Differentially expressed genes in orbital adipose/connective tissue of thyroid-associated orbitopathy.

Background: Thyroid-associated orbitopathy (TAO) is a disease associated with autoimmune thyroid disorders and it can lead to proptosis, diplopia, and vision-threatening compressive optic neuropathy. To comprehensively understand the molecular mechanisms underlying orbital adipogenesis in TAO, we characterize the intrinsic molecular properties of orbital adipose/connective tissue from patients with TAO and control individuals. Methods: RNA sequencing analysis (RNA-seq) was performed to measure the gene expression of orbital adipose/connective tissues of TAO patients. Differentially expressed genes (DEGs) were detected and analyzed through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA). The protein-protein interaction (PPI) network was constructed using the STRING database, and hub genes were identified by the Cytoscape plug-in, cytoHubba. We validated several top DEGs through quantitative real-time polymerase chain reaction (qRT-PCR). Results: We identified 183 DEGs in adipose tissue between TAO patients (n = 3) and control patients (n = 3) through RNA sequencing, including 114 upregulated genes and 69 downregulated genes. The PPI network of these DEGs had 202 nodes and 743 edges. PCR-based validation results of orbital adipose tissue showed multiple top-ranked genes in TAO patients (n = 4) are immune and inflammatory response genes compared with the control individual (n = 4). They include ceruloplasmin isoform x3 (CP), alkaline tissue-nonspecific isozyme isoform x1 (ALPL), and angiotensinogen (AGT), which were overrepresented by 2.27- to 6.40-fold. Meanwhile, protein mab-21-like 1 (MAB21L1), phosphoinositide 3-kinase gamma-subunit (PIK3C2G), and clavesin-2 (CLVS2) decreased by 2.6% to 32.8%. R-spondin 1 (RSPO1), which is related to oogonia differentiation and developmental angiogenesis, was significantly downregulated in the orbital muscle tissues of patients with TAO compared with the control groups (P = 0.024). Conclusions: Our results suggest that there are genetic differences in orbital adipose-connective tissues derived from TAO patients. The upregulation of the inflammatory response in orbital fat of TAO may be consistent with the clinical phenotype like eyelid edema, exophthalmos, and excess tearing. Downregulation of MAB21L1, PIK3C2G, and CLVS2 in TAO tissue demonstrates dysregulation of differentiation, oxidative stress, and developmental pathways.

MiR-423-5p promotes Müller cell activation via targeting NGF signaling in diabetic retinopathy.

AIMS: Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus and one of the major causes of visual impairment and blindness in industrialized countries. The early neuro-glial perturbations, especially retinal Müller cells (rMC) activation, intimately associated with the vascular alterations. MicroRNAs (miRNAs) have been reported to play critical roles in the progression of DR. Here, we aimed to further explore the role and underlying mechanism of miR-423-5p in Müller cell activation in streptozotocin (STZ)-induced diabetic mice and oxygen-induced retinopathy (OIR) model. MATERIALS AND METHODS: Retinal histology, optical coherence tomography (OCT) and biochemical markers were assessed. KEY FINDINGS: Our data revealed that the expression of miR-423-5p was significantly increased under high-glucose environment. We also demonstrated that miR-423-5p overexpression markedly accelerated retinal vascular leakage, leukocytosis, and rMC activation. This response was ameliorated in animals pre-treated with the inhibition of miR-423-5p. Specifically, miR-423-5p bound to the nerve growth factor (NGF) 3' UTR region to induce its silencing. NGF inhibition significantly promoted retinal microvascular dysfunction. SIGNIFICANCE: These findings demonstrate that miR-423-5p is a critical miRNA that promotes microvascular dysfunction in DR.

Iron derived from NCOA4-mediated ferritinophagy causes cellular senescence via the cGAS-STING pathway.

Cellular senescence is a hallmark of aging and has been linked to age-related diseases. Age-related macular degeneration (AMD), the most common aging-related retinal disease, is prospectively associated with retinal pigment epithelial (RPE) senescence. However, the mechanism of RPE cell senescence remains unknown. In this study, tert-butyl hydroperoxide (TBH)-induced ARPE-19 cells and D-galactose-treated C57 mice were used to examine the cause of elevated iron in RPE cell senescence. Ferric ammonium citrate (FAC)-treated ARPE-19 cells and C57 mice were used to elucidated the mechanism of iron overload-induced RPE cell senescence. Molecular biology techniques for the assessment of iron metabolism, cellular senescence, autophagy, and mitochondrial function in vivo and in vitro. We found that iron level was increased during the senescence process. Ferritin, a major iron storage protein, is negatively correlated with intracellular iron levels and cell senescence. NCOA4, a cargo receptor for ferritinophagy, mediates degradation of ferritin and contributes to iron accumulation. Besides, we found that iron overload leads to mitochondrial dysfunction. As a result, mitochondrial DNA (mtDNA) is released from damaged mitochondria to cytoplasm. Cytoplasm mtDNA activates the cGAS-STING pathway and promotes inflammatory senescence-associated secretory phenotype (SASP) and cell senescence. Meanwhile, iron chelator Deferoxamine (DFO) significantly rescues RPE senescence and retinopathy induced by FAC or D-gal in mice. Taken together, these findings imply that iron derived from NCOA4-mediated ferritinophagy causes cellular senescence via the cGAS-STING pathway. Inhibiting iron accumulation may represent a promising therapeutic approach for age-related diseases such as AMD.

Smurf1: A possible therapeutic target in dry age-related macular degeneration.

Smad ubiquitylation regulatory factor-1 (Smurf1) is one of C2-WW-HECT domain E3 ubiquitin ligases, it can regulate BMP pathway by mediating ubiquitylation degradation of Smad1/Smad5. Many functions about Smurf1 also are still unknown, especially in retina. This research is about to explore the role of Smurf1 in retina degeneration. Tail vein injection of sodium iodate (NaIO3) in C57BL/6J mice was the animal model of retina degeneration. In NaIO3 model, Smurf1 had more expression than normal mice. Specific Smurf1 inhibitor, A01, was injected into vitreous cavity. Results showed that inhibiting Smurf1 could alleviate acute retina injury, such as keeping a better retina structure in living imaging and histologic sections, less cell death and inflammation activation. Tert-butyl hydroperoxide (TBH) was used to establish oxidative stress injury in human retinal pigments epithelial cell line (ARPE-19). Oxidative stress injury gradually caused co-upregulation of Smurf1, TGF-ß1 and phosphorylated NF-κB (pNF-κB). TGF-ß1 could directly induce Smurf1 expression. Inhibiting Smurf1 had an anti-epithelial mesenchymal transition (anti-EMT) function. Similarly, A01 also could inhibit the expression of pNF-κB, NLRP3 and IL-1ß. At last, after searching bioinformatics database, Smurf1 had a possible interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (ß-TrCP), another E3 ubiquitin ligases. ß-TrCP can mediate ubiquitination degradation of p-IκBα. Lentivirus-SMURF1 was used to overexpress Smurf1, and GS143 was used to inhibit ß-TrCP. The results showed Smurf1 could directly induce NF-κB, pNF-κB, and NLRP3 expression, and keep a stable ß-TrCP expression. However, inhibiting ß-TrCP could cause more NF-κB activation and NLRP3 expression. Therefore, ß-TrCP may play a negative role in NF-κB pathway activation. In summary, Smurf1 plays a role in exacerbating oxidative stress injury and inflammation in retina and may become a potential therapeutic target in ROS injury of retina.

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.

GRP75 Modulates Endoplasmic Reticulum-Mitochondria Coupling and Accelerates Ca2+-Dependent Endothelial Cell Apoptosis in Diabetic Retinopathy.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER-mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca2+) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER-mitochondria coupling was increased through activation of the IP3R1-GRP75-VDAC1 axis, which transferred Ca2+ from the ER to the mitochondria. Elevated mitochondrial Ca2+ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER-mitochondria coupling via the IP3R1-GRP75-VDAC1 axis and accelerated Ca2+-dependent cell apoptosis. Our results demonstrated that inhibition of ER-mitochondrial coupling, including inhibition of GRP75 or Ca2+ overload, may be a potential therapeutic target in DR.

PGE2 promotes macrophage recruitment and neovascularization in murine wet-type AMD models.

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is a leading cause of blindness worldwide. Activated macrophages recruited to the injured eyes greatly contribute to the pathogenesis of choroidal neovascularization (CNV) in exudative AMD (wet AMD). This study describes the effects of cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) signalling on the macrophage activation and CNV formation of wet AMD. In a mouse model of laser-induced wet AMD, the mice received an intravitreal injection of celecoxib (a selective COX2 inhibitor). Optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal histology of the CNV lesions, and biochemical markers were assessed. The level of PGE2 expression was high in the laser-induced CNV lesions. Macrophage recruitment and CNV development were significantly less after celecoxib treatment. E-prostanoid1 receptor (EP1R)/protein kinase C (PKC) signalling was involved in M2 macrophage activation and interleukin-10 (IL-10) production of bone marrow-derived macrophages (BMDMs) in vitro. In addition, IL-10 was found to induce the proliferation and migration of human choroidal microvascular endothelial cells (HCECs). Thus, the PGE2/EP1R signalling network serves as a potential therapeutic target for CNV of the wet-type AMD. Video abstract.

Inhibition of Drp1 ameliorates diabetic retinopathy by regulating mitochondrial homeostasis.

Diabetic retinopathy (DR) is a potentially blinding complication resulting from diabetes mellitus (DM). Retinal vascular endothelial cells (RMECs) dysfunction occupies an important position in the pathogenesis of DR, and mitochondrial disorders play a vital role in RMECs dysfunction. However, the detailed mechanisms underlying DR-induced mitochondrial disorders in RMECs remain elusive. In the present study, we used High glucose (HG)-induced RMECs in vitro and streptozotocin (STZ)-induced Sprague-Dawley rats in vivo to explore the related mechanisms. We found that HG-induced mitochondrial dysfunction via mitochondrial Dynamin-related protein 1(Drp1)-mediated mitochondrial fission. Drp1 inhibitor, Mdivi-1, rescued HG-induced mitochondrial dysfunction. Protein Kinase Cδ (PKCδ) could induce phosphorylation of Drp1, and we found that HG induced phosphorylation of PKCδ. PKCδ inhibitor (Go 6983) or PKCδ siRNA reversed HG-induced phosphorylation of Drp1 and further mitochondrial dysfunction. The above studies indicated that HG increases mitochondrial fission via promoting PKCδ/Drp1 signaling. Drp1 induces excessive mitochondrial fission and produces damaged mitochondrial, and mitophagy plays a key role in clearing damaged mitochondrial. Our study showed that HG suppressed mitophagy via inhibiting LC3B-II formation and p62 degradation. 3-MA (autophagy inhibitor) aggravated HG-induced RMECs damage, while rapamycin (autophagy agonist) rescued the above phenomenon. Further studies were identified that HG inhibited mitophagy by down-regulation of the PINK1/Parkin signaling pathway, and PINK1 siRNA aggravated HG-induced RMECs damage. Further in-depth study, we propose that Drp1 promotion of Hexokinase II (HK-II) separation from mitochondria, thus inhibiting HK-II-PINK1-mediated mitophagy. In vivo, we found that intraretinal microvascular abnormalities (IRMA), including retinal vascular leakage, acellular capillaries, and apoptosis were increased in STZ-induced DR rats, which were reversed by pretreatment with Mdivi-1 or Rapamycin. Altogether, our findings provide new insight into the mechanisms underlying the regulation of mitochondrial homeostasis and provide a potential treatment strategy for Diabetic retinopathy.

Decorin Protects Retinal Pigment Epithelium Cells from Oxidative Stress and Apoptosis via AMPK-mTOR-Regulated Autophagy.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss among the elderly worldwide with unidentified pathogenesis and limited therapeutic options. Oxidative stress-induced damage to the retinal pigment epithelium (RPE) is central in the development and progression of AMD. Decorin (DCN), a small leucine-rich proteoglycan, possesses powerful antifibrotic, anti-inflammatory, and antiangiogenic properties. DCN has also been reported to serve a cytoprotective role in various cell types, but its protective effects against H2O2-induced oxidative stress and apoptosis in ARPE-19 cells remain unclear. In this study, we showed that DCN significantly attenuated the increase in cell viability loss, apoptosis rate, and reactive oxygen species (ROS) levels in ARPE-19 cells induced by H2O2. Furthermore, DCN activated the AMPK/mTOR pathway to promote autophagy while genetic inhibition of autophagy-related gene 5 (ATG5) hindered autophagic process and diminished the protective role of DCN against oxidative stress in ARPE-19 cells. Collectively, these results suggest that DCN could protect RPE cells from H2O2-induced oxidative stress and apoptosis via autophagy promotion, thus providing the therapeutic potential for AMD prevention and treatment.

MiR-143 Targets IGF-1R to Suppress Autoimmunity in Thyroid-Associated Ophthalmopathy.

Objective: Thyroid-associated ophthalmopathy (TAO) is an autoimmune disease that involves the remodeling of orbit and periorbital tissues. Thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF-1R) may stimulate the activation of autoimmunity in TAO, but the exact mechanism is unclear. We investigated whether IGF-1R/TSHR modulation in TAO may involve microRNA regulation. Methods: We conducted microarray analysis using RNA from the orbital connective tissue samples of 3 healthy and 3 patients with TAO. The involvement of differentially regulated microRNA in IGF-1R/TSHR modulation in TAO was evaluated in orbital fibroblasts (OFs) and female BALB/c mice. Results: Using hierarchical cluster analysis, we identified that miR-143 was downregulated in TAO. The expression levels of miR-143 in OFs were significantly reduced under IL-1B stimulation. However, OF proliferation and inflammatory responses decreased when miR-143 is overexpressed. In contrast, the suppression of miR-143 increased levels of inflammatory markers (IL-6, IL-8, MCP1) and hyaluronan accumulation. Moreover, overexpression of miR-143 significantly lowers levels of IGF-1R and TSHR. A luciferase assay indicated that miR-143 targets the 3'-UTR of IGF-1R. Increases in the expression of IGF-1R increased the expression of the inflammasome marker NLRP3 and apoptotic marker cleaved caspase-1; however, miR-143 overexpression decreased levels of IGF-1R, TSHR, NLRP3, cleaved caspase 1, IL-1B, and IL-18. In a mouse model of TAO, overexpression of miR-143 significantly reduced levels of IGF-1R and attenuated the adipogenesis associated with TAO. Conclusion: We found that miR-143 directly targets IGF-1R to alleviate the inflammatory response in TAO by indirectly decreasing levels of TSHR and inactivating NLRP3.

Tetramethylpyrazine inhibits the proliferation, invasiveness and migration of cervical cancer C33A cells by retarding the Hedgehog signaling pathway.

Cervical carcinoma (CC) ranks among the top four most common cancers in women worldwide. Over the last 10 years, several studies have confirmed the inhibitory effects of tetramethylpyrazine (TMP) on numerous types of cancer. To investigate the inhibitory effect of TMP on the CC C33A cell line, MTT and colony formation assays were performed to determine how TMP affects C33A cell survival and proliferation. Proliferation-, migration- and hedgehog (Hh) signaling pathway-related protein expression levels were analyzed via western blotting. Wound-healing and Transwell assays were used to detect the migration and invasion abilities of C33A cells, respectively. The results indicated that TMP markedly reduced the C33A cell survival rate compared with the cervical epithelial Ect1 cell line, which was unaffected by TMP treatment. C33A cell proliferation was downregulated by TMP treatment in a dose-dependent manner. TMP treatment also significantly inhibited C33A cell migration and invasiveness in a dose-dependent manner. Furthermore, TMP inhibited the Hh signaling pathway, as demonstrated by a dose-dependent reduction in Hh-related protein expression levels following TMP treatment. Subsequently, treatment with smoothened agonist increased the proliferation, invasiveness and migration abilities of TMP-treated C33A cells. In conclusion, TMP inhibited the proliferation, migration and invasiveness of CC cells via inhibition of the Hh signaling pathway.

Interferon-γ induces retinal pigment epithelial cell Ferroptosis by a JAK1-2/STAT1/SLC7A11 signaling pathway in Age-related Macular Degeneration.

Retinal pigment epithelium (RPE) cell damage is implicated in the pathogenesis of age-related macular degeneration (AMD). An increase of interferon-γ (IFN-γ) levels was observed in patients with AMD, but whether inflammatory factors are causally related to AMD progression is unclear. Here, we demonstrate a direct causal relationship between IFN-γ and RPE cell death. IFN-γ induced human retinal pigment epithelial cell (ARPE-19) death accompanied by increases in Fe2+ , reactive oxygen species, lipid peroxidation, and glutathione (GSH) depletion, which are main characteristics of ferroptosis. Mechanistically, IFN-γ upregulates the level of intracellular Fe2+ through inhibiting Fe2+ efflux protein SLC40A1 and induces GSH depletion by blocking cystine/glutamate antiporter, System xc-. At the same time, treatment with IFN-γ decreases the level of glutathione peroxidase 4 (GPx4), rendering the cells more sensitive to ferroptosis. JAK1/2 and STAT1 inhibitors could reverse the reduction of SLC7A11, GPx4 and GSH expression induced by IFN-γ, indicating IFN-γ induces ARPE-19 cell ferroptosis via activation of the JAK1-2/STAT1/SLC7A11 signaling pathway. The above results were largely confirmed in IFN-γ-treated mice in vivo. Finally, we used sodium iodate (NaIO3 )-induced retinal degeneration to further explore the role of ferroptosis in AMD in vivo. Consistent with the role of IFN-γ, treatment with NaIO3 decreased SLC7A11, GPx4 and SLC40A1 expressions. NaIO3 -induced RPE damage was accompanied by increased iron, lipid peroxidation products (4-hydroxynonenal, malondialdehyde), and GSH depletion, and ferroptosis inhibitors could reverse the above phenomenon. Taken together, our findings suggest that inhibiting ferroptosis or reducing IFN-γ may serve as a promising target for AMD.

Hyperglycemia-induced increasing of RELB/circ_0008590 in NF-κB pathway is repressed by miR-1243 in human retinal microvascular endothelial cells.

BACKGROUND: To investigate the abnormal expression of circ_0008590 and its parent gene, reticuloendotheliosis viral oncogene related B (RELB) in human retinal microvascular endothelial cells (hRECs) in hyperglycemia and the potential mechanism. METHODS: The levels of RELB, circ_0008590, and miR-1243 in hRECs or clinical samples were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Dual-luciferase reporter assay was used to test the interaction between RELB/circ_0008590 and miR-1243. Cell Counting Kit-8 (CCK-8), Transwell, flow cytometry (FCM), wound healing, and tube formation assays were used for the physiological investigation. The interaction between human RELB and circ_0008590 was studied in streptozotocin (STZ) induced diabetic retinopathy (DR) C57BL/6 mice. RESULTS: The levels of circ_0008590 and RELB were increased in hRECs in hyperglycemia; during the progression of DR, the levels of circ_0008590 and RELB messenger RNA (mRNA) in aqueous humor were first decreased and then increased, whereas miR-1243 showed an opposite trend. Both RELB 3'-untranslated region (UTR) and circ_0008590 shared a similar binding site for miR-1243. Further, miR-1243 mimic suppressed the proliferation and migration of hRECs, promoting the apoptosis ratio, which could be rescued by the overexpression of circ_0008590. In STZ-induced DR mice, miR-1243 agomir rescued the effects of the overexpression of human RELB. CONCLUSIONS: In hyperglycemia, high expression of RELB/circ_0008590 could be suppressed by miR-1243, and the nuclear factor-κB (NF-κB) pathway is subsequently affected.

LXA4 protects against blue-light induced retinal degeneration in human A2E-laden RPE cells and Balb-c mice.

BACKGROUND: Age-related macular degeneration (AMD) is one of the leading causes of permanent visual impairment in the elderly. Blue light (BL) has been reported to cause retinal damage and contribute to the onset and development of severe AMD. N-retinylidene-N-retinylethanolamine (A2E), a lipofuscin fluorophore, accumulates with ageing in the retinal pigment epithelium (RPE) cells. Once exposed to BL, A2E easily oxidizes to A2E-epoxides, causing oxidative-stress injury to the retina. Lipoxin A4 (LXA4), an endogenous anti-antioxidant lipid, plays a key role in multiple organs by binding to the formyl-peptide receptor-like 1 (FPRL1). This study examined the protective effects of LXA4 on oxidative-stress injury induced by BL exposure, and clarified the underlying mechanisms in cultured RPE cells and Balb-c mice. METHODS: LXA4 diluent was orally administered to mice before retinal degeneration was established. Optical coherence tomography, retinal histology, and RPE cell injury were assessed. RESULTS: LXA4 administration significantly ameliorated retinal damage as evidenced by the thicknesses of the retinal layers and the tight junctions of RPE cells in vivo. LXA4 inhibited BL-induced reactive oxygen species (ROS) production, reduced tight junctions, and the death of A2E-laden RPE cells. LXA4 also potently increased the expression of haem oxygenase-1 (HO1) and NAD(P)H quinone oxidoreductase 1 (NQO1), probably by decreasing the association between nuclear factor erythroid 2-related factor 2 (NRF2) and Kelch-like ECH (Epichlorohydrin) -associated protein 1 (Keap1), and ameliorating NRF2 nuclear translocation and the antioxidant response element (ARE) deoxyribonucleic acid (DNA) binding activity. CONCLUSIONS: Our results showed that LXA4 ameliorated retinal degeneration, and should be considered in the prevention and treatment of AMD.

Protective roles of the TIR/BB-loop mimetic AS-1 in alkali-induced corneal neovascularization by inhibiting ERK phosphorylation.

Hydrocinnamoyl-L-valylpyrrolidine (AS-1), a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), inhibits inflammation by disrupting the interaction between the interleukin-1 receptor (IL-1R) and MyD88. Here, we describe the effects of AS-1 on injury-induced increases in inflammation and neovascularization in mouse corneas. Mice were administered a subconjunctival injection of 8 µL AS-1 diluent before or after corneal alkali burn, followed by evaluation of corneal resurfacing and corneal neovascularization (CNV) by slit-lamp biomicroscopy and clinical assessment. Corneal inflammation was assessed by whole-mount CD45+ immunofluorescence staining, and corneal hemangiogenesis and lymphangiogenesis following injury were evaluated by immunostaining for the vascular markers isolectin B4 (IB4) and the lymphatic vascularized marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), respectively. Additionally, corneal tissues were collected to determine the expression of 35 cytokines, and we detected activation of IL-1RI, MyD88, and mitogen-activated protein kinase (MAPK). The results showed that alkali conditions increased the number of CD45+ cells and expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, and LYVE1 in corneas, with these levels decreased in the AS-1-treated group. Moreover, AS-1 effectively prevented alkali-induced cytokine production, blocked interactions between IL-1RI and MyD88, and inhibited MAPK activation post-alkali burn. These results indicated that AS-1 prevented alkali-induced corneal hemangiogenesis and lymphangiogenesis by blocking IL-1RI-MyD88 interaction, as well as extracellular signal-regulated kinase phosphorylation, and could be efficacious for the prevention and treatment of corneal alkali burn.

Prostaglandin E2 promotes pathological retinal neovascularisation via EP4R-EGFR-Gab1-AKT signaling pathway.

Proliferative retinopathies, such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are major causes of visual impairment and blindness in industrialized countries. Prostaglandin E2 (PGE2) is implicated in cellular proliferation and migration via E-prostanoid receptor (EP4R). The aim of this study was to investigate the role of PGE2/EP4R signaling in the promotion of retinal neovascularisation. In a streptozotocin (STZ)-induced diabetic model and an oxygen-induced retinopathy (OIR) model, rats received an intravitreal injection of PGE2, cay10598 (an EP4R agonist) or AH23848 (an EP4R antagonist). Optical coherence tomography, retinal histology and biochemical markers were assessed. Treatment with PGE2 or cay10598 accelerated pathological retinal angiogenesis in STZ and OIR-induced rat retina, which was ameliorated in rats pretreated with AH23848. Serum VEGF-A was upregulated in the PGE2-treated diabetic rats vs non-treated diabetic rats and significantly downregulated in AH23848-treated diabetic rats. PGE2 or cay10598 treatment also significantly accelerated endothelial tip-cell formation in new-born rat retina. In addition, AH23848 treatment attenuated PGE2-or cay10598-induced proliferation and migration by repressing the EGF receptor (EGFR)/Growth factor receptor bound protein 2-associated binder protein 1 (Gab1)/Akt/NF-κB/VEGF-A signaling network in human retinal microvascular endothelial cells (hRMECs). PGE2/EP4R signaling network is thus a potential therapeutic target for pathological intraocular angiogenesis.

Identification of prognostic alternative splicing signatures in uveal melanoma.

PURPOSE: Alternative splicing (AS) events were reportedly associated with the development of multiple cancers. The study was designed to provide a comprehensive analysis of AS events and explore their potential prognostic value in uveal melanoma (UM). METHODS: The prognostic AS events, identified based on the data of 80 UM patients obtained from The Cancer Genome Atlas, were further screened and analyzed for construction of prognostic signatures by using LASSO regression and multivariate Cox model. Kaplan-Meier survival analysis was used to evaluate the prognostic value. The AS events-related functional pathways were explored by gene set enrichment analysis (GSEA). The difference between two subgroups in terms of treatment options was investigated. The regulatory network between prognostic AS events and splicing factors (SFs) was then constructed. RESULTS: A total of 1014 AS events were identified as prognostic AS events. Five prognostic AS events were involved in the construction of prognostic signatures, including AKAP2/87175/AP, RGMA/32575/ES, DNASE1L1/90581/ES, BIN1/55198/ES and ERCC2/50430/AT. UM patients were then divided into two subgroups. Prognostic AS signatures had an excellent performance in predicting the survival of UM patients, with an area under curve (AUC) of 0.962. GSEA results suggested several splicing-associated mechanisms, including cellular metabolic process and apoptosis. Low-risk subgroup could be more sensitive to drugs. A higher expression of immune checkpoint genes was observed in high-risk group than in low-risk group. SFs-AS regulatory network also revealed significant association between AS events and SFs. CONCLUSIONS: Aberrant AS events in UM patients might serve as prognostic predictors.

TGR5 Activation Ameliorates Mitochondrial Homeostasis via Regulating the PKCδ/Drp1-HK2 Signaling in Diabetic Retinopathy.

Background: Diabetic retinopathy (DR) is one of the most important microvascular diseases of diabetes. Our previous research demonstrated that bile acid G-protein-coupled membrane receptor (TGR5), a novel cell membrane receptor of bile acid, ameliorates the vascular endothelial cell dysfunction in DR. However, the precise mechanism leading to this alteration remains unknown. Thus, the mechanism of TGR5 in the progress of DR should be urgently explored. Methods: In this study, we established high glucose (HG)-induced human retinal vascular endothelial cells (RMECs) and streptozotocin-induced DR rat in vitro and in vivo. The expression of TGR5 was interfered through the specific agonist or siRNA to study the effect of TGR5 on the function of endothelial cell in vitro. Western blot, immunofluorescence and fluorescent probes were used to explore how TGR5 regulated mitochondrial homeostasis and related molecular mechanism. The adeno-associated virus serotype 8-shTGR5 (AAV8-shTGR5) was performed to evaluate retinal dysfunction in vivo and further confirm the role of TGR5 in DR by HE staining, TUNEL staining, PAS staining and Evans Blue dye. Results: We found that TGR5 activation alleviated HG-induced endothelial cell apoptosis by improving mitochondrial homeostasis. Additionally, TGR5 signaling reduced mitochondrial fission by suppressing the Ca2+-PKCδ/Drp1 signaling and enhanced mitophagy through the upregulation of the PINK1/Parkin signaling pathway. Furthermore, our result indicated that Drp1 inhibited mitophagy by facilitating the hexokinase (HK) 2 separation from the mitochondria and HK2-PINK1/Parkin signaling. In vivo, intraretinal microvascular abnormalities, including retinal vascular leakage, acellular capillaries and apoptosis, were poor in AAV8-shTGR5-treated group under DR, but this effect was reversed by pretreatment with the mitochondrial fission inhibitor Mdivi-1 or autophagy agonist Rapamycin. Conclusion: Overall, our findings indicated that TGR5 inhibited mitochondrial fission and enhanced mitophagy in RMECs by regulating the PKCδ/Drp1-HK2 signaling pathway. These results revealed the molecular mechanisms underlying the protective effects of TGR5 and suggested that activation of TGR5 might be a potential therapeutic strategy for DR.

Altered Corneal Nerves in Chinese Thyroid-Associated Ophthalmopathy Patients Observed by In Vivo Confocal Microscopy.

BACKGROUND Thyroid-associated ophthalmopathy (TAO) is a common endocrine autoimmune disease. The present study explored corneal nerve changes in TAO patients. MATERIAL AND METHODS Thirty-eight Chinese TAO patients and 20 healthy individuals were included in the study. Central corneal subbasal nerve density and morphology were evaluated with in vivo laser scanning confocal microscopy and quantified using automated CCmetrics software. RESULTS The values of the central corneal subbasal nerve plexus parameters of both active and inactive TAO patients were significantly decreased compared with those of controls, including corneal nerve fiber density (CNFD) (P<0.001 for both), corneal nerve branch density (CNBD) (P<0.001 for both), corneal nerve fiber length (CNFL) (P<0.001 for both), corneal nerve fiber total branch density (CTBD) (P<0.001 for both), corneal nerve fiber area (CNFA) (P<0.001 for both), corneal nerve fiber width (CNFW) (P=0.046, P=0.027, respectively), and corneal nerve fiber fractal dimension (ACNFrD) (P<0.001 for both). In addition, CNFD and ACNFrD values were significantly lower in the active TAO patients compared with those in the inactive TAO patients (P=0.020, P=0.002, respectively). There were significant correlations between CNFD, CNBD, CNFL, CTBD, CNFA, and ACNFrD and the ocular surface parameters and activity assessment items. CONCLUSIONS Abnormal corneal subbasal nerves were observed in both active and inactive Chinese TAO patients, suggesting that nerve degeneration is associated with the disease. However, the exact underlying mechanisms remain to be elucidated.