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Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

ABSTRACT: The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaired closure of an epithelial defect and accelerated infiltration of neutrophils into the wound periphery as compared to the response in WT tissue. Expression of wound healing-related proinflammatory and reparative components, i.e., interleukin-6, transforming growth factor β, matrix metalloproteinases, were unaffected by the loss of tenascin X expression. Marked accumulation of malondialdehyde (a lipid peroxidation-derived product) was observed in KO healing epithelia as compared with its WT counterpart. Neutropenia induced by systemic administration of a specific antibody rescued the impairment of epithelial healing in KO corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels.

SUBMITTER: Sumioka T

PROVIDER: S-EPMC8137452 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

Sumioka Takayoshi T Iwanishi Hiroki H Okada Yuka Y Miyajima Masayasu M Ichikawa Kana K Reinach Peter S PS Matsumoto Ken-Ichi KI Saika Shizuya S

Laboratory investigation; a journal of technical methods and pathology 20210329 6

The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaire ...[more]

PMID: 33782532

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Project description:RationaleThe cornea is a unique structure that maintains its clarity by remaining avascular. Corneal injuries can lead to neovascularisation (CNV) and fibrosis and are the third most common cause of blindness worldwide.ObjectiveCorneal injuries induce an immune cell infiltration to initiate reparative processes. However, inflammation caused by sustained immune cell infiltration is known to be detrimental and can delay the healing process. This study was designed to understand the potential role of neutrophil and epithelial cell crosstalk in post-injury CNV.Methods and resultsWestern blotting and immunostaining assays demonstrated that neutrophils infiltrated corneas and underwent pyroptosis following acute alkali injury. In vivo studies showed that genetic ablation of Gasdermin D (GsdmD), a key effector of pyroptosis, enhanced corneal re-epithelialisation and suppressed post-injury CNV. In vitro co-culture experiments revealed that interleukin-1β (IL-1β) was released from pyroptotic neutrophils which suppressed migration of murine corneal epithelial cells. Real-time RT-PCR and immunostaining assays identified two factors, Wnt5a and soluble fms-like tyrosine kinase-1 (sflt-1), highly expressed in newly healed epithelial cells. sflt-1 is known to promote corneal avascularity. Bone marrow transplantation, antibody mediated neutrophil depletion, and pharmacological inhibition of pyroptosis promoted corneal wound healing and inhibited CNV in an in vivo murine corneal injury model.ConclusionTaken together, our study reveals the importance of neutrophil/epithelium crosstalk and neutrophil pyroptosis in response to corneal injuries. Inhibition of neutrophil pyroptosis may serve as a potential treatment to promote corneal healing without CNV.Key pointsNeutrophil pyroptosis delays re-epithelialization after corneal injury Compromised re-epithelialization promotes corneal neovascularization after injury Inhibition of post-injury pyroptosis could be an effective therapy to promote corneal wound healing.

Project description:Acellular cornea derived hydrogels provide significant advantages in preserving native corneal stromal keratocyte cells and endothelial cells. However, for clinical application, hydrogel physical properties need to be improved, and their role in corneal epithelial wound healing requires further investigation. In this study, an acellular porcine corneal stroma (APCS) hydrogel (APCS-gel) was successfully prepared from 20 mg/ml APCS, demonstrated optimal light transmittance and gelation kinetic properties and retained critical corneal ECM of collagens and growth factors. Compared with fibrin gel, the APCS-gel had a higher porosity ratio and faster nutrition diffusion with an accompanying improvement in the proliferation of primary rabbit corneal epithelial cells (RCECs) and stromal cells (RCSCs). These corneal cell types also displayed improved viability and cellular infiltration. Furthermore, the APCS-gel provides significant advantages in the preservation of RCECs stemness and enhancement of corneal wound healing in vitro and in vivo. After 7 days of culture, 3-4 layers of RCECs were formed on the APCS-gel in vitro, while only 1-2 layers were found on the fibrin gel. More corneal stem/progenitor cell phenotypes (K12-, p63+, ABCG2+) and proliferation phenotypes (Ki67+) were detected on the APCS-gel than fibrin gel. Using a corneal epithelial wound healing model, we also found faster reepithelization in corneas that received APCS-gel compared to fibrin gel. Additionally, our APCS-gel demonstrated better physical and biological properties when compared to Tisseel, a clinically used type of fibrin gel. In conclusion, our APCS-gel provided better corneal epithelial and stromal cell biocompatibility to fibrin gels and due to its transparency and faster gelation time could potentially be superior for clinical purposes. STATEMENT OF SIGNIFICANCE: Extracellular matrix (ECM) can be used to provide tissue specific physical microstructure and biochemical cues for tissue regeneration. Here, we produced an ECM hydrogel derived from acellular porcine cornea stroma (APCS-gel) that retained critical biological characteristics of the native tissue and provided significant transparency and fast gelation time. Our data demonstrated that the APCS-gel was superior to clinically used fibrin gel, as the APCS-gel showed high porosity and permeability, better corneal stromal keratocytes infiltration, increased cellular proliferation and retention of corneal epithelial cells stemness. The APCS-gel improved corneal wound healing in vitro and in vivo. This APCS-gel may have clinical utility for corneal diseases, and the more general approach used to make this hydrogel might be used in other tissues.

Dataset Information

Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

Publications

Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

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