Injured Corneas Research Articles

Development and characterization of a preclinical mouse model of alkali-induced limbal stem cell deficiency

PurposeLimbal stem cell deficiency (LSCD) secondary to ocular surface alkali burn is a blinding condition that features corneal conjunctivalization. Mechanistic insights into its pathophysiology are lacking. Here, we developed a mouse model that recapitulates human disease to comprehensively delineate the clinicopathological features of a conjunctivalized cornea. MethodsLSCD was induced in the right eyes of 6-8-week-old C57BL/6 male and female mice (n = 151) by topical administration of 0.25N sodium hydroxide on the cornea. Uninjured left eyes served as controls. Clinical, histological, phenotypic, molecular, and immunological assessments were performed at multiple time-points over 6-months. ResultsClinically, alkali burn caused persistent corneal opacity (p = 0.0014), increased punctate staining (p = 0.0002), and reduced epithelial thickness (p = 0.0082) compared to controls. Total LSCD was confirmed in corneal whole mounts by loss of K12 protein (p < 0.0001) and mRNA expression (p = 0.0090). Instead, K8+, K13+, K15+ and MUC5AC+ conjunctival epithelia prevailed. 20 % of injured corneas developed islands of K12+ epithelia, suggesting epithelial transdifferentiation. Squamous metaplasia was detected in 50 % of injured corneas. Goblet cell density peaked early post-injury but decreased over time (p = 0.0047). Intraepithelial corneal basal nerve density remained reduced even at 6-months post-injury (p = 0.0487). ConclusionsWe developed and comprehensively characterized a preclinical mouse model of alkali-induced LSCD. Understanding the pathophysiological processes that transpire on the ocular surface in LSCD is key to discovering, testing, and advancing biological and pharmacological interventions that can be dispensed prior to or in conjunction with stem cell therapy to rehabilitate the cornea and restore vision.

BENEFICIAL EFFECTS OF ANGIOSTATIN K1-3 AND LACTOFERRIN IN ALKALI-BURNED RABBIT CORNEA: A COMPARATIVE STUDY

Corneal injury is associated with hypoxia-induced neovascularization, which interferes optical transparency of the cornea, resulting in vision loss and blindness. The treatment of corneal damage remains a ignificant unmet medical need. Lactoferrin (Lf) and angiostatins (AS) are naturally occurring antiinflammatory and antiangiogenic proteins, which play important roles in eye physiology and can be used as protectors against corneal diseases. The aim of the study was to evaluate and compare the effects of plasminogen fragment AS K1-3 and Lf (as a referent preparation) on the levels of the principal markers of angiogenesis, apoptosis, and autophagy in the burn-injured cornea. Additionally, effects of AS on the metabolic activity of macrophages have been studied. Materials and methods. Experimental model of corneal burn was induced by NaOH application to rabbit’s eye surface. One of the group of animals with injured cornea topically received AS, which were applied as eye drops (1 μM solution) daily for 14 days after injury, another one was treated by the equimolar solution of Lf. The levels of protein markers of angiogenesis (VEGF), apoptosis (caspase-3), and autophagy (beclin-1) were evaluated in corneal lysates by western blot. Histological analysis was performed by hematoxylin/eosin staining of corneal slices followed by light microscopy. Effects of AS in the range of concentrations 10-200 nM on the activity of phagocytic cells were assessed in murine macrophage cell line J744.2 by the test with nitroblue tetrazolium (NBT). The quantitative results were analysed with the use of Mann-Whitney U-test. The difference between group mean values was considered significant at P &lt; 0.05. Results. Dramatically increased levels of protein markers related to angiogenesis, apoptosis, and utophagy were found in the corneas with burn-induced injury. AS or Lf-based treatment reduced burninduced overexpression of VEGF, caspase-3, and beclin-1. It is important to note that AS, along with anti-VEGF activity, appeared to more effectively suppress apoptosis and autophagy by decreasing tested markers near the control level, as compared with Lf. Histochemical examination revealed typical signs of fibrotic and necrotic changes in the injured corneas, which were associated with excessive inflammatory infiltration and intense neovascularization. Treatment of burn-induced injury with AS alleviated histopathological changes in cornea, which is evidenced by improving epithelial regeneration, reducing neovascularization, nd moderating leucocyte infiltration. These observations are in agreement with the ata of NBT test suggesting that AS in the concentrations &gt;50 nM is able to moderately inhibit metabolic activity of macrophages up to 30% as compared with intact cells (P&lt;0.05). Conclusions. Obtained results indicate that AS may serve as an effective treatment option to relief alkali-induced corneal injury with the efficacy comparable or even higher than that of firmly established eye protective protein Lf.

Reversed Corneal Fibroblasts Therapy Restores Transparency of Mouse Cornea after Injury.

Cell-based therapies using corneal stromal stem cells (CSSC), corneal keratocytes, or a combination of both suppress corneal scarring. The number of quiescent keratocytes in the cornea is small; it is difficult to expand them in vitro in quantities suitable for transplantation. This study examined the therapeutic effect of corneal fibroblasts reversed into keratocytes (rCF) in a mouse model of mechanical corneal injury. The therapeutic effect of rCF was studied in vivo (slit lamp, optical coherence tomography) and ex vivo (transmission electron microscopy and immunofluorescence staining). Injection of rCF into the injured cornea was accompanied by recovery of corneal thickness, improvement of corneal transparency, reduction of type III collagen in the stroma, absence of myofibroblasts, and the improvement in the structural organization of collagen fibers. TEM results showed that 2 months after intrastromal injection of cells, there was a decrease in the fibril density and an increase in the fibril diameter and the average distance between collagen fibrils. The fibrils were well ordered and maintained the short-range order and the number of nearest-neighbor fibrils, although the averaged distance between them increased. Our results demonstrated that the cell therapy of rCF from ReLEx SMILe lenticules promotes the recovery of transparent corneal stroma after injury.

Trimebutine prevents corneal inflammation in a rat alkali burn model

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1β expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization.

PAX6/CXCL14 regulatory axis promotes the repair of corneal injury by enhancing corneal epithelial cell proliferation

BackgroundCorneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6’s influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment.MethodsTo investigate the PAX6/CXCL14 regulatory axis’s role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis’s function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis’s mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries.ResultsOur study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14’s receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14’s therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models.ConclusionCCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries.

Replace or Regenerate? Diverse Approaches to Biomaterials for Treating Corneal Lesions.

The inner structures of the eye are protected by the cornea, which is a transparent membrane exposed to the external environment and subjected to the risk of lesions and diseases, sometimes resulting in impaired vision and blindness. Several eye pathologies can be treated with a keratoplasty, a surgical procedure aimed at replacing the cornea with tissues from human donors. Even though the success rate is high (up to 90% for the first graft in low-risk patients at 5-year follow-up), this approach is limited by the insufficient number of donors and several clinically relevant drawbacks. Alternatively, keratoprosthesis can be applied in an attempt to restore minimal functions of the cornea: For this reason, it is used only for high-risk patients. Recently, many biomaterials of both natural and synthetic origin have been developed as corneal substitutes to restore and replace diseased or injured corneas in low-risk patients. After illustrating the traditional clinical approaches, the present paper aims to review the most innovative solutions that have been recently proposed to regenerate the cornea, avoiding the use of donor tissues. Finally, innovative approaches to biological tissue 3D printing and xenotransplantation will be mentioned.

Investigation of healing strategies in a rat corneal opacity model with polychromatic light and stem cells injection

Corneal opacities are a major cause of vision loss worldwide. However, the current therapies are suboptimal to manage the corneal wound healing process. Therefore, there is an obvious need to develop new treatment strategies that are efficient in promoting wound healing in patients with severe corneal disorders. In this study, we investigated and compared the efficacy of adipose-derived mesenchymal stem cells (ADMSCs) and photobiomodulation (PBM) with polychromatic light in the NIR (600–1200 nm) alone and in combination, on corneal opacity, inflammatory response, and tissue architecture in a rat corneal opacity model created by mechanical injury. All animals were divided into four groups randomly following the injury: injury only (no treatment), ADMSCs treatment, PBM treatment and combined (ADMSCs+PBM) treatment (n = 12 eyes per group). At the 10th and 30th day following injury, corneal opacity formation, neovascularization, and corneal thickness were assessed. On the 30th day the harvested corneas were analyzed by transmission electron microscopy (TEM), histological evaluation, immunohistochemical (IHC) staining and real-time polymerase chain reaction (RT-PCR). On day 30, the corneal opacity score, neovascularization grade, and corneal thickness in all treatment groups were significantly lower in comparison with the untreated injured corneas. The TEM imaging and H&E staining together clearly revealed a significant enhancement in corneal regeneration with improved corneal microenvironment and reduced vascularization in the combined administration of PBM and ADMSCs compared to treatment of PBM and ADMSCs alone. In addition, the IHC staining, and RT-PCR analysis supported our hypothesis that combining ADMSCs therapy with PBM alleviated the inflammatory response, and significantly decreased scar formation compared to either ADMSCs or PBM alone during the corneal wound healing.

Multiple effects of angiostatins in injured cornea

Prolonged inflammation and excessive neovascularization of the cornea due to severe injury can impair optical clarity and lead to vision impairment. Plasminogen kringle (K) fragments, known as angiostatins (AS), play a well-established role as inhibitors of neovascularization by suppressing pro-angiogenic signaling­. However, AS effects in the cornea, beyond inhibiting the angiogenesis, are still unexplored. In this study, we estimate the protective effect of two AS variants (K1-3 and K5) against alkali burn injury induced in rabbit and rat corneas. AS K1-3 in the single doses of 0.075 or 0.75 μg (0.1 or 1.0 μM, respectively) or 0.3 μg of AS K5 (1.0 μM) were applied locally as eye drops daily for 14 days after the injury. A significant regression of corneal vessels in-growth in injured eyes treated with AS was revealed. Western blot analysis of corneal tissue lysates revealed that injury-induced overexpression of protein markers­ of hypoxia (HIF-1α), angiogenesis (VEGF), tissue remodeling and fibrosis (MMP-9), autophagy (beclin-1) and endoplasmic reticulum stress (GRP-78) was significantly reduced under AS treatment. Besides, the level of tight junctions protein ZO-1 was shown to be up-regulated after the treatment of the damaged cornea with AS K1-3. Summarizing, our study uncovered novel biological functions of the kringle-containing plasminogen fragments indicating its beneficial effects during corneal healing in the experimental model of alkali burn. The data obtained can be helpful for the development of novel efficient formulations to manage complications of ocular surface injuries.

Critical Role of CD55 in Controlling Wound Healing.

How reparative processes are coordinated following injury is incompletely understood. In recent studies, we showed that autocrine C3a and C5a receptor (C3ar1 and C5ar1) G protein-coupled receptor signaling plays an obligate role in vascular endothelial growth factor receptor 2 growth signaling in vascular endothelial cells. We documented the same interconnection for platelet-derived growth factor receptor growth signaling in smooth muscle cells, epidermal growth factor receptor growth signaling in epidermal cells, and fibroblast growth factor receptor signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. In this study, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 (also known as decay accelerating factor), which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is heightened C3ar1/C5ar1 signaling resulting from the loss of CD55's restraint jointly potentiating growth factor production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury (burn, corneal denudation, ear lobe puncture, and reengraftment of autologous skin) showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In wild-type mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking Ab (against CD55's active site) to wounds accelerated the healing rate by 40-70%. To our knowledge, these results provide new insights into mechanisms that underlie wound repair and open up a new tool for accelerating healing.

The application of a 4D-printed chitosan-based stem cell carrier for the repair of corneal alkali burns

BackgroundCorneal alkali burns can lead to ulceration, perforation, and even corneal blindness due to epithelial defects and extensive cell necrosis, resulting in poor healing outcomes. Previous studies have found that chitosan-based in situ hydrogel loaded with limbal epithelium stem cells (LESCs) has a certain reparative effect on corneal alkali burns. However, the inconsistent pore sizes of the carriers and low cell loading rates have resulted in suboptimal repair outcomes. In this study, 4D bioprinting technology was used to prepare a chitosan-based thermosensitive gel carrier (4D-CTH) with uniform pore size and adjustable shape to improve the transfer capacity of LESCs.MethodsPrepare solutions of chitosan acetate, carboxymethyl chitosan, and β-glycerophosphate sodium at specific concentrations, and mix them in certain proportions to create a pore-size uniform scaffold using 4D bioprinting technology. Extract and culture rat LESCs (rLESCs) in vitro, perform immunofluorescence experiments to observe the positivity rate of deltaNp63 cells for cell identification. Conduct a series of experiments to validate the cell compatibility of 4D-CTH, including CCK-8 assay to assess cell toxicity, scratch assay to evaluate the effect of 4D-CTH on rLESCs migration, and Calcein-AM/PI cell staining experiment to examine the impact of 4D-CTH on rLESCs proliferation and morphology. Establish a severe alkali burn model in rat corneas, transplant rLESCs onto the injured cornea using 4D-CTH, periodically observe corneal opacity and neovascularization using a slit lamp, and evaluate epithelial healing by fluorescein sodium staining. Assess the therapeutic effect 4D-CTH-loaded rLESCs on corneal alkali burn through histological evaluation of corneal tissue paraffin sections stained with hematoxylin and eosin, as well as immunofluorescence staining of frozen sections.ResultsUsing the 4D-CTH, rLESCs were transferred to the alkali burn wounds of rats. Compared with the traditional treatment group (chitosan in situ hydrogel encapsulating rLESCs), the 4D-CTH-rLESC group had significantly higher repair efficiency of corneal injury, such as lower corneal opacity score (1.2 ± 0.4472 vs 0.4 ± 0.5477, p < 0.05) and neovascularization score (5.5 ± 1.118 vs 2.6 ± 0.9618, p < 0.01), and significantly higher corneal epithelial wound healing rate (72.09 ± 3.568% vs 86.60 ± 5.004%, p < 0.01).ConclusionIn summary, the corneas of the 4D-CTH-rLESC treatment group were similar to the normal corneas and had a complete corneal structure. These findings suggested that LESCs encapsulated by 4D-CTH significantly accelerated corneal wound healing after alkali burn and can be considered as a rapid and effective method for treating epithelial defects.Graphical abstract

Anti-Angiogenic and Anti-Scarring Dual Effect of Galectin-3 Inhibition in Mouse Models of Corneal Wound Healing

Corneal scarring is the third leading cause of global blindness. Neovascularization of ocular tissues is a major predisposing factor for scar development. Although corneal transplantation is effective at restoring vision, some patients are at high risk of graft rejection due to the presence of blood vessels in the injured cornea. Current treatment options to control corneal scarring are limited and their outcomes are typically poor. In this study, using mouse models of corneal wound healing, we demonstrate that topical application of a small molecule inhibitor of galectin-3, GB1265: reduces (i) corneal angiogenesis, (ii) corneal fibrosis, (iii) infiltration of immune cells and (iv) expression of proinflammatory cytokine, IL-1β, in injured corneas. Four independent techniques (RNA-seq, NanoString, RT-qPCR, and Western blot analyses) all demonstrated that decrease in corneal opacity in the galectin-3 inhibitor-treated corneas is associated with a decrease in a number of genes and signaling pathways that are known to promote fibrosis. These findings allow us to place a high level of confidence in our conclusion that galectin-3 inhibition by a small molecule inhibitor, GB1265, has dual anti-angiogenic and anti-scarring effect. Targeting galectin-3 by GB1265, is thus, an attractive strategy for development of innovative treatment of myriad of ocular and nonocular diseases characterized by pathological angiogenesis and fibrosis.

Single Cell Transcriptomics Define Cell Heterogeneity in Limbal/Corneal Cells Relevant to Nerve Regeneration

Balanced interplay of cell-type specific transcriptomic regulation is critical for nerve regeneration in the central and peripheral nervous system. As a model, we use the cornea, where we recently discovered the RvD6-isomer and its function as a regulator of nerve regeneration, critical for dry eye in aging. Single-RNA-sequencing (snRNASeq) and spatial transcriptomics were used to interrogate cell populations and gene expression in central cornea/limbus from male, 3-month, C57BL/6 wild-type (WT) and injured mice cornea. Isolated nuclei from cornea/limbus of WT mice (N=12) were processed by 10×Genomics pipeline Cellranger. Central mice corneas were damaged with 2mm trephine, and epithelium and anterior stroma removed with corneal rust ring remover (Algerbrush II). Two days post-injury, corneas were excised, formalin-fixed paraffin-embedded, and 5µm sections prepared for Visium CytAssist mediated probe hybridization, library construction, sequencing, data analysis using Spaceranger, data visualization and cell cluster annotation using Loupe Browser. Cell populations were distinguished by t-distributed stochastic neighbor embedding (t-SNE). RNAscope® assay was used to validate two genes, ALDH3A1 and SPARC.

Angiostatins modulate ACE2 and GFAP levels in injured rat cornea and do not affect viability of retinal pigment epithelial cells

Angiostatins modulate ACE2 and GFAP levels in injured rat cornea and do not affect viability of retinal pigment epithelial cells

Photocurable and Temperature-Sensitive Bioadhesive Hydrogels for Sutureless Sealing of Full-Thickness Corneal Wounds.

Penetrating corneal wounds can cause severe vision impairment and require prompt intervention to restore globe integrity and minimize the risk of infection. Tissue adhesives have emerged as a promising alternative to suturing for mitigating postoperative complications. However, conventional water-soluble adhesives suffer formidable challenges in sealing penetrating corneal wounds due to dilution or loss in a moist environment. Inspired by the robust adhesion of mussels in aquatic conditions, an injectable photocurable bioadhesive hydrogel (referred to as F20HD5) composed of polyether F127 diacrylate and dopamine-modified hyaluronic acid methacrylate is developed for sutureless closure of corneal full-thickness wounds. F20HD5 exhibits high transparency, wound-sealing ability, proper viscosity, biodegradability, and excellent biocompatibility. It allows in situ cross-linking via visible light, thereby providing sufficient mechanical strength and adhesiveness. In vivo, the adhesive hydrogel effectively closed penetrating linear corneal incisions and corneal injuries with minimal tissue loss in rabbits. During the 56-day follow-up, the hydrogel facilitates the repair of the injured corneas, resulting in more symmetrical curvatures and less scarring in distinction to the untreated control. Thus, bioinspired hydrogel holds promise as an effective adhesive for sealing full-thickness corneal wounds.

A suture-free, shape self-adaptive and bioactive PEG-Lysozyme implant for Corneal stroma defect repair and rapid vision restoration.

Corneal transplantation is a prevailing treatment to repair injured cornea and restore vision but faces the limitation of donor tissue shortage clinically. In addition, suturing-needed transplantation potentially causes postoperative complications. Herein, we design a PEG-Lysozyme injective hydrogel as a suture-free, shape self-adaptive, bioactive implant for corneal stroma defect repair. This implant experiences a sol-gel phase transition via an in situ amidation reaction between 4-arm-PEG-NHS and lysozyme. The physicochemical properties of PEG-Lysozyme can be tuned by the components ratio, which confers the implant mimetic corneal modulus and provides tissue adhesion to endure increased intraocular pressure. In vitro tests prove that the implant is beneficial to Human corneal epithelial cells growth and migration due to the bioactivity of lysozyme. Rabbit lamellar keratoplasty experiment demonstrates that the hydrogel can be filled into defect to form a shape-adaptive implant adhered to native stroma. The implant promotes epithelialization and stroma integrity, recovering the topology of injured cornea to normal. A newly established animal forging behavior test prove a rapid visual restoration of rabbits when use implant in a suture free manner. In general, this work provides a promising preclinical practice by applicating a self-curing, shape self-adaptive and bioactive PEG-Lysozyme implant for suture-free stroma repair.

Sympathetic Nerves Coordinate Corneal Epithelial Wound Healing by Controlling the Mobilization of Ly6Chi Monocytes From the Spleen to the Injured Cornea.

This study aims to investigate the potential involvement of spleen-derived monocytes in the repair process following corneal epithelial abrasion. A corneal epithelial abrasion model was established in male C57BL/6J mice, and the dynamic changes of monocyte subpopulations in the injured cornea were analyzed using flow cytometry. The effects of Ly6Chi monocyte depletion and local adoptive transfer of purified Ly6Chi monocytes on wound closure and neutrophil recruitment to the injured cornea were observed. The effect of sympathetic nerves on the recruitment of spleen-derived Ly6Chi monocytes to the injured cornea was also investigated using multiple methods. The emigration of fluorescence-labeled monocytes to the injured cornea was validated through intravital microscopy. Finally, differential genes between different groups were identified through high-throughput RNA sequencing and analyzed for functional enrichment, followed by verification by quantitative PCR. Ly6Chi monocytes were present in large numbers in the injured cornea prior to neutrophil recruitment. Predepletion of Ly6Chi monocytes significantly inhibited neutrophil recruitment to the injured cornea. Furthermore, surgical removal of the spleen significantly reduced the number of Ly6Chi monocytes in the injured cornea. Further observations revealed that sympathetic blockade significantly reduced the number of Ly6Chi monocytes recruited to the injured cornea. In contrast, administration of the β2-adrenergic receptor agonist significantly increased the number of Ly6Chi monocytes recruited to the injured cornea in animals treated with sympathectomy and catecholamine synthesis inhibition. Our results suggest that spleen-derived Ly6Chi monocytes, under the control of the sympathetic nervous system, play a critical role in the inflammatory response following corneal injury.

Sulfated motifs in heparan sulfate inhibit Streptococcus pneumoniae adhesion onto fibronectin and attenuate corneal infection.

A large number of bacterial pathogens bind to host extracellular matrix (ECM) components. For example, many Gram-negative and Gram-positive pathogens express binding proteins for fibronectin (FN) on their cell surface. Mutagenesis studies of bacterial FN-binding proteins have demonstrated their importance in pathogenesis in preclinical animal models. However, means to draw on these findings to design therapeutic approaches that specifically target FN-bacteria interactions have not been successful because bacterial pathogens can elaborate several FN-binding proteins and also because FN is an essential protein and likely a nondruggable target. Here we report that select heparan compounds potently inhibit Streptococcus pneumoniae infection of injured corneas in mice. Using intact heparan sulfate (HS) and heparin (HP), heparinase-digested fragments of HS, HP oligosaccharides, and chemically or chemoenzymatically modified heparan compounds, we found that inhibition of S. pneumoniae corneal infection by heparan compounds is not mediated by simple charge effects but by a selective sulfate group. Removal of 2-O-sulfates significantly inhibited the ability of HP to inhibit S. pneumoniae corneal infection, whereas the addition of 2-O-sulfates to heparosan (H) significantly increased H's ability to inhibit bacterial corneal infection. Proximity ligation assays indicated that S. pneumoniae attaches directly to FN fibrils in the corneal epithelial ECM and that HS and HP specifically inhibit this binding interaction in a 2-O-sulfate-dependent manner. These data suggest that heparan compounds containing 2-O-sulfate groups protect against S. pneumoniae corneal infection by inhibiting bacterial attachment to FN fibrils in the subepithelial ECM of injured corneas. Moreover, 2-O-sulfated heparan compounds significantly inhibited corneal infection in immunocompromised hosts, by a clinical keratitis isolate of S. pneumoniae, and also when topically administered in a therapeutic manner. These findings suggest that the administration of nonanticoagulant 2-O-sulfated heparan compounds may represent a plausible approach to the treatment of S. pneumoniae keratitis.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. Invitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

The corneal fibroblast: The Dr. Jekyll underappreciated overseer of the responses to stromal injury

PurposeTo review the functions of corneal fibroblasts in wound healing. MethodsLiterature review. ResultsCorneal fibroblasts arise in the corneal stroma after anterior, posterior or limbal injuries and are derived from keratocytes. Transforming growth factor (TGF) β1 and TGFβ2, along with platelet-derived growth factor (PDGF), are the major modulators of the keratocyte to corneal fibroblast transition, while fibroblast growth factor (FGF)-2, TGFβ3, and retinoic acid are thought to regulate the transition of corneal fibroblasts back to keratocytes. Adequate and sustained levels of TGFβ1 and/or TGFβ2, primarily from epithelium, tears, aqueous humor, and corneal endothelium, drive the development of corneal fibroblasts into myofibroblasts. Myofibroblasts have been shown in vitro to transition back to corneal fibroblasts, although apoptosis of myofibroblasts has been documented as a major contributor to the resolution of fibrosis in several in situ corneal injury models. Corneal fibroblasts, aside from their role as a major progenitor to myofibroblasts, also perform many critical functions in the injured cornea, including the production of critical basement membrane (BM) components during regeneration of the epithelial BM and Descemet's membrane, production of non-basement membrane-associated stromal collagen type IV to control and downregulate TGFβ effects on stromal cells, release of chemotactic chemokines that attract bone marrow-derived cells to the injured stroma, production of growth factors that modulate regeneration and maturation of the overlying epithelium, and production of collagens and other ECM components that contribute to stromal integrity after injury. ConclusionsCorneal fibroblasts are major contributors to and overseers of the corneal response to injuries.

Mesenchymal Stem Cell Exosomes as Immunomodulatory Therapy for Corneal Scarring.

Corneal scarring is a leading cause of worldwide blindness. Human mesenchymal stem cells (MSC) have been reported to promote corneal wound healing through secreted exosomes. This study investigated the wound healing and immunomodulatory effects of MSC-derived exosomes (MSC-exo) in corneal injury through an established rat model of corneal scarring. After induction of corneal scarring by irregular phototherapeutic keratectomy (irrPTK), MSC exosome preparations (MSC-exo) or PBS vehicle as controls were applied to the injured rat corneas for five days. The animals were assessed for corneal clarity using a validated slit-lamp haze grading score. Stromal haze intensity was quantified using in-vivo confocal microscopy imaging. Corneal vascularization, fibrosis, variations in macrophage phenotypes, and inflammatory cytokines were evaluated using immunohistochemistry techniques and enzyme-linked immunosorbent assays (ELISA) of the excised corneas. Compared to the PBS control group, MSC-exo treatment group had faster epithelial wound closure (0.041), lower corneal haze score (p = 0.002), and reduced haze intensity (p = 0.004) throughout the follow-up period. Attenuation of corneal vascularisation based on CD31 and LYVE-1 staining and reduced fibrosis as measured by fibronectin and collagen 3A1 staining was also observed in the MSC-exo group. MSC-exo treated corneas also displayed a regenerative immune phenotype characterized by a higher infiltration of CD163+, CD206+ M2 macrophages over CD80+, CD86+ M1 macrophages (p = 0.023), reduced levels of pro-inflammatory IL-1β, IL-8, and TNF-α, and increased levels of anti-inflammatory IL-10. In conclusion, topical MSC-exo could alleviate corneal insults by promoting wound closure and reducing scar development, possibly through anti-angiogenesis and immunomodulation towards a regenerative and anti-inflammatory phenotype.