Transcriptional patterns of human retinal pigment epithelial cells under protracted high glucose.

BACKGROUND: The retinal pigment epithelium (RPE) is essential for retinal homeostasis. Comprehensively exploring the transcriptional patterns of diabetic human RPE promotes the understanding of diabetic retinopathy (DR). METHODS AND RESULTS: A total of 4125 differentially expressed genes (DEGs) were screened out from the human primary RPE cells subjected to prolonged high glucose (HG). The subsequent bioinformatics analysis is divided into 3 steps. In Step 1, 21 genes were revealed by intersecting the enriched genes from the KEGG, WIKI, and Reactome databases. In Step 2, WGCNA was applied and intersected with the DEGs. Further intersection based on the enrichments with the GO biological processes, GO cellular components, and GO molecular functions databases screened out 12 candidate genes. In Step 3, 13 genes were found to be simultaneously up-regulated in the DEGs and a GEO dataset involving human diabetic retinal tissues. VEGFA and ERN1 were the 2 starred genes finally screened out by overlapping the 3 Steps. CONCLUSION: In this study, multiple genes were identified as crucial in the pathological process of RPE under protracted HG, providing potential candidates for future researches on DR. The current study highlights the importance of RPE in DR pathogenesis.

Establishing chronic models of age-related macular degeneration via long-term iron ion overload.

Age-related macular degeneration (AMD) is characterized by the degenerative senescence in the retinal pigment epithelium (RPE) and photoreceptors, which is accompanied by the accumulation of iron ions in the aging retina. However, current models of acute oxidative stress are still insufficient to simulate the gradual progression of AMD. To address this, we established chronic injury models by exposing the aRPE-19 cells, 661W cells, and mouse retina to iron ion overload over time. Investigations at the levels of cell biology and molecular biology were performed. It was demonstrated that long-term treatment of excessive iron ions induced senescence-like morphological changes, decreased cell proliferation, and impaired mitochondrial function, contributing to apoptosis. Activation of the mitogen-activated protein kinase (MAPK) pathway and the downstream molecules were confirmed both in the aRPE-19 and 661W cells. Furthermore, iron ion overload resulted in dry AMD-like lesions and decreased visual function in the mouse retina. These findings suggest that chronic exposure to overloading iron ions plays a significant role in the pathogenesis of retinopathy and provide a potential model for future studies on AMD.

DNA cytosine deamination is associated with recurrent Somatic Copy Number Alterations in stomach adenocarcinoma.

Stomach Adenocarcinoma (STAD) is a leading cause of death worldwide. Somatic Copy Number Alterations (SCNAs), which result in Homologous recombination (HR) deficiency in double-strand break repair, are associated with the progression of STAD. However, the landscape of frequent breakpoints of SCNAs (hotspots) and their functional impacts remain poorly understood. In this study, we aimed to explore the frequency and impact of these hotspots in 332 STAD patients and 1,043 cancer cells using data from the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE). We studied the rates of DSB (Double-Strand Breaks) loci in STAD patients by employing the Non-Homogeneous Poisson Distribution (λ), based on which we identified 145 DSB-hotspots with genes affected. We further verified DNA cytosine deamination as a critical process underlying the burden of DSB in STAD. Finally, we illustrated the clinical impact of the significant biological processes. Our findings highlighted the relationship between DNA cytosine deamination and SCNA in cancer was associated with recurrent Somatic Copy Number Alterations in STAD.

Silibinin Prevents TGFß-Induced EMT of RPE in Proliferative Vitreoretinopathy by Inhibiting Stat3 and Smad3 Phosphorylation.

Purpose: The purpose of this study was to investigate the effects of silibinin on epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) and proliferative vitreoretinopathy (PVR) formation, as well as its underlying molecular mechanism. Methods: Cellular morphological change and EMT molecular markers were evaluated by using phase contrast imaging, qPCR, and Western blot (WB) to investigate the impact of silibinin on the EMT of ARPE-19 cells. Scratch assay and transwell assay were used to study the effect of silibinin on cell migration. An intravitreally injected RPE-induced rat PVR model was used to assess the effect of silibinin on PVR in vivo. RNA-seq was applied to study the molecular mechanism of silibinin-mediated PVR prevention. Results: Silibinin inhibited TGFß1-induced EMT and migration of RPE in a dose-dependent manner in vitro. Moreover, silibinin prevented proliferative membrane formation in an intravitreal injected RPE-induced rat PVR model. In line with these findings, RNA-seq revealed a global suppression of TGFß1-induced EMT and migration-related genes by silibinin in RPEs. Mechanistically, silibinin reduced TGFß1-induced phosphorylation levels of Smad3 and Stat3, and Smad3 nuclear translocation in RPE. Conclusions: Silibinin inhibits the EMT of RPE cells in vitro and prevents the formation of PVR membranes in vivo. Mechanistically, silibinin inhibits Smad3 phosphorylation and suppresses Smad3 nuclear translocation through the inhibition of Stat3 phosphorylation. These findings suggest that silibinin may serve as a potential treatment for PVR.

Protective effect of ginsenoside Rg1 on 661W cells exposed to oxygen-glucose deprivation/reperfusion via keap1/nrf2 pathway.

AIM: To construct an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) induced injury to the optic nerve and to study the oxidative damage mechanism of ischemia-reperfusion (I/R) injury in 661W cells and the protective effect of ginsenoside Rg1. METHODS: The 661W cells were treated with different concentrations of Na2S2O4 to establish OGD/R model in vitro. Apoptosis, intracellular reactive oxygen species (ROS) levels and superoxide dismutase (SOD) levels were measured at different time points during the reperfusion injury process. The injury model was pretreated with graded concentrations of ginsenoside Rg1. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of cytochrome C (cyt C)/B-cell lymphoma-2 (Bcl2)/Bcl2 associated protein X (Bax), heme oxygenase-1 (HO-1), caspase9, nuclear factor erythroid 2-related factor 2 (nrf2), kelch-like ECH-associated protein 1 (keap1) and other genes. Western blot was used to detect the expression of nrf2, phosphorylated nrf2 (pnrf2) and keap1 protein levels. RESULTS: Compared to the untreated group, the cell activity of 661W cells treated with Na2S2O4 for 6 and 8h decreased (P<0.01). Additionally, the ROS content increased and SOD levels decreased significantly (P<0.01). In contrast, treatment with ginsenoside Rg1 reversed the cell viability and SOD levels in comparison to the Na2S2O4 treated group (P<0.01). Moreover, Rg1 reduced the levels of caspase3, caspase9, and cytC, while increasing the Bcl2/Bax level. These differences were all statistically significant (P<0.05). Western blot analysis showed no significant difference in the protein expression levels of keap1 and nrf2 with Rg1 treatment, however, Rg1 significantly increased the ratio of pnrf2/nrf2 protein expression compared to the Na2S2O4 treated group (P<0.001). CONCLUSION: The OGD/R process is induced in 661W cells using Na2S2O4. Rg1 inhibits OGD/R-induced oxidative damage and alleviates the extent of apoptosis in 661W cells through the keap1/nrf2 pathway. These results suggest a potential protective effect of Rg1 against retinal I/R injury.

MAP4K4 is involved in the neuronal development of retinal photoreceptors.

Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is a potential regulator of photoreceptor development. To investigate the mechanisms underlying MAP4K4 during the neuronal development of retinal photoreceptors, we generated knockout models of C57BL/6j mice in vivo and 661 W cells in vitro. Our findings revealed homozygous lethality and neural tube malformation in mice subjected to Map4k4 DNA ablation, providing evidence for the involvement of MAP4K4 in early stage embryonic neural formation. Furthermore, our study demonstrated that the ablation of Map4k4 DNA led to the vulnerability of photoreceptor neurites during induced neuronal development. By monitoring transcriptional and protein variations in mitogen-activated protein kinase (MAPK) signaling pathway-related factors, we discovered an imbalance in neurogenesis-related factors in Map4k4 -/- cells. Specifically, MAP4K4 promotes jun proto-oncogene (c-JUN) phosphorylation and recruits other factors related to nerve growth, ultimately leading to the robust formation of photoreceptor neurites. These data suggest that MAP4K4 plays a decisive role in regulating the fate of retinal photoreceptors through molecular modulation and contributes to our understanding of vision formation.

Qihuang Granule protects the retinal pigment epithelium from oxidative stress via regulation of the alternative complement pathway.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of vision loss in elderly people, and dry AMD is the most common type of AMD. Oxidative stress and alternative complement pathway activation may play essential roles in the pathogenesis of dry AMD. There are no available drugs for dry AMD. Qihuang Granule (QHG) is an herbal formula for the treatment of dry AMD, and it achieves a good clinical effect in our hospital. However, its potential mechanism is unclear. Our study investigated the effects of QHG on oxidative stress-associated retinal damage to reveal its underlying mechanism. METHODS: Oxidative stress models were established using H2O2 and NaIO3 in ARPE-19 cells and C57BL/6 mice. Cell apoptosis and viability were assessed using phase contrast microscopy and flow cytometry, respectively. Alterations in the mouse retinal structure were evaluated using Masson staining and transmission electron microscopy (TEM). The expression of complement factor H (CFH), complement component 3a (C3a) and complement component 5a (C5a) in retinal pigment epithelium (RPE) cells and mice was measured using RT‒PCR, Western blot analysis and ELISA. RESULTS: Pretreatment with QHG significantly prevented cell apoptosis and disorder of the RPE and inner segment/outer segment (IS/OS) in H2O2-treated RPE cells and NaIO3-injected mice. QHG alleviated mitochondrial damage in mouse RPE cells, as shown by TEM. QHG also promoted CFH expression and inhibited the expression of C3a and C5a. CONCLUSIONS: The results suggest that QHG protects the retinal pigment epithelium from oxidative stress, likely by regulating the alternative complement pathway.

Truncation mutations in MYRF underlie primary angle closure glaucoma.

Mutations in myelin regulatory factor (MYRF), a gene mapped to 11q12-q13.3, are responsible for autosomal dominant high hyperopia and seem to be associated with angle closure glaucoma, which is one of the leading causes of irreversible blindness worldwide. Whether there is a causal link from the MYRF mutations to the pathogenesis of primary angle-closure glaucoma (PACG) remains unclear at this time. Six truncation mutations, including five novel and one previously reported, in MYRF are identified in seven new probands with hyperopia, of whom all six adults have glaucoma, further confirming the association of MYRF mutations with PACG. Immunofluorescence microscopy demonstrates enriched expression of MYRF in the ciliary body and ganglion cell layer in humans and mice. Myrfmut/+ mice have elevated IOP and fewer ganglion cells along with thinner retinal nerve fiber layer with ganglion cell layer than wild-type. Transcriptome sequencing of Myrfmut/+ retinas shows downregulation of Dnmt3a, a gene previously associated with PACG. Co-immunoprecipitation demonstrates a physical association of DNMT3A with MYRF. DNA methylation sequencing identifies several glaucoma-related cell events in Myrfmut/+ retinas. The interaction between MYRF and DNMT3A underlies MYRF-associated PACG and provides clues for pursuing further investigation into the pathogenesis of PACG and therapeutic target.

Multifunctional Interleukin-24 Resolves Neuroretina Autoimmunity via Diverse Mechanisms.

IL-24 is a multifunctional cytokine that regulates both immune cells and epithelial cells. Although its elevation is associated with a number of autoimmune diseases, its tolerogenic properties against autoreactive T cells have recently been revealed in an animal model of central nervous system (CNS) autoimmunity by inhibiting the pathogenic Th17 response. To explore the potential of IL-24 as a therapeutic agent in CNS autoimmunity, we induced experimental autoimmune uveitis (EAU) in wildtype mice and intravitreally injected IL-24 into the inflamed eye after disease onset. We found that the progression of ocular inflammation was significantly inhibited in the IL-24-treated eye when compared to the control eye. More importantly, IL-24 treatment suppressed cytokine production from ocular-infiltrating, pathogenic Th1 and Th17 cells. In vitro experiments confirmed that IL-24 suppressed both Th1 and Th17 differentiation by regulating their master transcription factors T-bet and RORγt, respectively. In addition, we found that intravitreal injection of IL-24 suppressed the production of proinflammatory cytokines and chemokines from the retinas of the EAU-inflamed eyes. This observation appears to be applicable in humans, as IL-24 similarly inhibits human retinal pigment epithelium cells ARPE-19. In conclusion, we report here that IL-24, as a multifunctional cytokine, is capable of resolving ocular inflammation in EAU mice by targeting both uveitogenic T cells and RPE cells. This study sheds new light on IL-24 as a potential therapeutic candidate for autoimmune uveitis.

Oxidative stress-induced lncRNA CYLD-AS1 promotes RPE inflammation via Nrf2/miR-134-5p/NF-κB signaling pathway.

Oxidative stress-induced damage to and dysfunction of retinal pigment epithelium (RPE) cells are important pathogenetic factors of age-related macular degeneration (AMD); however, the underlying molecular mechanism is not fully understood. Long noncoding RNAs (lncRNAs) have important roles in various biological processes. In this study, using an oxidative damage model in RPE cells, we identified a novel oxidation-related lncRNA named CYLD-AS1. We further revealed that the expression of CYLD-AS1 was increased in RPEs during oxidative stress. Depletion of CYLD-AS1 promoted cell proliferation and mitochondrial function and protected RPE cells against hydrogen peroxide (H2 O2 )-induced damage. Mechanistically, CYLD-AS1 also regulated the expression of NRF2, which is related to oxidative stress, and NF-κB signaling pathway members, which are related to inflammation. Remarkably, these two signaling pathways were mediated by the CYLD-AS1 interactor miR-134-5p. Moreover, exosomes secreted by CYLD-AS1 knockdown RPE cells had a lower proinflammatory effect than those secreted by control cells. In summary, our study revealed that CYLD-AS1 affects the oxidative stress-related and inflammatory functions of RPE cells by sponging miR-134-5p to mediate NRF2/NF-κB signaling pathway activity, suggesting that targeting CYLD-AS1 could be a promising strategy for the treatment of AMD and related diseases.

Arbutin Protects Retinal Pigment Epithelium Against Oxidative Stress by Modulating SIRT1/FOXO3a/PGC-1α/ß Pathway.

Age-related macular degeneration (AMD), which is the leading cause of blindness among the elderly in western societies, is majorly accompanied by retinal pigment epithelium (RPE) degeneration. Because of the irreversible RPE cell loss among oxidative stress, it is crucial to search for available drugs for atrophic (dry) AMD. RNA-Seq analysis revealed that genes related to aging and mitochondrial health were differentially expressed under Arbutin treatment, whereas compared to oxidative injury, our study demonstrated that Arbutin substantially abrogated oxidative stress-induced cell senescence and apoptosis linked to intracellular antioxidant enzyme system homeostasis maintenance, restored mitochondrial membrane potential (MMP), and reduced the SA-ß-GAL accumulation in RPE. Furthermore, Arbutin alleviated oxidative stress-mediated cell apoptosis and senescence via activation of SIRT1, as evidenced by the increase of the downstream FoxO3a and PGC-1α/ß that are related to mitochondrial biogenesis, and the suppression of NF-κB p65 inflammasome, whereas rehabilitation of oxidative stress by SIRT1 inhibitor attenuated the protective effect of Arbutin. In conclusion, we validated the results in an in vivo model constructed by NAIO3-injured mice. OCT and HE staining showed that Arbutin sustained retinal integrity in the case of oxidative damage in vivo, and the disorder of RPE cytochrome was alleviated through fundus observation. In summary, our findings identified that oxidative stress-induced mitochondrial malfunction and the subsequent senescence acceleration in RPE cells, whereas Arbutin inhibited TBHP-induced RPE degeneration via regulating the SIRT1/Foxo3a/PGC-1α/ß signaling pathway. These findings suggested that Arbutin is a new agent with potential applications in the development of AMD diseases.

Salvianolic Acid B Suppresses ER Stress-Induced NLRP3 Inflammasome and Pyroptosis via the AMPK/FoxO4 and Syndecan-4/Rac1 Signaling Pathways in Human Endothelial Progenitor Cells.

Mounting evidence demonstrates uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which generally results in endothelial dysfunction, a major pathogenetic factor of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), produced by Radix Salviae, exerts antioxidative and anti-inflammatory activities in multiple cell types. However, SalB's effects on ER stress-related inflammasome and endothelial dysfunction remain unknown. Here, we showed SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation, with declined intracellular reactive oxygen species (ROS) amounts and restored mitochondrial membrane potential (MMP), as well as increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). ER stress suppression by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Additionally, SalB alleviated ER stress-mediated pyroptotic cell death via the suppression of TXNIP/NLRP3 inflammasome, as evidenced by reduced cleavage of caspase-1 and interleukin- (IL-) 1ß and IL-18 secretion levels. Furthermore, this study provided a mechanistic basis that AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathway modulation by SalB substantially prevented BM-EPCs damage associated with ER stress by decreasing intracellular ROS amounts and inducing NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress triggered mitochondrial ROS release and NLRP3 generation in BM-EPCs, while SalB inhibits NLRP3 inflammasome-mediated pyroptotic cell death by regulating the AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 pathways. The current findings reveal SalB as a potential new candidate for the treatment of atherosclerotic heart disease.

Erianin Controls Collagen-Mediated Retinal Angiogenesis via the RhoA/ROCK1 Signaling Pathway Induced by the alpha2/beta1 Integrin-Collagen Interaction.

Purpose: Erianin has been reported to inhibit tumor activity by suppressing the expression of integrins. It is hypothesized that erianin can inhibit retinal neovascularization in collagen by suppressing the expression of integrins. With an aim to test this hypothesis, the regulation of erianin on collagen-mediated retinal angiogenesis via the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway induced by α2 and ß1 integrin-collagen interactions was investigated. Methods: The effects of erianin on human retinal vascular endothelial cells (HRVECs) were assessed in vitro using a hypoxia model in a three-dimensional cell culture induced by cobalt (II) chloride (CoCl2). A hypoxia-induced retinopathy model in adult zebrafish and zebrafish embryos was established to assess the antiangiogenic effect of erianin with and without vitreous collagen in vivo. The expression of α2 and ß1 integrin and RhoA/ROCK1 pathway in HRVECs and zebrafish retinas were analyzed. Results: In vitro, collagen improved the angiogenic potential of HRVECs, including migration, adhesion, and tube formation, in a three-dimensional cell culture model. Erianin suppressed the angiogenic processes of the CoCl2-induced hypoxia HRVEC model in a concentration-dependent manner. In vivo, erianin reduced retinal angiogenesis in the hypoxia-induced retinopathy model in adult and embryo zebrafish. Erianin inhibited the expression of α2 and ß1 integrin and RhoA/ROCK1 in a hypoxia-induced model in vitro in three-dimensional cell culture and in vivo in adult zebrafish. Conclusions: Collagen-mediated retinal angiogenesis may be regulated by erianin via the RhoA/ROCK1 signaling pathway induced by α2 and ß1 integrin-collagen interactions. These findings suggest that erianin has the therapeutic potential on intraocular collagen-mediated retinal angiogenesis.

Missense mutation in the PAX6 gene can cause a complex mild variable phenotype predominated by concomitant strabismus.

PURPOSE: We aimed to reveal the underlying genetic defect in a multigenerational Chinese family with autosomal dominant concomitant strabismus complicated by multiple ocular developmental abnormalities. METHODS: Comprehensive ophthalmic examinations were performed in 14 patients and 24 healthy family members. Whole exome sequencing was performed, and Sanger sequencing was used to confirm the probable mutation in all the family members. RESULTS: Concomitant strabismus was the predominant phenotype in the affected family members, although the patients also exhibited variable phenotypes, including nystagmus, mild iris abnormalities, myopia, cataract, and coloboma. An R208W mutation in PAX6 was identified as the pathogenic mutation in the affected family members. CONCLUSIONS: We recommend considering PAX6 as a candidate gene in the diagnostic screen for familial concomitant strabismus in order to avoid missed diagnosis of the mild ocular abnormalities. Careful examinations of mild ocular phenotypes are necessary for an accurate diagnosis of varied ocular abnormalities in the families with the PAX6 mutation, and proper diagnosis can facilitate genetic and clinical counseling for affected patients.

METTL3-Mediated m6A RNA Modification Regulates Corneal Injury Repair.

Limbal stem cells are essential for continuous corneal regeneration and injury repair. METTL3-catalyzed N6-methyladenosine (m6A) mRNA modifications are involved in many biological processes and play a specific role in stem cell regeneration, while the role of m6A modifications in corneal injury repair remains unknown. In this study, we generated a limbal stem cell-specific METTL3 knockout mouse model and studied the role of m6A in repairing corneal injury caused by alkali burn. The results showed that METTL3 knockout in the limbal stem cells promotes the in vivo cell proliferation and migration, leading to the fast repair of corneal injury. In addition, m6A modification profiling identified stem cell regulatory factors AHNAK and DDIT4 as m6A targets. Our study reveals the essential functions of m6A RNA modification in regulating injury repair and provides novel insights for clinical therapy of corneal diseases.

Maintaining blood retinal barrier homeostasis to attenuate retinal ischemia-reperfusion injury by targeting the KEAP1/NRF2/ARE pathway with lycopene.

Retinal ischemia-reperfusion (I/R) often results in intractable visual impairments, where blood retinal barrier (BRB) homeostasis mediated by retinal pigment epithelium (RPE) and retinal microvascular endothelium (RME) is crucial. However, strategies targeting the BRB are limited. Thus, we investigated the inconclusive effect of lycopene (LYC) in retinal protection under I/R. LYC elevated cellular viability and reversed oxidative stress in aRPE-19 cells/hRME cells under I/R conditions based on oxygen-glucose deprivation (OGD) in vitro. Molecular analysis showed that LYC promoted NRF2 expression and enhanced the downstream factors of the KEAP1/NRF2/ARE pathway: LYC increased the activities of antioxidants, including SOD and CAT, whereas it enhanced the mRNA expression of HO-1 (ho-1) and NQO-1 (nqo-1). The activation resulted in restrained ROS and MDA. On the other hand, LYC ameliorated the damage to retinal function and morphology in a mouse I/R model, which was established by unilateral ligation of the left pterygopalatine artery/external carotid artery and reperfusion. LYC promoted the expression of NRF2 in both the neural retina and the RPE choroid in vivo. This evidence revealed the potential of LYC in retinal protection under I/R, uncovering the pharmacological effect of the KEAP1/NRF2/ARE pathway in BRB targeting. The study generates new insights into scientific practices in retinal research.

Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation.

Lung cancer with loss-of-function of the LKB1 tumor suppressor is a common aggressive subgroup with no effective therapies. LKB1-deficiency induces constitutive activation of cAMP/CREB-mediated transcription by a family of three CREB-regulated transcription coactivators (CRTC1-3). However, the significance and mechanism of CRTC activation in promoting the aggressive phenotype of LKB1-null cancer remain poorly characterized. Here, we observed overlapping CRTC expression patterns and mild growth phenotypes of individual CRTC-knockouts in lung cancer, suggesting functional redundancy of CRTC1-3. We consequently designed a dominant-negative mutant (dnCRTC) to block all three CRTCs to bind and co-activate CREB. Expression of dnCRTC efficiently inhibited the aberrantly activated cAMP/CREB-mediated oncogenic transcriptional program induced by LKB1-deficiency, and specifically blocked the growth of human and murine LKB1-inactivated lung cancer. Collectively, this study provides direct proof for an essential role of the CRTC-CREB activation in promoting the malignant phenotypes of LKB1-null lung cancer and proposes the CRTC-CREB interaction interface as a novel therapeutic target.

Lateral rectus muscle differentiation potential in paralytic esotropia patients.

PURPOSE AND BACKGROUND: Recently, we found that maximal medial rectus recession and lateral rectus resection in patients with complete lateral rectus paralysis resulted in a partial restoration of abduction. In an attempt to understand some of the mechanisms involved with this effect we examined gene expression profiles of lateral recti from these patients, with our focus being directed to genes related to myogenesis. MATERIALS AND METHODS: Lateral recti resected from patients with complete lateral rectus paralysis and those from concomitant esotropia (controls) were collected. Differences in gene expression profiles between these two groups were examined using microarray analysis and quantitative Reverse-transcription PCR (qRT-PCR). RESULTS: A total of 3056 differentially expressed genes (DEGs) were identified between these two groups. Within the paralytic esotropia group, 2081 genes were up-regulated and 975 down-regulated. The results of RT-PCR revealed that PAX7, MYOG, PITX1, SIX1 and SIX4 showed higher levels of expression, while that of MYOD a lower level of expression within the paralytic esotropia group as compared with that in the control group (p < 0.05). CONCLUSION: The decreased expression of MYOD in the paralytic esotropia group suggested that extraocular muscle satellite cell (EOMSCs) differentiation processes were inhibited. Whereas the high expression levels of PAX7, SIX1/4 and MYOG, suggested that the EOMSCs were showing an effective potential for differentiation. The stimulation resulting from muscle surgery may induce EOMSCs to differentiate and thus restore abduction function.

METTL3 attenuates proliferative vitreoretinopathy and epithelial-mesenchymal transition of retinal pigment epithelial cells via wnt/ß-catenin pathway.

Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT-PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE-19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE-19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE-19 cells, total m6A abundance and the METTL3 expression were down-regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFß1 to trigger EMT by regulating wnt/ß -catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE-19 cells. In vivo, intravitreal injection of METTL3-overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE-19 cells in vitro and suppresses the PVR process in vivo.

Lidocaine Inhibits Myoblast Cell Migration and Myogenic Differentiation Through Activation of the Notch Pathway.

PURPOSE: To assess the cellular and molecular effects of lidocaine on muscles/myoblasts. METHODS: Cultured myogenic precursor (C2C12) cells were treated with varying concentrations of lidocaine. RESULTS: Cell viability of C2C12 cells was inhibited by lidocaine in a concentration-dependent manner, with concentrations ≥0.08%, producing a dramatic reduction in cell viability. These ≥0.08% concentrations of lidocaine arrested cell cycles of C2C12 cells in the G0/G1 phase. Moreover, lidocaine inhibited cell migration and myogenic processes in C2C12 cells at low concentrations. Results from QRT-PCR assays revealed that following treatment with lidocaine, Notch1, Notch2, Hes1, Csl and Dll4 all showed higher levels of expression, while no changes were observed in Mmal1, Hey1, Dll1 and Jag1. CONCLUSION: This work provides the first description of the effects of lidocaine upon the regeneration of muscles and maintenance of satellite cells at the cellular and molecular levels. In specific, we found that the Dll4-Notch-Csl-Hes1 axis was up-regulated suggesting that the Notch signaling pathway was involved in producing these effects of lidocaine. These findings provide a new and important foundation for future investigations into the effects of drug therapies in muscle diseases.