The Role of Ubiquitination and the E3 Ligase Nedd4 in Regulating Corneal Epithelial Wound Healing.

Purpose: Ubiquitination serves as a fundamental post-translational modification in numerous cellular events. Yet, its role in regulating corneal epithelial wound healing (CEWH) remains elusive. This study endeavored to determine the function and mechanism of ubiquitination in CEWH. Methods: Western blot and immunoprecipitation were used to discern ubiquitination alterations during CEWH in mice. Interventions, including neuronally expressed developmentally downregulated 4 (Nedd4) siRNA and proteasome/lysosome inhibitor, assessed their impact on CEWH. In vitro analyses, such as the scratch wound assay, MTS assay, and EdU staining, were conducted to gauge cell migration and proliferation in human corneal epithelial cells (HCECs). Moreover, transfection of miR-30/200 coupled with a luciferase activity assay ascertained their regulatory mechanism on Nedd4. Results: Global ubiquitination levels were markedly increased during the mouse CEWH. Importantly, the application of either proteasomal or lysosomal inhibitors notably impeded the healing process both in vivo and in vitro. Furthermore, Nedd4 was identified as an essential E3 ligase for CEWH. Nedd4 expression was significantly upregulated during CEWH. In vivo studies revealed that downregulation of Nedd4 substantially delayed CEWH, whereas further investigations underscored its role in regulating cell proliferation and migration, through the Stat3 pathway by targeting phosphatase and tensin homolog (PTEN). Notably, our findings pinpointed miR-30/200 family members as direct regulators of Nedd4. Conclusions: Ubiquitination holds pivotal significance in orchestrating CEWH. The critical E3 ligase Nedd4, under the regulatory purview of miR-30 and miR-200, facilitates CEWH through PTEN-mediated Stat3 signaling. This revelation sheds light on a prospective therapeutic target within the realm of CEWH.

Joint CB1 and NGF Receptor Activation Suppresses TRPM8 Activation in Etoposide-Resistant Retinoblastoma Cells.

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.

Unraveling the Intricate Network of lncRNAs in Corneal Epithelial Wound Healing: Insights Into the Regulatory Role of linc17500.

Purpose: Epigenetic mechanisms orchestrate a harmonious process of corneal epithelial wound healing (CEWH). However, the precise role of long non-coding RNAs (lncRNAs) as key epigenetic regulators in mediating CEWH remains elusive. Here, we aimed to elucidate the functional contribution of lncRNAs in regulating CEWH. Methods: We used a microarray to characterize lncRNA expression profiling during mouse CEWH. Subsequently, the aberrant lncRNAs and their cis-associated genes were subjected to comprehensive Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blot analyses were performed to determine the expression profiles of key markers during CEWH. The in vivo effects of linc17500 on this process were investigated through targeted small interfering RNA (siRNA) injection. Post-siRNA treatment, corneal re-epithelialization was assessed, alongside the expression of cytokeratins 12 and 14 (Krt12 and Krt14) and Ki67. Effects of linc17500 on mouse corneal epithelial cell (TKE2) proliferation, cell cycle, and migration were assessed by multicellular tumor spheroids (MTS), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and scratch-wound assay, respectively. Results: Microarray analysis revealed dysregulation of numerous lncRNA candidates during CEWH. Bioinformatic analysis provided valuable annotations regarding the cis-associated genes of these lncRNAs. In vivo experiments demonstrated that knockdown of linc17500 resulted in delayed CEWH. Furthermore, the knockdown of linc17500 and its cis-associated gene, CDC28 protein kinase regulatory subunit 2 (Cks2), was found to impede TKE2 cell proliferation and migration. Notably, downregulation of linc17500 in TKE2 cells led to suppression of the activation status of Akt and Rb. Conclusions: This study sheds light on the significant involvement of lncRNAs in mediating CEWH and highlights the regulatory role of linc17500 on TKE2 cell behavior. Translational Relevance: These findings provide valuable insights for future therapeutic research aimed at addressing corneal wound complications.

METTL3-Mediated RNA m6A Modification Regulates the Angiogenic Behaviors of Retinal Endothelial Cells by Methylating MMP2 and TIE2.

Purpose: N6-methyladenosine (m6A) is a commonly occurring modification of mRNAs, catalyzed by a complex containing methyltransferase like 3 (METTL3). Our research aims to explore how METTL3-dependent m6A modification affects the functions of retinal endothelial cells (RECs). Methods: An oxygen-induced retinopathy (OIR) mouse model was established, and RECs were isolated using magnetic beads method. Human retinal microvascular endothelial cells (HRMECs) were treated with normoxia (21% O2) or hypoxia (1% O2). Dot blot assay determined m6A modification levels. Quantitative RT-PCR and Western blot detected the mRNA and protein expression levels of the target candidates, respectively. Genes were knocked down by small interfering RNA transfection. Matrigel-based angiogenesis and transwell assays evaluated the abilities of endothelial tube formation and migration, respectively. Methylated RNA immunoprecipitation-qPCR determined the levels of m6A modification in the target genes. Results: The m6A modification levels were significantly upregulated in the retinas and RECs of OIR mice. Exposure to hypoxia significantly elevated both METTL3 expression and m6A modification levels in HRMECs. METTL3 knockdown curtailed endothelial tube formation and migration in vitro under both normoxic and hypoxic conditions. Concurrently, this knockdown in HRMECs resulted in reduced m6A modification levels of MMP2 and TIE2 transcripts, subsequently leading to a decrease in their respective protein expressions. Notably, knockdown of MMP2 and TIE2 also markedly inhibited the angiogenic activities of HRMECs. Conclusions: METTL3-mediated m6A modification promotes the angiogenic behaviors of RECs by targeting MMP2 and TIE2, suggesting its significance in retinal angiogenesis and METTL3 as a potential therapeutic target.

Engineered Knockout of TRPA1 Inhibits Laser-Induced Choroidal Neovascularization Along With Associated TGFß1 Expression and Neutrophil Infiltration.

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser-induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow-derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor ß1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases.

L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type 1 Activation in Human Corneal Epithelial Cells.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1-3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.

Chronic Mg2+ Deficiency Does Not Impair Insulin Secretion in Mice.

Magnesium is an essential mediator of a vast number of critical enzymatic cellular reactions in the human body. Some clinical epidemiological studies suggest that hypomagnesemia accounts for declines in insulin secretion in patients with type 2 diabetes (T2D); however, the results of various experimental studies do not support this notion. To address this discrepancy, we assessed the short- and long-term effects of hypomagnesemia on ß-cell function and insulin secretion in primary mouse islets of Langerhans and in a mouse model of hypomagnesemia known as Trpm6Δ17 /fl;Villin1-Cre mice. We found that lowering the extracellular Mg2+ concentration from 1.2 mM to either 0.6 or 0.1 mM remarkably increased glucose-induced insulin secretion (GIIS) in primary islets isolated from C57BL/6 mice. Similarly, both the plasma insulin levels and GIIS rose in isolated islets of Trpm6Δ17 /fl;Villin1-Cre mice. We attribute these rises to augmented increases in intracellular Ca2+ oscillations in pancreatic ß-cells. However, the glycemic metabolic profile was not impaired in Trpm6Δ17 /fl;Villin1-Cre mice, suggesting that chronic hypomagnesemia does not lead to insulin resistance. Collectively, the results of this study suggest that neither acute nor chronic Mg2+ deficiency suppresses glucose-induced rises in insulin secretion. Even though hypomagnesemia can be symptomatic of T2D, such deficiency may not account for declines in insulin release in this disease.

Tenascins and osteopontin in biological response in cornea.

The structural composition, integrity and regular curvature of the cornea contribute to the maintenance of its transparency and vision. Disruption of its integrity caused by injury results in scarring, inflammation and neovascularization followed by losses in transparency. These sight compromising effects is caused by dysfunctional corneal resident cell responses induced by the wound healing process. Upregulation of growth factors/cytokines and neuropeptides affect development of aberrant behavior. These factors trigger keratocytes to first transform into activated fibroblasts and then to myofibroblasts. Myofibroblasts express extracellular matrix components for tissue repair and contract the tissue to facilitate wound closure. Proper remodeling following primary repair is critical for restoration of transparency and visual function. Extracellular matrix components contributing to the healing process are divided into two groups; a group of classical tissue structural components and matrix macromolecules that modulate cell behaviors/activities besides being integrated into the matrix structure. The latter components are designated as matricellular proteins. Their functionality is elicited through mechanisms which modulate the scaffold integrity, cell behaviors, activation/inactivation of either growth factors or cytoplasmic signaling regulation. We discuss here the functional roles of matricellular proteins in mediating injury-induced corneal tissue repair. The roles are described of major matricellular proteins, which include tenascin C, tenascin X and osteopontin. Focus is directed towards dealing with their roles in modulating individual activities of wound healing-related growth factors, e. g., transforming growth factor ß (TGF ß). Modulation of matricellular protein functions could encompass a potential novel strategy to improve the outcome of injury-induced corneal wound healing.

Epigenetic Activation of Circadian Clock Genes Elicits Inflammation in Experimental Murine Dry Eye.

PURPOSE: To explore whether circadian clock genes contribute to elicit inflammation in experimental dry eye (EDE). METHODS: RNA sequencing analyzed mRNA expression patterns in EDE model. RT-qPCR and/or Western blot determined the expression of inflammatory factors and circadian genes during EDE. MethylTarget™ assays determined the promoter methylation levels of Per genes in vivo. Per2 or Per3 knockdown assessed their effects on inflammatory factors in vitro. RESULTS: We utilized an intelligently controlled environmental system (ICES) to establish a mouse EDE model. The significant upregulated genes were enriched for circadian rhythms. Therein lied oscillatory and time-dependent upregulation of PER2 and PER3, as well as their promoter hypomethylation during EDE. Silencing PER2 or PER3 significantly decreased inflammatory factor expression and also reversed such increased inflammatory response in azacitidine (AZA) treatment in vitro model. CONCLUSIONS: Our findings suggest that DNA methylation mediated the upregulation of PER2 and PER3, leading to inflammatory response in EDE.

The RNA m5C Methylase NSUN2 Modulates Corneal Epithelial Wound Healing.

Purpose: The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH. Methods: RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH. Results: The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration. Conclusions: NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.

Calcipotriol Attenuates Form Deprivation Myopia Through a Signaling Pathway Parallel to TGF-ß2-Induced Increases in Collagen Expression.

Purpose: To determine the role of calcipotriol, a vitamin D3 analogue, in myopia development and altering the expression of scleral α1 chain of type I collagen (Col1α1) in mice. We also aimed to identify if the signaling pathway mediating the above changes is different from the one involved in transforming growth factor ß2 (TGF-ß2)-mediated increases of COL1A1 in cultured human scleral fibroblasts (HSFs). Methods: C57BL/6J mice were either intraperitoneally injected with calcipotriol and subjected to form deprivation (FD) or exposed to normal refractive development for 4 weeks. Scleral vitamin D receptor (Vdr) expression was knocked down using a Sub-Tenon's capsule injection of an adeno-associated virus-packaged short hairpin RNA (AAV8-shRNA). Refraction and biometric measurements evaluated myopia development. A combination of knockdown and induction strategies determined the relative contributions of the vitamin D3 and the TGF-ß2 signaling pathways in modulating COL1A1 expression in HSFs. Results: Calcipotriol injections suppressed FD-induced myopia (FDM), but it had no significant effect on normal refractive development. AAV8-shRNA injection reduced Vdr mRNA expression by 42% and shifted the refraction toward myopia (-3.15 ± 0.99D, means ± SEM) in normal eyes. In HSFs, VDR knockdown reduced calcipotriol-induced rises in COL1A1 expression, but it did not alter TGF-ß2-induced increases in COL1A1 expression. Additionally, TGF-ß2 augmented calcipotriol-induced rises in COL1A1 expression. TGF-ß receptor (TGFBRI/II) knockdown blunted TGF-ß2-induced increases in COL1A1 expression, whereas calcipotriol-induced increases in VDR and COL1A1 expression levels were unaltered. Conclusions: Scleral vitamin D3 inhibits myopia development in mice, potentially by activating a VDR-dependent signaling pathway and increasing scleral COL1A1 expression levels.

TRPM7 kinase is required for insulin production and compensatory islet responses during obesity.

Most overweight individuals do not develop diabetes due to compensatory islet responses to restore glucose homeostasis. Therefore, regulatory pathways that promote ß cell compensation are potential targets for treatment of diabetes. The transient receptor potential cation channel subfamily M member 7 protein (TRPM7), harboring a cation channel and a serine/threonine kinase, has been implicated in controlling cell growth and proliferation. Here, we report that selective deletion of Trpm7 in ß cells disrupted insulin secretion and led to progressive glucose intolerance. We indicate that the diminished insulinotropic response in ß cell-specific Trpm7-knockout mice was caused by decreased insulin production because of impaired enzymatic activity of this protein. Accordingly, high-fat-fed mice with a genetic loss of TRPM7 kinase activity displayed a marked glucose intolerance accompanied by hyperglycemia. These detrimental glucoregulatory effects were engendered by reduced compensatory ß cell responses because of mitigated protein kinase B (AKT)/ERK signaling. Collectively, our data identify TRPM7 kinase as a potentially novel regulator of insulin synthesis, ß cell dynamics, and glucose homeostasis under obesogenic diet.

Sirt1 Regulates Corneal Epithelial Migration by Deacetylating Cortactin.

Purpose: Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+) dependent deacetylase, which plays an essential role in cellular metabolism, autophagy, and chromatin accessibility. Our study aimed to determine its role in controlling corneal epithelial wound healing (CEWH). Methods: Corneal epithelial (CE)-specific Sirt1 deletion mice were created using the Cre-lox system. CE debridement was used to create a CEWH model. Corneal epithelial cells (CECs) were collected with an Algerbrush. Western blot analysis and RT-qPCR were performed to determine protein and mRNA expression levels. SiRNA transfection technology knocked down SIRT1 and cortactin expression levels in human corneal epithelial cells. Scratch wound assay, MTS assay, and TUNEL assay determined cell migratory, proliferative, and apoptotic behavior, respectively. Co-immunoprecipitation probed for SIRT1 and cortactin interaction. Immunofluorescence staining evaluated the location and expression levels of SIRT1, cortactin, acetylated-cortactin, and F-actin. Results: During CEWH, increases in SIRT1 mRNA and protein expression levels accompanied the downregulation of acetylated lysine in non-histone proteins. The loss of SIRT1 function reduced cell migration and, in turn, delayed CEWH. SIRT1 bound to and deacetylated cortactin in vitro and in vivo. Loss of either SIRT1 or cortactin suppressed wound edge lamellipodia formation, which is consistent with migration retardation. Conclusions: During CEWH, SIRT1 upregulation and its modification of cortactin boost CEC migration by increasing the development of lamellipodia at the wound edge. Therefore SIRT1 may serve as a potential target to enhance CEWH.

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice.

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood. It remains elusive whether corneal lesion is caused by exposure to keratitis (lagophthalmos) of EOB phenotype or the loss of Spns2 directly perturbs the corneal tissue morphogenesis and intra-eyelid structures. Therefore, we investigated differences between the cornea and ocular adnexa morphogenesis in KO and wild-type (WT) embryos and adults as well. The loss of Spns2 perturbs cornea morphogenesis during embryonic development as early as E16.5 besides EOB phenotype. Histology showed that the corneal stroma was thinner with less extracellular matrix accumulation, e.g., collagen and keratocan in the KO mouse. Epithelial stratification, expression of keratin 12 and formation of desmosomes and hemidesmosomes were also perturbed in these KO corneas. Lacking Spns2 impaired morphogenesis of the Meibomian glands and of orbicularis oculi muscles. KO glands were labeled for ELOVL4 and PPARγ and were Oil-Red O-positive, suggesting KO acinar cells possessed functionality as the glands. This is the first report on the roles of Spns2 in corneal and Meibomian gland morphogenesis. Corneal tissue destruction in an adult KO mouse might be due to not only lagophthalmos but also to an impaired morphogenesis of cornea, Meibomian glands, and orbicularis oculi muscle.

NSUN2-mediated RNA m5C modification modulates uveal melanoma cell proliferation and migration.

RNA 5-methylcytosine (m5C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m5C in UM. Initially, we identified significantly elevated global RNA m5C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m5C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m5C-RIP-qPCR, and luciferase assay identified ß-Catenin (CTNNB1) as a direct target of NSUN2-mediated m5C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m5C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.

Inhibition of CD146 lessens uveal melanoma progression through reducing angiogenesis and vasculogenic mimicry.

PURPOSE: Anti-angiogenesis drug therapy is ineffective in treating uveal melanoma since it only targets angiogenesis leaving vasculogenic mimicry aside. There is no effective clinical strategy targeting vasculogenic mimicry, yet. We show here that CD146 is a novel target to inhibit uveal melanoma progression since it regulates both uveal melanoma angiogenesis and vasculogenic mimicry activity. METHODS: CD146 inhibition was achieved with its specific siRNAs or antibody AA98. Tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation, migration, invasion of uveal melanoma cells were evaluated after CD146 inhibition. The underlying mechanisms were investigated by Western blot and immunofluorescence. Finally, uveal melanoma cells were injected subretinally into the eyes of nude mice and AA98 was administrated. Tumor size was revealed by H&E staining, and angiogenesis and vasculogenic mimicry were evaluated with CD31-PAS staining. RESULTS: CD146 inhibition induced declines in tube formation and migration of primary human retinal microvascular endothelial cells and tube-like structure formation of uveal melanoma cells. CD146 mediated VEGFR/AKT/p38/NF-κB and FAK/VE-cadherin signal cascades were partially responsible for these biological effects. CD146 blockade by siRNA or AA98 also resulted in inhibition of migration and invasion as well as EMT process of uveal melanoma cells. The physiological relevance of such declines was confirmed by showing that AA98 treatment markedly suppressed the tumor growth, angiogenesis and vasculogenic mimicry induced by implantation of uveal melanoma cells into the eyes of nude mice. CONCLUSIONS: CD146 is a novel mediator of both angiogenesis and vasculogenic mimicry in uveal melanoma. Its antibody AA98 has the potency to be developed as a new antibody drug for treating uveal melanoma. Our results warrant further assessment of CD146 as a potential target to improve therapeutic management of uveal melanoma in a clinical setting.

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients.

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1ß expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3' UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1ß expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1ß tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1ß expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1ß secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

Form-deprivation myopia downregulates calcium levels in retinal horizontal cells in mice.

The process of eye axis lengthening in myopic eyes is regulated by multiple mechanisms in the retina, and horizontal cells (HCs) are an essential interneuron in the visual regulatory system. Wherein intracellular Ca2+ plays an important role in the events involved in the regulatory role of HCs in the retinal neural network. It is unknown if intracellular Ca2+ regulation in HCs mediates changes in the retinal neural network during myopia progression. We describe here a novel calcium fluorescence indicator system that monitors HCs' intracellular Ca2+ levels during form-deprivation myopia (FDM) in mice. AAV injection of GCaMP6s, as a protein calcium sensor, into a Gja10-Cre mouse monitored the changes in Ca2+signaling in HC that accompany FDM progression in mice. An alternative Gja10-Cre/Ai96-GCaMP6s mouse model was created by cross mating Gja10-Cre with Ai96 mice. Immunofluorescence imaging and live imaging of the retinal cells verified the identity of these animal models. Changes in retinal horizontal cellular Ca2+ levels were resolved during FDM development. The numbers of GCaMP6s and the proportion of HCs were tracked based on profiling changes in GCaMP6s+calbindin+/calbindin+ coimmunostaining patterns. They significantly decreased more after either two days (P < 0.01) or two weeks (P < 0.001) in form deprived eyes than in the untreated fellow eyes. These decreases in their proportion reached significance only in the retinal central region rather than also in the retinal periphery. A novel approach employing a GCaMP6s mouse model was developed that may ultimately clarify if HCs mediate Ca2+ signals that contribute to controlling FDM progression in mice. The results indicate so far that FDM progression is associated with declines in HC Ca2+ signaling activity.

TRPV4 Stimulation Level Regulates Ca2+-Dependent Control of Human Corneal Endothelial Cell Viability and Survival.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.