molecular sponge and inhibits hypoxia-induced human retinal microvascular endothelial cells proliferation. In an oxygen-induced retinopathy (OIR) model and a streptozotocin-induced diabetes model, Egfl8-overexpression treatment reduces diabetes-related reactive gliosis, inflammation, and acellular capillaries and attenuates the development of pathological neovascularization. In addition, PPT2-EGFL8 targeting miR-423-5p plays an important role in hypoxia-induced peroxisome proliferator-activated receptor-ß/δ (PPARD)/angiopoietin-like 4 (ANGPTL4) signaling activation, especially the expression of the C-terminal ANGPTL4 fragment. Finally, ANGPTL4 significantly induces retinal vessel breakage in the inner limiting membrane and facilitates retinal vessel sprouting into the vitreous in the OIR mice. Thus, either new biomarkers or new therapeutic targets may be identified with translation of these findings.

BCL6B Contributes to Ocular Vascular Diseases via Notch Signal Silencing.

BACKGROUND: Endothelial cell activation is tightly controlled by the balance between VEGF (vascular endothelial cell growth factor) and Notch signaling pathway. VEGF destabilizes blood vessels and promotes neovascularization, which are common features of sight-threatening ocular vascular disorders. Here, we show that BCL6B (B-cell CLL/lymphoma 6 member B protein), also known as BAZF, ZBTB28, and ZNF62, plays a pivotal role in the development of retinal edema and neovascularization. METHODS: The pathophysiological physiological role of BCL6B was investigated in cellular and animal models mimicking 2 pathological conditions: retinal vein occlusion and choroidal neovascularization. An in vitro experimental system was used in which human retinal microvascular endothelial cells were supplemented with VEGF. Choroidal neovascularization cynomolgus monkey model was generated to investigate the involvement of BCL6B in the pathogenesis. Mice lacking BCL6B or treated with BCL6B-targeting small-interfering ribose nucleic acid were examined for histological and molecular phenotypes. RESULTS: In retinal endothelial cells, the BCL6B expression level was increased by VEGF. BCL6B-deficient endothelial cells showed Notch signal activation and attenuated cord formation via blockage of the VEGF-VEGFR2 signaling pathway. Optical coherence tomography images showed that choroidal neovascularization lesions were decreased by BCL6B-targeting small-interfering ribose nucleic acid. Although BCL6B mRNA expression was significantly increased in the retina, BCL6B-targeting small-interfering ribose nucleic acid suppressed ocular edema in the neuroretina. The increase in proangiogenic cytokines and breakdown of the inner blood-retinal barrier were abrogated in BCL6B knockout (KO) mice via Notch transcriptional activation by CBF1 (C promotor-binding factor 1) and its activator, the NICD (notch intracellular domain). Immunostaining showed that Müller cell activation, a source of VEGF, was diminished in BCL6B-KO retinas. CONCLUSIONS: These data indicate that BCL6B may be a novel therapeutic target for ocular vascular diseases characterized by ocular neovascularization and edema.

YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

BACKGROUND: Ocular neovascularization is a leading cause of blindness. Retinal microglia have been implicated in hypoxia-induced angiogenesis and vasculopathy, but the underlying mechanisms are not entirely clear. Lactylation is a novel lactate-derived posttranslational modification that plays key roles in multiple cellular processes. Since hypoxia in ischemic retinopathy is a precipitating factor for retinal neovascularization, lactylation is very likely to be involved in this process. The present study aimed to explore the role of lactylation in retinal neovascularization and identify new therapeutic targets for retinal neovascular diseases. RESULTS: Microglial depletion by the colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX3397 suppresses retinal neovascularization in oxygen-induced retinopathy. Hypoxia increased lactylation in microglia and accelerates FGF2 expression, promoting retinal neovascularization. We identify 77 sites of 67 proteins with increased lactylation in the context of increased lactate under hypoxia. Our results show that the nonhistone protein Yin Yang-1 (YY1), a transcription factor, is lactylated at lysine 183 (K183), which is regulated by p300. Hyperlactylated YY1 directly enhances FGF2 transcription and promotes angiogenesis. YY1 mutation at K183 eliminates these effects. Overexpression of p300 increases YY1 lactylation and enhances angiogenesis in vitro and administration of the p300 inhibitor A485 greatly suppresses vascularization in vivo and in vitro. CONCLUSIONS: Our results suggest that YY1 lactylation in microglia plays an important role in retinal neovascularization by upregulating FGF2 expression. Targeting the lactate/p300/YY1 lactylation/FGF2 axis may provide new therapeutic targets for proliferative retinopathies.

m6A modifications of circular RNAs in ischemia-induced retinal neovascularization.

Ischemia-induced pathological neovascularization in the retina is a leading cause of blindness in various age groups. The purpose of the current study was to identify the involvement of circular RNAs (circRNAs) methylated by N6-methyladenosine (m6A), and predict their potential roles in oxygen-induced retinopathy (OIR) in mice. Methylation assessment via microarray analysis indicated that 88 circRNAs were differentially modified by m6A methylation, including 56 hyper-methylated circRNAs and 32 hypo-methylated circRNAs. Gene ontology enrichment analysis predicted that the enriched host genes of the hyper-methylated circRNAs were involved in cellular process, cellular anatomical entity, and protein binding. Host genes of the hypo-methylated circRNAs were enriched in the regulation of cellular biosynthetic process, the nucleus, and binding. According to the Kyoto Encyclopedia of Genes and Genomes analysis, those host genes were involved in the pathways of selenocompound metabolism, salivary secretion, and lysine degradation. MeRIP-qPCR verified significant alterations in m6A methylation levels of mmu_circRNA_33363, mmu_circRNA_002816, and mmu_circRNA_009692. In conclusion, the study revealed the m6A modification alterations in OIR retinas, and the findings above shed light on the potential roles of m6A methylation in circRNA regulatory functions in the pathogenesis of ischemia-induced pathological retinal neovascularization.

Molecular Mediators and Regulators of Retinal Angiogenesis.

BACKGROUND: Retinal neovascularization is the major cause of vision loss that affects both adults and young children including premature babies. It has been a major pathology in several retinal diseases like age-related macular degeneration (AMD), diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Current treatment modalities such as anti-VEGF therapy, laser are not suitable for every patient and response to these therapies is highly variable. Thus, there is a need to investigate newer therapeutic targets for DR, ROP and AMD, based on a clear understanding of disease pathology and regulatory mechanisms involved. METHOD: Appropriate articles published till February 2021 were extracted from PUBMED using keywords like ocular angiogenesis, DR, ROP, AMD, miRNA, mRNA, and cirMiRNA and containvaluable information regarding the involvement of miRNA in causing neovascularization. After compiling the list of miRNA regulating mRNA expression in angiogenesis and neovascularaization, their interactions were studied using online available tool MIENTURNET (http://userver.bio.uniroma1.it/apps/mienturnet/). The pathways involved in these processes were also predicted using the same tool. RESULTS: Most of the studies have explored potential targets like HIF1-α, PDGF, TGFß, FGF, etc., for their involvement in pathological angiogenesis in different retinal diseases. The regulatory role of microRNA (miRNA) has also been explored in various retinal ocular pathologies. This review highlights regulatory mechanism of cellular and circulatory miRNAs and their interactions with the genes involved in retinal neovascularization. The role of long noncoding RNA (ncRNA) in the regulation of genes involved in different pathways is also noteworthy and discussed in this review. CONCLUSION: This review highlights the potential regulatory mechanism/pathways involved in retinal neovascularization and its implications in retinal diseases and for identifying new drug targets.

Connexin 43 (Cx43) regulates high-glucose-induced retinal endothelial cell angiogenesis and retinal neovascularization.

Diabetic retinopathy (DR) is an important microvascular complication of type 1 and type 2 diabetes mellitus (DM) and a major cause of blindness. Retinal neovascularization plays a critical role in the proliferative DR. In this study, high glucose-induced connexin 43 (Cx43) expression in human retinal endothelial cells (hRECs) in a dose-dependent manner. Compared with hRECs under normal culture conditions, high-glucose (HG)-stimulated hRECs showed promoted tubule formation, increased ROS release, and elevated levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), vascular endothelial growth factor A (VEGFA), and intercellular adhesion molecule 1 (ICAM-1) in the culture medium. HG-induced alterations were further magnified after Cx43 overexpression, whereas partially eliminated after Cx43 knockdown. Finally, in the DR mouse model, impaired retinal structure, increased CD31 expression, and elevated mRNA levels of TNF-α, IL-1ß, VEGFA, and ICAM-1 were observed; in-vivo Cx43 knockdown partially reversed these phenomena. Conclusively, Cx43 knockdown could inhibit hREC angiogenesis, therefore improving DR in the mouse model.

MiR-375 mitigates retinal angiogenesis by depressing the JAK2/STAT3 pathway.

Aberrant neovascularization in the retina is an important threat to vision and closely related to several retinal diseases, such as wet form of age-related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. However, the pathogenesis remains largely unknown. MicroRNAs (miRNAs) have been demonstrated to play critical regulatory roles in angiogenesis. Therefore, we aimed to identify the key miRNAs that regulate retinal neovascularization and elucidate the potential underlying mechanisms. In the present study, we performed RNA sequencing of microRNAs in the retina and found that miR-375 was significantly downregulated in the retina of oxygen-induced retinopathy mice. In retinal microvascular endothelial cells (RMECs), overexpression of miR-375 inhibited cell proliferation and angiogenesis. Conversely, inhibition of miR-375 had the opposite effects. Moreover, our results showed that miR-375 negatively regulated the protein expression of JAK2 by inhibiting its translation. The promoting effects of anti-miR-375 on cell proliferation and angiogenesis were attenuated by an inhibitor of STAT3. These results indicate that miR-375 mitigates cell proliferation and angiogenesis, at least in part, through the JAK2/STAT3 pathway in RMECs, which implies an important underlying mechanism of retinal angiogenesis and provides potential therapeutic targets for retinal microangiopathy.

miR-429 negatively regulates the progression of hypoxia-induced retinal neovascularization by the HPSE-VEGF pathway.

Heparanase (HPSE) and vascular endothelial growth factor (VEGF) are believed to play a vital role in hypoxia-induced retinal neovascularization (RNV). HPSE is a target gene of miR-429. Our study aimed to investigate the effect of the miR-429-HPSE-VEGF pathway on hypoxia-induced RNV. The gene and protein expression of miR-429, HPSE and VEGF in human retinal endothelial cells and retinas was determined by real-time PCR and Western blot assays. The effects of miR-429 on human retinal endothelial cells and retinal neovascularization under hypoxia condition were verified by in vitro and in vivo experiments. First, we studied the effect of the miR-429-HPSE-VEGF pathway in HRECs under hypoxic conditions. HREC functions such as migration and tube formation were enhanced under hypoxic conditions. Overexpression of miR-429 in HRECs reversed these changes. Then, we investigated the effect of miR-429 on hypoxia-induced RNV in vivo. When miR-429 agomirs were injected into the vitreous cavity of mice with oxygen-induced retinopathy to overexpress miR-429, the mRNA and protein expression of VEGF was significantly reduced. In addition, indicators of retinal neovascularization, such as the retinal avascular area, and morphology of vessels, were reduced significantly in the miR-429 overexpression group. In this study, our data showed that miR-429 plays an important role by inhibiting the HPSE-VEGF pathway in hypoxia-induced retinopathy.