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.

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.

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.

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.

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.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression.

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, -3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-ß2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, ß-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Canonical Wnt Signaling Drives Myopia Development and Can Be Pharmacologically Modulated.

Purpose: The purpose of this study was to evaluate the role of the canonical Wnt signaling in the development of the myopia. Methods: Plasma from adult patients with myopia, myopic animal models including the adenomatous polyposis coli (APC) gene mutation mouse model, and the form deprivation (FD) induced mouse model of myopia were used. Niclosamide, a canonical Wnt pathway inhibitor, was orally administrated in animal models. Plasma levels of DKK-1 were determined by using enzyme-linked immunosorbent assay. Refraction, vitreous chamber depth (VCD), axial length (AL), and other parameters, were measured at the end of the FD treatment. Canonical Wnt signaling changes were evaluated by Western blot analysis and immunostaining analysis. Results: Plasma level of Wnt inhibitor DKK-1 was markedly decreased in patients with myopia. Meanwhile, the canonical Wnt pathway was progressively activated during myopia development in mice. Moreover, inhibition of canonical Wnt signaling by niclosamide in mouse models markedly reduced lens thickness (LT), VCD, and AL elongation, resulting in myopia inhibition. Conclusions: Dysregulation of canonical Wnt signaling is a characteristic of myopia and targeting Wnt signaling pathways has potential as a therapeutic strategy for myopia.

Phenotypic and transcriptomic changes in the corneal epithelium following exposure to cigarette smoke.

Cigarette smoke extract (CSE), a complex mixture of compounds, contributes to a range of eye diseases; however, the underlying pathophysiological responses to tobacco smoke remain ambiguous. The purpose of the present study was to evaluate the cigarette smoke-induced phenotypic and transcriptomic changes in the corneal epithelium with a view to elucidating the likely underlying mechanism. Accordingly, for the first time, we characterized the genome-wide effects of CSE on the corneal epithelium. The ocular surface of the mice in the experimental groups was exposed to CSE for 1 h per day for a period of one week, while mice in the control group were exposed to preservative-free artificial tears. Corneal fluorescein staining, in vivo confocal microscopy and scanning electron microscopy were performed to examine the corneal ultrastructure. Transcriptome sequencing and bioinformatics analysis were performed followed by RT-qPCR to validate gene expression changes. The results indicate that CSE exposure disrupted the structural integrity of the superficial epithelium, decreased the density of microvilli, and compromised the corneal epithelial barrier intactness. RNA-seq revealed 667 differentially expressed genes, and functional analysis highlighted the enhancement of several biological processes such as antioxidant activity and the response to oxidative stress. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that glutathione metabolism and drug metabolism cytochrome P450 were the most relevant pathways contributing to the effects of CSE on the corneal epithelium. Protein-protein interaction (PPI) network analysis illustrated that GCLC, NQO1, and HMOX1 were the most relevant nodes. In conclusion, the present study indicates that CSE exposure induces changes in the phenotype and genotype of the corneal epithelium. The antioxidant response element is essential for counteracting the effects of cigarette smoke on this tissue layer. These results shed novel insights into how cigarette smoke damages this ocular surface.

Onion Epithelial Membrane Scaffolds Transfer Corneal Epithelial Layers in Reconstruction Surgery.

Plants and their extracts have been used especially in China for more than ten centuries for preventing and treating disease. However, there are only few reports describing their use in animal cell culture and tissue transplantation. In this study, onion epithelial membranes (OEM) is used as scaffolds to support cultures of a variety of cells such as fibroblasts and epithelial cells notably; they maintain the phenotypic characteristics of corneal epithelial cells. This improvement includes preservation of the proliferative potential and stemness of rabbit corneal epithelial cells (RCECs). Such an outcome suggests that this cost-effective technology warrants further evaluation to determine if OEM is a viable candidate for use as scaffolds in corneal epithelial transplantation surgery. To test this possibility, rabbit corneal epithelial cells expanded on OEM are transplanted to treat corneal epithelial defects in limbal stem cell deficient rabbits. This procedure is successful because it shortens the time required for wound healing to restore losses in corneal epithelial integrity, and forms a more compact and stratified epithelium framework than the untreated group. Ultimately, should they be proven to be effective in other relevant animal model systems, their usefulness for treating wounds in a clinical setting warrants consideration.

Up-Regulation of Matrix Metalloproteinase-2 by Scleral Monocyte-Derived Macrophages Contributes to Myopia Development.

Myopia is a leading cause of visual impairment worldwide. This sight-compromising condition is associated with scleral thinning, extracellular matrix remodeling, and inappropriate optical axial length elongation. Although macrophages are present in the sclera, their involvement in this condition is unknown. By using a form-deprivation myopia (FDM) mouse model, we found that both the scleral macrophage density and their matrix metalloproteinase-2 (MMP-2) expression levels increased in myopic eyes. Partial scleral macrophage depletion by clodronate shifted the refraction toward hyperopia in both the form-deprived and the untreated fellow eyes compared with their respective counterparts in the vehicle-injected control mice. However, this procedure did not alter susceptibility to FDM. FDM development was 59% less in the macrophage-specific Mmp2 deletion (LysMCreMmp-2fl/fl) mice than in their Cre-negative littermates (Mmp2fl/fl mice). Moreover, the expression of scleral C-C motif chemokine ligand-2 (CCL2), which is a potent monocyte chemoattractant recruiting monocytes to tissue sites, was increased during myopia progression. However, the increase in the density of scleral macrophages and myopia development were suppressed in fibroblast-specific Ccl2 deletion mice. These declines suggested that the increase in scleral macrophage density in myopic eyes stems from the up-regulation of scleral Ccl2 expression in fibroblasts, which, in turn, promotes monocytes recruitment. In summary, scleral monocyte-derived macrophages contribute to myopia development through enhancing MMP-2 expression in mice.

RNA m6 A methylation regulates uveal melanoma cell proliferation, migration, and invasion by targeting c-Met.

N6 -methyladenosine (m6 A) is a novel epitranscriptomic marker that contributes to regulating diverse biological processes through controlling messenger RNA metabolism. However, it is unknown if m6 A RNA methylation affects uveal melanoma (UM) development. To address this question, we probed its function and molecular mechanism in UM. Initially, we demonstrated that global RNA m6 A methylation levels were dramatically elevated in both UM cell lines and clinical specimens. Meanwhile, we found that METTL3, a main m6 A regulatory enzyme, was significantly increased in UM cells and specimens. Subsequently, cycloleucine (Cyc) or METTL3 targeted small interfering RNA was used to block m6 A methylation in UM cells. We found that Cyc or silencing METTL3 significantly suppressed UM cell proliferation and colony formation through cell cycle G1 arrest, as well as migration and invasion by functional analysis. On the other hand, overexpression of METTL3 had the opposite effects. Furthermore, bioinformatics and methylated RNA immunoprecipitation-quantitative polymerase chain reaction identified c-Met as a direct target of m6 A methylation in UM cells. In addition, western blot analysis showed that Cyc or knockdown of METTL3 downregulated c-Met, p-Akt, and cell cycle-related protein levels in UM cells. Taken together, our results demonstrate that METTL3-mediated m6 A RNA methylation modulates UM cell proliferation, migration, and invasion by targeting c-Met. Such a modification acts as a critical oncogenic regulator in UM development.

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line.

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 µM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 µM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell-cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell-cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.

miR-142-3p suppresses uveal melanoma by targeting CDC25C, TGFßR1, GNAQ, WASL, and RAC1.

Purpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis. Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGFß signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGFßR1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGFßR1, GNAQ, WASL, or RAC1 accounts for declines in cell migration. Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse.

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-ß1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-ß1 mRNA expression levels in WT tissue were blunted in the TRPA1-/- counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.

SIRT1 Deletion Impairs Retinal Endothelial Cell Migration Through Downregulation of VEGF-A/VEGFR-2 and MMP14.

Purpose: Silent information regulator protein 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that is abundantly expressed in vascular endothelial cells (VECs), and it has an essential role in angiogenesis. However, its contribution to retinal vascular development remains unclear. Here we characterize its involvement in regulating this process under both physiological and pathologic conditions. Methods: Endothelium-specific Sirt1 knockout mice were established using the Cre-lox system. VECs were isolated using magnetic beads and identified by immunostaining. Retinal whole-mount staining analyzed the retinal vascular patterns. SIRT1 was knocked down or overexpressed in human retinal microvascular endothelial cells (HRMECs) using small interfering RNA (siRNA) or lentivirus infection, respectively. Scratch assay, Transwell, and Matrigel angiogenesis assay evaluated cell migration and tube formation, respectively. Quantitative RT-PCR analyzed genes regulating VEC migration. Western blotting determined protein expression. Coimmunoprecipitation detected the interaction of hypoxia-inducible factor 1α (HIF-1α) and SIRT1 as well as acetylation status of HIF-1α. Results: Specific deletion of Sirt1 in VECs dramatically delayed retinal vessel expansion and reduced vessel density. In the oxygen-induced retinopathy (OIR) mouse model, Sirt1 ablation markedly suppressed retinal revascularization and consequently increased retinal avascularity. SIRT1 downregulation in HRMECs inhibited cell migration and tube formation, while overexpression of SIRT1 had the opposite effects. Vascular endothelial growth factor-A (VEGF-A)/VEGF receptor-2 (VEGFR-2), and matrix metalloproteinases 14 (MMP14) expression significantly declined in Sirt1-null VECs, as well as SIRT1 siRNA-transfected HRMECs. SIRT1 downregulation upregulated the HIF-1α acetylation status. Conversely, SIRT1 overexpression decreased this response. Conclusions: SIRT1 contributes to both physiological and pathologic retinal angiogenesis through promoting retinal VEC migration. Its underlying molecular mechanism involves SIRT1-mediated deacetylation of HIF-1α and subsequent upregulation of VEGF-A/VEGFR-2 and MMP14 expression.

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells.

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 µM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 µM menthol and 20 µM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 µM) but not by a highly selective TRPM8 blocker, AMTB (20 µM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 µM) and menthol (100 µM) increased the whole-cell currents, whereas 20 µM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.

Corneal Collagen Cross-Linking With Riboflavin and UVA Regulates Hemangiogenesis and Lymphangiogenesis in Rats.

Purpose: The purpose of this study was to determine whether corneal collagen crosslinking (CXL) inhibits hemangiogenesis and lymphangiogenesis during acute corneal inflammation in an in vivo rat model. Methods: Inflammatory corneal neovascularization was induced by suture placement into a rat cornea. At day 3 after suture, a CXL protocol using riboflavin and UVA was administered after mechanical epithelial debridement. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically. CD45 and CD68 immunostaining evaluated corneal leucocyte and macrophage immune cell infiltration, respectively. A TUNEL assay detected stromal cell apoptosis. Quantitative RT-PCR analysis identified angiogenic and lymphangiogenic genes as well as proinflammatory cytokine expression. Western blot analysis characterized vascular endothelial cell CD31 and lymphatic vessel endothelial hyaluronan receptor (LYVE-1) protein expression. Results: CXL treatment significantly reduced corneal pathologic suture-induced hemangiogenesis and lymphangiogenesis 7 days after suture emplacement, but this procedure failed to affect hemangiogenesis and lymphangiogenesis 14 days after suture. Increased cell apoptosis and reduced CD45+ and CD68+ cell infiltration were evident in CXL-treated rats on days 7 and 14 after suture emplacement. CXL treatment significantly decreased angiogenic and lymphangiogenic mRNA expression levels and both CD31 and LYVE-1 protein expression levels, whereas it increased proinflammatory cytokine levels on day 7 after suture emplacement. However, on day 14 after corneal neovascularization, angiogenic and lymphangiogenic mRNA gene expression levels were upregulated along with hematic CD31 and lymphatic LYVE-1 protein expression. Conclusions: CXL treatment only temporarily inhibits corneal inflammatory-associated hemangiogenesis and lymphangiogenesis in vivo. Such insight suggests that future studies are warranted to develop novel CXL strategies with longer-lasting effectiveness in attenuating hemantic- and lymphatic-related corneal diseases.

Cause and Effect Relationship between Changes in Scleral Matrix Metallopeptidase-2 Expression and Myopia Development in Mice.

Myopia is a serious sight-compromising condition in which decreases in scleral biomechanical strength are associated with protease up-regulation resulting in thinning of its collagenous framework and changes in the extracellular matrix composition. Matrix metallopeptidase (MMP)-2 is one of the known proteases mediating these alterations. To determine whether MMP-2 up-regulation precedes myopia development, the direct effects of gain and loss in Mmp2 gene function were evaluated on refractive development and form deprivation myopia in mice. Four weeks after injecting an adeno-associated virus serotype 8 packaged Mmp2 overexpression vector (AAV8-Mmp2), scleral MMP-2 up-regulation was accompanied by significant myopia in a normal visual environment. In contrast, AAV8 packaging with shRNA targeting Mmp2 inhibited rises in MMP-2 expression induced by form deprivation by 54% and reduced myopia development by 23% compared with eyes injected with an irrelevant scrambled sequence. Because opposing changes in MMP-2 protein expression levels had corresponding effects on myopia progression, up-regulation of this protease contributes to inducing this condition. This notion of a cause-and-effect relationship between MMP-2 up-regulation and myopia development is supported by showing that form-deprived myopia development was attenuated by 27% in fibroblast-specific Mmp2 deletion (S100a4creMmp2fl/fl) mice relative to Cre-negative littermates (Mmp2fl/fl). Therefore, MMP-2 is a potential drug target for inhibiting myopia progression.

Continuous-light versus pulsed-light accelerated corneal crosslinking with ultraviolet-A and riboflavin.

PURPOSE: To determine whether the pulsed-light ultraviolet-A (UVA) accelerated corneal crosslinking (CXL) procedure is more efficacious and selective than its continuous-light counterpart in rabbits. SETTING: School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China. DESIGN: Experimental study. METHODS: Fifty-four rabbits were divided into 2 groups. Group 1 had continuous-light accelerated CXL using 9 mW/cm2 UVA for 10 minutes (5.4 J/cm2). Group 2 had pulsed-light accelerated CXL by exposing them to 9 mW/cm2 UVA for 20 minutes (1 second on/1 second off). Corneal stromal demarcation line depth, in vivo confocal microscopic analysis, biomechanical stiffness, endothelial cell density, and keratocyte apoptosis were measured after performing these CXL procedures. RESULTS: The mean stromal demarcation line depth was 254.7 µm ± 47.4 (SD) in Group 1 and 341.1 ± 36.1 µm in Group 2 (P < .01). One day after CXL, confocal analysis and histological staining identified keratocyte apoptotic fragments in the anterior stroma in the Group 2 corneas whereas all cells were obliterated in Group1. Seven days after treatment, the thicknesses in Group 1 were significantly greater than those in Group 2 (P < .05). Endothelial cell losses were reversible; however, in Group 1, some losses were still evident on day 7. Increases in both the stress-strain relationship and tangent modulus in Group 2 were greater than those in Group 1. CONCLUSION: The pulsed-light accelerated CXL protocol was less injurious and more efficacious at inducing CXL than the continuous-light accelerated CXL protocol in rabbit corneas.