Dataset Information

Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn.

ABSTRACT: Corneal wound healing involves a complex cascade of cytokine-controlled cellular events, including inflammatory and angiogenesis responses that are regulated by transcriptional chromatin remodeling. Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate (NUCKS) is a key chromatin modifier and transcriptional regulator of metabolic signaling. In this study, we investigated the role of NUCKS in corneal wound healing by comparing its effects on corneal alkali burn in NUCKS knockout (NKO) and NUCKS wild-type (NWT) mice. Our data showed that following alkali-injury, inhibition of NUCKS (NKO) accelerated ocular resurfacing and suppressed neovascularization; the cytokine profile of alkali burned corneas in NKO mice showed suppressed expression of inflammation cytokines (IL1A &IL1B); upregulated expression of antiangiogenic factor (Pigment Epithelium-derived Factor; PEDF); and downregulated expression of angiogenic factor (Vascular Endothelial Growth Factor, VEGF); in vitro, following LPS-induced NF?B activation, NKO corneal cells showed reduced expression of IL6, IP10 and TNF?. In vitro, corneal epithelial cells showed reduced NF-?b activation on silencing of NUCKS and corresponding NF?B-mediated cytokine expression was reduced. Here, we illustrate that inhibition of NUCKS played a role in cytokine modulation and facilitated corneal recovery. This reveals a potential new effective strategy for ocular burn treatment.

SUBMITTER: Poon MW

PROVIDER: S-EPMC5247723 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn.

Poon Ming-Wai MW Jiang Dan D Qin Peng P Zhang Yuelin Y Qiu Beiying B Chanda Sumit S Tergaonkar Vinay V Li Qing Q Wong Ian Y IY Yu Zhendong Z Tse Hung-Fat HF Wong David S H DS Lian Qizhou Q

Scientific reports 20170120

Corneal wound healing involves a complex cascade of cytokine-controlled cellular events, including inflammatory and angiogenesis responses that are regulated by transcriptional chromatin remodeling. Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate (NUCKS) is a key chromatin modifier and transcriptional regulator of metabolic signaling. In this study, we investigated the role of NUCKS in corneal wound healing by comparing its effects on corneal alkali burn in NUCKS knockout (NKO) a ...[more]

PMID: 28106169

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Project description:BackgroundCorneal neovascularization (CNV) is a sight-threatening condition that necessitates epigenetic control. The role of lysine-specific demethylase 1 (LSD1) in CNV remains unclear, despite its established significance in tumor angiogenesis regulation.MethodsAn alkali burn-induced CNV mouse model was used in vivo. The effects of LSD1 inhibitor tranylcypromine hydrochloride (TCP) were examined through slit lamp, histological staining, and immunofluorescence. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed in corneal tissues. Oxidative stress and ferrous ion expression during CNV were determined using 4-HNE, GPX4, and FerroOrange staining. In vitro, a hypoxia-reoxygenation (H/R) model was established using human umbilical vein endothelial cells (HUVECs) to study LSD1 or hypoxia-inducible factor (HIF-1α) knockdown and lentiviral overexpression of HIF-1α. The effects on HUVECs migration, invasion, and angiogenesis were evaluated through cell scratching assay, transwell migration assay and tube formation assay. The role of ferroptosis was investigated using ROS staining, FerroOrange staining, and key ferroptosis proteins. Further, The JAK2/STAT3 pathway's involvement in CNV regulation was explored through in vivo experiments with subconjunctival injection of AG490.ResultsThe results showed a substantial correlation between corneal damage and LSD1 levels. In addition, HIF-1α expression was also elevated after alkali burns, and subconjunctival injection of TCP reduced corneal inflammation and neovascularization. Corneal alkali burns increased ROS levels and reduced antioxidative stress indicators, accompanied by elevated ferrous ion levels, which were reversed by TCP injection. In vitro, TCP or siRNAs inhibited H/R-induced ferroptosis and angiogenesis in HUVECs by affecting specific protein expressions and MDA, SOD, and GSH levels. HIF-1α levels, associated with ROS production, ferroptosis, and angiogenesis, increased during H/R, but were reversed by TCP or siRNA administration. HIF-1α overexpression counteracted the effects of LSD1 inhibition. Additionally, AG490 injection effectively reduced HIF-1α and VEGFA expression in the CNV model.DiscussionThese findings suggest that LSD1 inhibition via the HIF-1α-driven pathway prevents angiogenesis, oxidative stress, and ferroptosis in corneal alkali burn-induced CNV, highlighting LSD1 as a potential therapeutic target.

Project description:PurposeSuture placement and alkali burn to the cornea are often used to induce inflammatory corneal neovascularization (CorNV) models in animals. This study compares the changes in genome-wide gene expression under these two CorNV conditions in mice.MethodsCorNV were induced in Balb/c mice by three interrupted 10-0 sutures placed at sites about 1 mm from the corneal apex, or by alkali burns that were 2 mm in size in the central area of the cornea. At the points in time when neovascularization progressed most quickly, some eyeballs were subjected to histological staining to examine CorNV and inflammatory cells infiltration, and some corneas were harvested to extract mRNA for microarray assay. After normalization and filtering, the microarray data were subject to statistical analysis using Significance Analysis of Microarray software, and interested genes were annotated using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) program. The expression change of classical proangiogenic molecule like vascular endothelial growth factor (VEGF) and antiangiogenic molecule like pigment epithelium-derived factor (PEDF) was further verified using western blotting.ResultsSuture placement induced CorNV in the areas between the suture and limbus, but did not affect the transparency of the yet unvasuclarized areas of the corneas. In contrast, alkali burn caused edema and total loss of transparency of the whole cornea. Histology showed that sutures only caused localized epithelial loss and inflammatory infiltration between the suture and limbus, but chemical burn depleted the whole epithelial layer of the central cornea and caused heavy cellular infiltration of the whole cornea. At day 5 after suture placement, 1,055 differentially expressed probes were identified, out of which 586 probes were upregulated and 469 probes were downregulated. At a comparable time point, namely on day 6 after the alkali burn to the corneas, 472 probes were upregulated and 389 probes were downregulated. Among these differentially expressed probes, a significant portion (530 probes in total, including 286 upregulated and 244 downregulated probes) showed a similar pattern of change in both models. Annotation (using DAVID) of the overlapping differential genes revealed that the significant enrichment gene ontology terms were "chemotaxis" and "immune response" for the upregulated genes, and "oxidation reduction" and "programmed cell death" for the downregulated genes. Some genes or gene families (e.g., S100A family or α-, β-, or γ-crystallin family) that had not been related to corneal pathogenesis or neovascularization were also revealed to be involved in CorNV. VEGF was upregulated and PEDF was stable as shown with western blotting.ConclusionsSutures and alkali burn to the corneas produced types of damage that affected transparency differentially, but gene profiling revealed similar patterns of changes in gene expression in these two CorNV models. Further studies of the primary genes found to be involved in CorNV will supplement current understanding about the pathogenesis of neovascularization diseases.

Project description:PurposeCorneal neovascularization (CNV) impairs corneal transparency and visual acuity. The study aims to deepen our understanding of the molecules involved in CNV induced by alkali burns, facilitate a better grasp of CNV mechanisms, and uncover potential therapeutic targets.MethodsEighty-four mice were selected for establishing CNV models via alkali burns. On days 3, 7, and 14 after the burns, corneal observations and histological investigations were conducted. An integrated analysis of RNA sequencing (RNA-seq)-based transcriptomics and label-free quantitative proteomics was performed in both normal and burned corneas. Bioinformatics approaches, encompassing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were applied to discern differentially expressed genes (DEGs) and crucial signaling pathways. Four potentially CNV-related genes were validated using quantitative real-time PCR (qRT-PCR) and Western blot.ResultsSignificant CNV was observed on the seventh day. Forty-one genes were differentially expressed in neovascularized corneas, with 15 upregulated and 26 downregulated at both mRNA and protein levels. Bioinformatics analysis revealed that these DEGs participated in diverse biological processes, encompassing retinol and retinoic acid metabolism, neutrophil chemotaxis, and actin filament assembly, along with significant enrichment pathways like cytochrome P450, tyrosine, and phenylalanine metabolism. The upregulation of lymphocyte cytosolic protein 1 (LCP1) and cysteine and glycine-rich protein 2 (CSRP2) genes and the downregulation of transglutaminase 2 (TGM2) and transforming growth factor-beta-induced (TGFBI) genes were confirmed.ConclusionsWe analyzed gene expression differences in mouse corneas 7 days after alkali burns, finding 41 genes with altered expression. The exact role of these genes in CNV is not fully understood, but exploring angiogenesis-related molecules offers potential for CNV treatment or prevention.

Dataset Information

Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn.

Publications

Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn.

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