Cell-Free Biological Approach for Corneal Stromal Wound Healing.
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ABSTRACT:
SUBMITTER: Jhanji V
PROVIDER: S-EPMC8193853 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, ?-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits ?4, ?5, ?V, ?1 and ?3 whereas SFCM increased ?4, ?5, ?V, ?1 and ?5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.
| S-EPMC5855686 | biostudies-literature
Project description:Corneal stromal wound healing is a complex event that occurs to restore the transparency of an injured cornea. It involves immediate apoptosis of keratocytes followed by their activation, proliferation, migration, and trans-differentiation to myofibroblasts. Myofibroblasts contract to close the wound and secrete extracellular matrix and proteinases to remodel it. Released proteinases may degenerate the basement membrane allowing an influx of cytokines from overlying epithelium. Immune cells infiltrate the wound to clear cellular debris and prevent infections. Gradually basement membrane regenerates, myofibroblasts and immune cells disappear, abnormal matrix is resorbed, and transparency of the cornea is restored. Often this cascade deregulates and corneal opacity results. Factors that prevent corneal opacity after an injury have always intrigued the researchers. They hold clinical relevance as they can guide the outcomes of corneal surgeries. Studies in the past have shed light on the role of various factors in stromal healing. TGFβ (transforming growth factor-beta) signaling is the central player guiding stromal responses. Other major regulators include myofibroblasts, basement membrane, collagen fibrils, small leucine-rich proteoglycans, biophysical cues, proteins derived from extracellular matrix, and membrane channels. The knowledge about their roles helped to develop novel therapies to prevent corneal opacity. This article reviews the role of major regulators that determine the outcome of stromal healing. It also discusses emerging therapies that modulate the role of these regulators to prevent stromal opacity.
| S-EPMC9183221 | biostudies-literature
Project description:The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-β. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGβ1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.
| S-EPMC8910342 | biostudies-literature
Project description:Proper wound healing is vital for maintenance of corneal integrity and transparency. Corneal epithelial damage is one of the most frequently observed ocular disorders. Because clinical options are limited, further novel treatments are needed to improve clinical outcomes for this type of disease. In the present study, it was found that placental extract-derived dipeptide (JBP485) significantly increased the proliferation and migration of corneal epithelial cells (CECs). Moreover, JBP485 accelerated corneal epithelial wound healing in vivo without inflammation and neovascularization and was found to be effective for the treatment of corneal damage. These data indicate that JBP485 efficiently activates the viability of CECs and has potential as a novel treatment for various kinds of corneal epithelial disease.
| S-EPMC4589775 | biostudies-other
Project description:Neprilysin (NEP), an ectoenzyme that modulates inflammation by degrading neuropeptides, was recently identified in the human corneal epithelium. The cornea expresses many NEP substrates, but the function of NEP in homeostatic maintenance and wound healing of the cornea is unknown. We therefore investigated the role of this enzyme under naive and injured conditions using NEP-deficient (NEP-/-) and wild type (WT) control mice. In vivo ocular surface imaging and histological analysis of corneal tissue showed no differences in limbal vasculature or corneal anatomy between naive NEP-/- and WT mice. Histological examination revealed increased corneal innervation in NEP-/- mice. In an alkali burn model of corneal injury, corneal wound healing was significantly accelerated in NEP-/- mice compared to WT controls 3 days after injury. Daily intraperitoneal administration of the NEP inhibitor thiorphan also accelerated corneal wound healing after alkali injury in WT mice. Collectively, our data identify a previously unknown role of NEP in the cornea, in which pharmacologic inhibition of its activity may provide a novel therapeutic option for patients with corneal injury.
| S-EPMC6158251 | biostudies-literature
Project description:Although the wound healing effects of nitric oxide (NO) are known, the mechanism by which NO modulates corneal wound healing remains unclear. In this study, we investigated the effect of exogenous NO donor (NaNO2) on corneal wound healing. We found that NaNO2 (0.1 μM to 100 μM) increased human corneal epithelial cell (HCEC) viability and migration. It also modulated the phosphorylation of mitogen-activated protein kinases (MAPKs) in a time- dependent manner in those HCECs. Further, p38 MAPK phosphorylation increased at 6 h and normalized at 24 h, while the phosphorylation of extracellular signal regulated kinase (ERK) was increased both at 6 h and 24 h. Topical treatment with NaNO2 (10 μM) enhanced corneal epithelial healing and decreased corneal opacity in murine corneal alkali burn model by modulating inflammatory cytokines. Our findings suggest that NO increased HCEC proliferation and migration via time-dependent MAPK activation and eventually enhanced corneal recovery from the alkali burn.
| S-EPMC5556055 | biostudies-literature
Project description:Specific factors from the corneal epithelium underlying the stimulation of stromal fibrosis and myofibroblast formation in corneal wound healing have not been fully elucidated. Given that exosomes are known to transfer bioactive molecules among cells and play crucial roles in wound healing, angiogenesis, and cancer, we hypothesized that corneal epithelial cell-derived exosomes may gain access to the underlying stromal fibroblasts upon disruption of the epithelial basement membrane and that they induce signaling events essential for corneal wound healing. In the present study, exosome-like vesicles were observed between corneal epithelial cells and the stroma during wound healing after corneal epithelial debridement. These vesicles were also found in the stroma following anterior stromal keratectomy, in which surgical removal of the epithelium, basement membrane, and anterior stroma was performed. Exosomes secreted by mouse corneal epithelial cells were found to fuse to keratocytes in vitro and to induce myofibroblast transformation. In addition, epithelial cell-derived exosomes induced endothelial cell proliferation and ex vivo aortic ring sprouting. Our results indicate that epithelial cell-derived exosomes mediate communication between corneal epithelial cells and corneal keratocytes as well as vascular endothelial cells. These findings demonstrate that epithelial-derived exosomes may be involved in corneal wound healing and neovascularization, and thus, may serve as targets for potential therapeutic interventions.
| S-EPMC5292698 | biostudies-literature
Project description:Corneal transparency, dependent on the integrity of epithelial cells, is essential for vision. Corneal epithelial damage is one of the most commonly observed ocular conditions and proper wound healing is necessary for corneal transparency. Sirt6, a histone deacetylase, has been shown to regulate many cellular events including aging and inflammation. However, its specific role in corneal epithelial wound healing remains unknown. Here we demonstrated that Sirt6 was expressed in corneal epithelial cells and its expression decreased with age. In an in vivo corneal epithelial wound healing model, Sirt6 deficiency resulted in delayed and incomplete wound healing and was associated excessive inflammation in the corneal stroma and dysfunction of Notch signaling, leading to keratinization of the corneal epithelium and corneal opacity. Aging Sirt6-deficient mice spontaneously developed corneal keratitis with extensive infiltration of inflammatory cells into the cornea. In vitro experiments demonstrated that primary corneal epithelial cells with Sirt6 downregulation expressed increased basal levels of inflammatory genes and exhibited hyper-inflammatory reactivity to IL-1β and TNFα treatment. These results provide compelling evidence that Sirt6 is a critical regulator of inflammation in the cornea, and is responsible for corneal epithelial wound healing, thus contributing to the maintenance of epithelial integrity and corneal transparency.
| S-EPMC6128418 | biostudies-literature
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.
| S-EPMC8542601 | biostudies-literature
Project description:[This corrects the article DOI: 10.1186/s41038-019-0178-8.].
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