Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice.
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ABSTRACT:
SUBMITTER: Mutoji KN
PROVIDER: S-EPMC8199272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neutrophils are the first cells to migrate into the cornea in response to alkali burns, and excessive neutrophil infiltration is associated with inflammatory injury and a poorer prognosis. In an effort to understand the mechanisms underlying the inflammation mediated by neutrophils after alkali burns, we examined the role of alkali-activated neutrophils on human corneal epithelial cells (HCEs) proliferation and migration, as well as the effects of acetylsalicylic acid (ASA) and dexamethasone (DXM) on NETosis. We stimulated human neutrophils with sodium hydroxide (NaOH) and observed dose- and time-dependent neutrophil extracellular traps (NETs) formation. We also observed that ASA, but not DXM, significantly inhibited NaOH-induced NETosis. Furthermore, the activation of nuclear factor (NF)-κB, but not the production of reactive oxygen species, was involved in ASA-regulated NETosis. Moreover, NETs were found to be involved in alkali-activated neutrophils (ANs) induced neutrophil-HCE adhesion. ANs enhanced HCEs proliferation via phagocytosis. Meanwhile, ANs inhibited HCEs migration through the release of NETs, which was partially rescued by 5 mM ASA. In conclusion, ANs may interfere with HCEs proliferation and migration by phagocytosis and NETs formation, respectively. ASA may enhance HCEs migration by decreasing NETs formation through inhibition of NF-κB activation and could be a promising strategy for improving the prognosis of corneal alkali burns.
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Project description: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.
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Project description:Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.
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Project description:Alkali burn is a potentially blinding corneal injury. During the progression of alkali burn-induced injury, overwhelmed oxidative stress in the cornea triggers cell damage, including oxidative changes in cellular macromolecules and lipid peroxidation in membranes, leading to impaired corneal transparency, decreased vision, or even blindness. In this study, we identified that ferroptosis, a type of lipid peroxidation-dependent cell death, mediated alkali burn-induced corneal injury. Ferroptosis-targeting therapy protected the cornea from cell damage and neovascularization. However, the specific ferroptosis inhibitor ferrostatin-1 (Fer-1) is hydrophobic and cannot be directly applied in the clinic. Therefore, we developed Fer-1-loaded liposomes (Fer-1-NPs) to improve the bioavailability of Fer-1. Our study demonstrated that Fer-1-NPs exerted remarkable curative effects regarding corneal opacity and neovascularization in vivo. The efficacy was comparable to that of dexamethasone, but without appreciable side effects. The significant suppression of ferroptosis (induced by lipid peroxidation and mitochondria disruption), inflammation, and neovascularization might be the mechanisms underlying the therapeutic effect of Fer-1-NPs. Moreover, the Fer-1-NPs treatment showed no signs of cytotoxicity, hematologic toxicity, or visceral organ damage, which further confirmed the biocompatibility. Overall, Fer-1-NPs provide a new prospect for safe and effective therapy for corneal alkali burn.
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Project description:Netrins are secreted molecules involved in axon guidance and angiogenesis. However, the role of netrins in the vasculature remains unclear. Netrin-4 and netrin-1 have been found to be either pro- or antiangiogenic factors. Previously, we found that netrin-1 acts as an anti-angiogenic factor in rats by inhibiting alkali burn-induced corneal neovascularization. Here, we further investigate the effects of netrin-4, another member of the same netrin family, on neovascularization in vitro and in vivo. We found that netrin-4 functions similarly as netrin-1 in angiogenesis. In vitro angiogenesis assay shows that netrin-4 affected human umbilical vein endothelial cell (HUVEC) tube formation, viability and proliferation, apoptosis, migration, and invasion in a dose-dependent manner. Netrin-4 was topically applied in vivo to alkali-burned rat corneas on day 0 (immediately after injury) and/or day 10 post-injury. Netrin-4 subsequently suppressed and reversed corneal neovascularization. Netrin-4 inhibited corneal epithelial and stromal cell apoptosis, inhibited vascular endothelial growth factor (VEGF), but promoted pigment epithelium-derived factor (PEDF) expression, decreased NK-KB p65 expression, and inhibits neutrophil and macrophage infiltration. These results indicate that netrin-4 shed new light on its potential roles in treatmenting for angiogenic diseases that affect the ocular surface, as well as other tissues.
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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.
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Project description:The extracellular matrix (ECM) is a highly dynamic structural network and plays an essential role in cell behavior and regulation during metabolic homeostasis and obesity progression. Abnormal ECM remodeling impairs adipocyte plasticity required for diverse cellular functions. Collagen XV (ColXV) is a proteoglycan localized to the outermost layer of basement membranes (BMs) and forms a bridge between the BMs and the fibrillar collagen matrix. Nevertheless, how ColXV affects ECM composition and the reason for subsequent adipocyte apoptosis is still unclear. This report found, through RNA-seq data, that ColXV is linked to cell growth and ECM remodeling. Findings show that, in response to excessive expression of extracellular ColXV, the AMPK/mTORC1 pathway is strongly activated and triggers a cascade of mitochondrial apoptosis. This is the first study to make use of ECM three-dimensional reconstruction, based on decellularization in the adipose tissues and the study reveals that ColXV is an activation factor that alters ECM remodeling in adipose tissues. It was also demonstrated that the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor 1 (FGFR1) axis involved in ECM remodeling is suppressed by ColXV due to reduction of FGF2 translocation to FGFR1. Furthermore, ColXV induced remodeling of ECM preceding apoptosis and continued to induce apoptosis in adipocytes. Collectively, our findings establish ColXV as a basement membrane collagen with homology to ColXVIII, indicating that it is one of the positive regulators for inducing ECM remodeling and further promoting adipocyte apoptosis.
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Project description:Clinical data identify age as a factor for severe liver fibrosis. We evaluate whether and how aging modulates the fibrotic response in a mouse model. Liver fibrosis was induced by CCl4 injections (thrice weekly for 2 weeks) in 7 weeks- and 15 months-old mice (young and old, respectively). Livers were analyzed for fibrosis, inflammation and remodeling 48 and 96 hours after the last injection. Old mice developed more severe fibrosis compared to young ones as evaluated by sirius red morphometry. Expression of pro-fibrogenic genes was equally induced in the two age-groups but enhanced fibrolysis in young mice was demonstrated by a significantly higher Mmp13 induction and collagenase activity. While fibrosis resolution occurred in young mice within 96 hours, no significant fibrosis attenuation was observed in old mice. Although recruitment of monocytes-derived macrophages was similar in young and old livers, young macrophages had globally a remodeling phenotype while old ones, a pro-fibrogenic phenotype. Moreover, we observed a higher proportion of thick fibers and enhanced expression of enzymes involved in collagen maturation in old mice.ConclusionImpaired fibrolysis of a matrix less prone to remodeling associated with a pro-inflammatory phenotype of infiltrated macrophages contribute to a more severe fibrosis in old mice.
| S-EPMC5310658 | biostudies-literature
Project description:Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs) have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs) were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-?), antiangiogenic cytokine (TSP-1) and decrease those promoting inflammation (TNF-?), chemotaxis (MIP-1? and MCP-1) and angiogenesis (VEGF and MMP-2). This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.
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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.
| S-EPMC10777872 | biostudies-literature