Lumican/Lumikine Promotes Healing of Corneal Epithelium Debridement by Upregulation of EGFR Ligand Expression via Noncanonical Smad-Independent TGFβ/TBRs Signaling.

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13: YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican-ALK5 binding.

SMAD4-Dependent Signaling Pathway Involves in the Pathogenesis of TGFBR2-Related CE-like Phenotype.

(1) Background: Our previous data indicated that disturbance of the Transforming Growth Factor beta (TGFB) signaling pathway via its Type-2 Receptor (TGFBR2) can cause a Corneal Ectasia (CE)-like phenotype. The purpose of this study is to elucidate whether the SMAD4-dependent signaling pathway is involved in the TGFBR2-related CE-like pathogenesis. (2) Methods: was designed to be conditionally knocked out from keratocytes.

Impaired healing in an incision wound in corneal stroma in a lumican-null mouse.

Purpose: We investigated healing pattern of an incisional wound in corneal stroma of lumican-null (KO) mice.

Methods: C57BL/6 mice (wild-type, WT) and lumican-null (knockout, KO) mice were used. A linear full-thickness incision was produced in one cornea of each mouse.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

Introduction: This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model.

Transforming growth factor beta receptor 2 (Tgfbr2) deficiency in keratocytes results in corneal ectasia.

Purpose: We hypothesized that Transforming growth factor beta receptor 2 (Tgfbr2) deletion in keratocyte (Tgfbr2), the corneal stroma cell, can result in corneal thinning and generate a potential model for Cornea Ectasia (CE).

Methods: Corneal thickness of Tgfbr2 and Tgfbr2 was examined with Optical Coherence Tomography (OCT) at post-natal (P) days 42 and 70, respectively. Histological H&E staining, transmission electron micrograph (TEM), and immunofluorescence staining (IFS) were harnessed to examine corneal cell morphology, proliferation, differentiation, and collagen fibrils.

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice.

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity.

Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks.

Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

Purpose: This study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development.

Methods: Bitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocytes).

Shp2-mediated MAPK pathway regulates ΔNp63 in epithelium to promote corneal innervation and homeostasis.

Corneal nerve fibers serving sensory, reflex, and neurotrophic functions sustain corneal homeostasis and transparency to promote normal visual function. It is not known whether corneal epithelium is also important for the corneal innervation. Herein, we generated a compound transgenic mouse strain, K14rtTA;tetO-Cre (TC);Shp2, in which Shp2 was conditionally knocked out from K14-positive cells including corneal epithelium (Shp2) upon doxycycline (dox) administration.

Repressed Wnt Signaling Accelerates the Aging Process in Mouse Eyes.

Purpose: Ocular aging is a natural process of functional decline in vision. When the process reaches a point that compromised vision affects normal daily activity, it manifests as age-related ocular diseases, such as age-related macular degeneration, cataracts, glaucoma, and pseudoexfoliation syndrome. We previously reported that repressed Wnt signaling accelerated the maturation of corneal epithelium during tissue development.

Aberrant expression of a stabilized β-catenin mutant in keratocytes inhibits mouse corneal epithelial stratification.

We previously reported that genetic deletion of β-catenin in mouse corneal keratocytes resulted in precocious corneal epithelial stratification. In this study, to strengthen the notion that corneal keratocyte-derived Wnt/β-catenin signaling regulates corneal epithelial stratification during mouse development, we examined the consequence of conditional overexpression of a stabilized β-catenin mutant (Ctnnb1) in corneal keratocytes via a doxycycline (Dox)-inducible compound transgenic mouse strain. Histological analysis showed that conditional overexpression of Ctnnb1 in keratocytes inhibited corneal epithelial stratification during postnatal development.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse.

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model.

Sensory nerve supports epithelial stem cell function in healing of corneal epithelium in mice: the role of trigeminal nerve transient receptor potential vanilloid 4.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue.

Impaired healing of cornea incision injury in a TRPV1-deficient mouse.

The present study attempts to elucidate the role of TRPV1 cation channel receptor on primary repair in an incision-wounded mouse cornea in vivo. Previous study revealed that blocking TRPV1 suppressed myofibroblast formation and expression of transforming growth factor β1 (TGFβ1) in cultured keratocytes or ocular fibroblasts. Male C57BL/6 (wild-type; WT) mice and male C57BL/6 Trpv1-null (KO) mice incurred a full-thickness incision injury (1.

Lack of plakoglobin impairs integrity and wound healing in corneal epithelium in mice.

We generated cornea-specific plakoglobin (Jup; junctional plakoglobin) knockout mice in order to investigate the function of plakoglobin on the maintenance of the homeostasis of corneal epithelium in mice. Cornea epithelium-specific conditional knockouts (Jup) (cKO) were obtained by breeding keratin12-Cre (Krt12-Cre) mice to Jup-floxed (Jup) mice. Light and transmission electron microscopic and immunohistochemical analyses were carried out to determine consequence of the loss of plakoglobin on maintaining corneal epithelium integrity under mechanical stress, e.

RNA nanoparticle distribution and clearance in the eye after subconjunctival injection with and without thermosensitive hydrogels.

Thermodynamically and chemically stable RNA nanoparticles derived from the three-way junction (3WJ) of the pRNA from bacteriophage phi29 DNA packaging motor were examined previously for ocular delivery. It was reported that, after subconjunctival injection, RNA nanoparticles with tri-way shape entered the corneal cells but not the retinal cells, whereas particle with four-way shape entered both corneal and retinal cells. The present study evaluated ocular delivery of RNA nanoparticles with various shapes and sizes, and assessed the effect of thermosensitive hydrogels (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid); PLGA-PEG-PLGA) for increasing the retention of RNA nanoparticles in the eye.

Generation and Characterization of a Novel Mouse Line, Keratocan-rtTA (KeraRT), for Corneal Stroma and Tendon Research.

Purpose: We created a novel inducible mouse line Keratocan-rtTA (KeraRT) that allows specific genetic modification in corneal keratocytes and tenocytes during development and in adults.

Methods: A gene-targeting vector (Kera- IRES2-rtTA3) was constructed and inserted right after the termination codon of the mouse Kera allele via gene targeting techniques. The resulting KeraRT mouse was crossed to tet-O-Hist1H2B-EGFP (TH2B-EGFP) to obtain KeraRT/TH2B-EGFP compound transgenic mice, in which cells expressing Kera are labeled with green fluorescence protein (GFP) by doxycycline (Dox) induction.

The role of corneal stroma: A potential nutritional source for the cornea.

Corneal stroma plays a pivotal role in normal visual function. Anatomically, it is located between the outer epithelium and the inner endothelium and is the thickest layer of the cornea. Keratocytes in the stroma produce a variety of cellular products, including growth factors/cytokines, extracellular matrix (ECM) components, and kinases.

Ectodysplasin A regulates epithelial barrier function through sonic hedgehog signalling pathway.

Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive.

Mouse Corneal Stroma Fibroblast Primary Cell Culture.

This protocol is developed for primary cell culture of cornea stromal keratocytes isolated from neonatal mouse eyeballs. It provides an optimal condition to isolate stromal keratocytes which maintain high viability for cell culture.

Wakayama symposium: role of canonical Notch signaling in conjucntival goblet cell differentiation and dry eye syndrome.

This review summarizes a recent finding regarding the intrinsic canonical Notch signaling pathway in regulating normal ocular surface morphogenesis and its role in the pathogenesis of goblet cell deficiency-associated keratoconjunctivitis sicca (KCS, or dry eye). Specifically, we used novel transgenic mice to investigate the mechanism of how the Notch1 activation may serve as the upstream control of expression of transcription factors Krüppel-like factors 4 or 5 (Klf4 or Klf5) which in turn controls goblet cell differentiation and activates mucin 5/ac synthesis during ocular surface morphogenesis.

Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development.

The development of organs with an epithelial parenchyma relies on reciprocal mesenchymal-epithelial communication. Mouse corneal epithelium stratification is the consequence of a coordinated developmental process based on mesenchymal-epithelial interactions. The molecular mechanism underlying these interactions remains unclear.

Perturbed meibomian gland and tarsal plate morphogenesis by excess TGFα in eyelid stroma.

Transforming growth factor alpha (TGFα) belongs to the epidermal growth factor (EGF) family and is known to play an important role during eyelid morphogenesis. In this study, we showed that ectopic expression of TGFα in the stroma of Kera-rtTA/tet-O-TGFα bitransgenic mice results in precocious eye opening, abnormal morphogenesis of the meibomian gland, tendon and tarsal plate malformation and epithelium hyperplasia. TGFα did not change proliferation and differentiation of meibocytes, but promoted proliferation and inhibited differentiation of the tarsal plate tenocytes.

Corneal Epithelial Wound Healing.

The cornea is important for a clear vision by refracting light onto the lens, which in turn focusing on the retina. To maintain a smooth optical surface, corneal epithelium has to continuously renew itself to function as a barrier so that it protects the eye from various environmental insults. The adult corneal epithelium is maintained homeostatically by an integrated process of cell proliferation, migration, differentiation, stratification, and desquamation/apoptosis.