ABSTRACT: 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.
Project description:With the immunoregulation potential, mesenchymal stem cells (MSCs) have been used for tissue regeneration by relieving inflammation in the injured tissues. When this repair process is interfered by immune disorders or pathological angiogenesis, the delays in corneal epithelial wound healing can lead to a persistent epithelial defect. Stem cell-derived extracellular vesicles (EVs), which carry abundant bioactive molecules from stem cells, have provided an alternative to regeneration therapy. In this study, we aimed to investigate if EVs from human placenta-derived MSCs (hP-MSCs) could ameliorate alkali injury of the cornea in the mouse model. 33.33??g/?L EVs in 10??L PBS were applied to the cornea. Repeat application three times, and 100??g EVs (in 30??L PBS) in total were administrated per day for two weeks. Our results revealed that EVs from hP-MSCs had preferable functions including enhancing proliferation and anti-inflammation and suppressing apoptosis of corneal epithelial cells. Furthermore, hP-MSC-derived EVs ameliorated mouse corneal wound healing by inhibiting angiogenesis and inflammation. Taken together, our current data suggested that hP-MSC-derived EVs have the beneficial effects of corneal wound healing, which provide alternative cell-free therapy with great practical value.
Project description:Tumor necrosis factor (TNF)-? is upregulated in eyes following corneal alkali injury and contributes to corneal and also retinal damage. Prompt TNF-? inhibition by systemic infliximab ameliorates retinal damage and improves corneal wound healing. However, systemic administration of TNF-? inhibitors carries risk of significant complications, whereas topical eye-drop delivery is hindered by poor ocular bioavailability and the need for patient adherence. This study investigates the efficacy of subconjunctival delivery of TNF-? antibodies using a polymer-based drug delivery system (DDS).The drug delivery system was prepared using porous polydimethylsiloxane/polyvinyl alcohol composite fabrication and loaded with 85 ?g of infliximab. Six Dutch-belted pigmented rabbits received ocular alkali burn with NaOH. Immediately after the burn, subconjunctival implantation of anti-TNF-? DDS was performed in three rabbits while another three received sham DDS (without antibody). Rabbits were followed with photography for 3 months.After 3 months, the device was found to be well tolerated by the host and the eyes exhibited less corneal damage as compared to eyes implanted with a sham DDS without drug. The low dose treatment suppressed CD45 and TNF-? expression in the burned cornea and inhibited retinal ganglion cell apoptosis and optic nerve degeneration, as compared to the sham DDS treated eyes. Immunolocalization revealed drug penetration in the conjunctiva, cornea, iris, and choroid, with residual infliximab in the DDS 3 months after implantation.This reduced-risk biologic DDS improves corneal wound healing and provides retinal neuroprotection, and may be applicable not only to alkali burns but also to other inflammatory surgical procedures such as penetrating keratoplasty and keratoprosthesis implantation.
Project description:Corneal alkali burns are a serious clinical problem that often leads to permanent visual impairment. In this process, transforming growth factor (Tgf)-beta1 is upregulated and involved in the response to corneal injury and the process of corneal stromal scarring. To develop an efficient compound to inhibit Tgf-beta1 in the cornea, we designed GB1201, a pyrrole-imidazole (PI) polyamide targeting rat Tgf-beta1 gene promoter to the activator protein-1 (AP-1) binding site. GB1201 showed a high binding affinity to the target DNA sequence in the gel mobility shift and Biacore assays. GB1201 significantly inhibited the rat Tgf-beta1 gene promoter activity in HEK (human embryonic kidney) 293 cells in a concentration-dependent manner. Topically administrated GB1201 was distributed immediately to the nuclei of all cell layers of the cornea and remained for 24 hours. A corneal alkali burn model in rats was used to evaluate the therapeutic efficacy of GB1201. GB1201 suppressed the upregulation of Tgf-beta1 in the burned cornea, both in the mRNA and protein levels. Moreover, daily treatment with GB1201 for a week significantly improved the corneal tissue wound healing, reduced corneal stromal scarring, and prevented corneal haze formation. Our data suggest that PI polyamide may open new opportunities for therapeutic intervention in the treatment of chemically burned corneas.
Project description:In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and ?-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor ?, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.
Project description:Purpose:Corneal injury that occurs after burning with alkali initiates wound-healing processes, including inflammation, neovascularization, and fibrosis. Excessive reactions to injury can reduce corneal transparency and thereby compromise vision. The NADPH oxidase (Nox) enzyme complex is known to be involved in cell signaling for wound-healing angiogenesis, but its role in corneal neovascularization has been little studied. Methods:The center corneas of wild-type and Nox4 knockout (KO) mice were injured with 3 µL 1 M NaOH, while the contralateral corneas remained untouched. On day 7, mRNA expression levels of NADPH oxidase isoforms, the proangiogenic factors VEGF-A and TGF?1, and proinflammatory genes ICAM-1 and VCAM-1 were determined. Corneal neovascularization and fibrosis were visualized using PECAM-1 antibody and picrosirius red staining, respectively, on the same day. Results:Expressions of both Nox2 and Nox4 gene isoforms as well as the above genes were markedly increased in the injured corneas at 7 days. Injured corneas showed neovascularization and fibrosis as well as an increase in clinical opacity score. All responses stimulated by alkali burn were abrogated in Nox4 KO mice. Conclusions:Nox4 could be a new target to treat pathologic corneal wound-healing responses and such targeting might prevent blindness caused by burn injuries.
Project description:The effects of each subtype-selective peroxisome proliferator activated receptor (PPAR) agonist (?, ?/?, ?) on corneal epithelial wound healing were investigated using a rat corneal alkali burn model. After the alkali burn, each PPAR agonist or vehicle ophthalmic solution was instilled topically onto the rat's cornea. Corneal epithelial healing processes were evaluated by fluorescein staining. Pathological analyses and real-time reverse transcription polymerase chain reactions were performed to evaluate Ki67 (proliferative maker) expression and inflammatory findings. The area of the corneal epithelial defect at 12 h and 24 h after the alkali burn was significantly smaller in each PPAR group than in the vehicle group. Ki67 mRNA expression was increased in the PPAR?/? group, whereas mRNA expressions of inflammatory cytokines were suppressed in all of the PPAR agonist groups. Nuclear factor kappa B (NF-?B) was the most suppressed in the PPAR? group. The accelerated corneal epithelial healing effects of each PPAR ligand were thought to be related to the promotion of proliferative capacity and inhibition of inflammation.
Project description:IkB kinase ? (IKK?) is a key signaling kinase for inflammatory responses, but it also plays diverse cell type-specific roles that are not yet fully understood. Here we investigated the role of IKK? in the cornea using Ikk?(?CS) mice in which the Ikk? gene was specifically deleted in the corneal stromal keratocytes. The Ikk?(?CS) corneas had normal morphology, transparency and thickness; however, they did not heal well from mild alkali burn injury. In contrast to the Ikk?(F/F) corneas that restored transparency in 2 weeks after injury, over 50% of the Ikk?(?CS) corneas failed to fully recover. They instead developed recurrent haze with increased stromal thickness, severe inflammation and apoptosis. This pathogenesis correlated with sustained myofibroblast transformation with increased ? smooth muscle actin (?-SMA) expression, higher levels of senescence ?-Gal activity and scar tissue formation at the late stage of wound healing. In addition, the Ikk?(?CS) corneas displayed elevated expression of hemo-oxygenase-1 (HO-1), a marker of oxidative stress, and activation of stress signaling pathways with increased JNK, c-Jun and SMAD2/3 phosphorylation. These data suggest that IKK? in keratocytes is required to repress oxidative stress and attenuate fibrogenesis and senescence in corneal wound healing.
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.
Project description:CB2R receptors have demonstrated beneficial effects in wound healing in several models. We therefore investigated a potential role of CB2R receptors in corneal wound healing. We examined the functional contribution of CB2R receptors to the course of wound closure in an in vivo murine model. We additionally examined corneal expression of CB2R receptors in mouse and the consequences of their activation on cellular signaling, migration and proliferation in cultured bovine corneal epithelial cells (CECs). Using a novel mouse model, we provide evidence that corneal injury increases CB2R receptor expression in cornea. The CB2R agonist JWH133 induces chemorepulsion in cultured bovine CECs but does not alter CEC proliferation. The signaling profile of CB2R activation is activating MAPK and increasing cAMP accumulation, the latter perhaps due to Gs-coupling. Lipidomic analysis in bovine cornea shows a rise in acylethanolamines including the endocannabinoid anandamide 1?h after injury. In vivo, CB2R deletion and pharmacological block result in a delayed course of wound closure. In summary, we find evidence that CB2R receptor promoter activity is increased by corneal injury and that these receptors are required for the normal course of wound closure, possibly via chemorepulsion.
Project description:Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and ?-smooth muscle actin (?-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGF?-mediated transcription of pro-fibrotic collagen I mRNA and ?-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.