Toxicity of nuclear-localized GFP in reporter mice.

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial-temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system.

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.

TGF-β-Based Therapies for Treating Ocular Surface Disorders.

The cornea is continuously exposed to injuries, ranging from minor scratches to deep traumas. An effective healing mechanism is crucial for the cornea to restore its structure and function following major and minor insults. Transforming Growth Factor-Beta (TGF-β), a versatile signaling molecule that coordinates various cell responses, has a central role in corneal wound healing.

Endogenous TSG-6 modulates corneal inflammation following chemical injury.

Purpose: Tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) is upregulated in various pathophysiological contexts, where it has a diverse repertoire of immunoregulatory functions. Herein, we investigated the expression and function of TSG-6 during corneal homeostasis and after injury.

Methods: Human corneas, eyeballs from BALB/c (TSG-6), TSG-6 and TSG-6 mice, human immortalized corneal epithelial cells and murine corneal epithelial progenitor cells were prepared for immunostaining and real time PCR analysis of endogenous expression of TSG-6.

Recent advances in age-related meibomian gland dysfunction (ARMGD).

Meibomian glands (MGs), located within the tarsal plate of the eyelid, secrete meibum which is the lipid-rich secretion necessary for stabilizing the tear film and preventing tear evaporation. Changes in the quality and quantity of meibum produced causes MG dysfunction (MGD), the leading cause of evaporative dry eye disease (EDED). MGD is an underdiagnosed disease and it is estimated that, in the US, approximately 70 % of the population over 60 have MGD.

Age related changes in hyaluronan expression leads to Meibomian gland dysfunction.

The prevalence of dry eye disease (DED) ranges from ∼5 to 50 % and its associated symptoms decrease productivity and reduce the quality of life. Approximately 85 % of all DED cases are caused by Meibomian gland dysfunction (MGD). As humans and mice age, their Meibomian glands (MGs) undergo age-related changes resulting in age related-MGD (ARMGD).

Characterization of the Limbal Epithelial Stem Cell Niche.

Purpose: Limbal epithelial stem cells (LESCs) reside within a LSC niche (LSCN). We recently identified that hyaluronan (HA) is a major constituent of the LSCN, and that HA is necessary for maintaining LESCs in the "stem cell" state, both in vitro and in vivo. Herein, we characterized the LSCN to identify key components of the HA-specific LSCN.

ROS-Mediated Fragmentation Alters the Effects of Hyaluronan on Corneal Epithelial Wound Healing.

A buildup of reactive oxygen species (ROS) occurs in virtually all pathological conditions. Hyaluronan (HA) is a major extracellular matrix component and is susceptible to oxidation by reactive oxygen species (ROS), yet the precise chemical structures of oxidized HA products (oxHA) and their physiological properties remain largely unknown. This study characterized the molecular weight (MW), structures, and physiological properties of oxHA.

Rational design and synthesis of lumican stapled peptides for promoting corneal wound healing.

Purpose: Lumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13 with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential.

Small leucine rich proteoglycans: Biology, function and their therapeutic potential in the ocular surface.

Small leucine rich proteoglycans (SLRPs) are the largest family of proteoglycans, with 18 members that are subdivided into five classes. SLRPs are small in size and can be present in tissues as glycosylated and non-glycosylated proteins, and the most studied SLRPs include decorin, biglycan, lumican, keratocan and fibromodulin. SLRPs specifically bind to collagen fibrils, regulating collagen fibrillogenesis and the biomechanical properties of tissues, and are expressed at particularly high levels in fibrous tissues, such as the cornea.

Meibomian gland development: Where, when and how?

The Meibomian gland (MG) is an indispensable adnexal structure of eye that produces meibum, an important defensive component for maintaining ocular homeostasis. Normal development and maintenance of the MGs is required for ocular health since atrophic MGs and disturbances in composition and/or secretion of meibum result in major ocular pathologies, collectively termed as Meibomian gland dysfunction (MGD). Currently available therapies for MGD merely provide symptomatic relief and do not treat the underlying deficiency of the MGs.

Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography.

Purpose: Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products.

In Vitro Study of Cytotoxic Mechanisms of Alkylphospholipids and Alkyltriazoles in Acute Lymphoblastic Leukemia Models.

This study investigates the efficacy of miltefosine, alkylphospholipid, and alkyltriazolederivative compounds against leukemia lineages. The cytotoxic effects and cellular and molecular mechanisms of the compounds were investigated. The inhibitory potential and mechanism of inhibition of cathepsins B and L, molecular docking simulation, molecular dynamics and binding free energy evaluation were performed to determine the interaction of cathepsins and compounds.

Hyaluronan Modulates the Biomechanical Properties of the Cornea.

Purpose: Hyaluronan (HA) is a major constituent of the extracellular matrix (ECM) that has high viscosity and is essential for maintaining tissue hydration. In the cornea, HA is enriched in the limbal region and is a key component of the limbal epithelial stem cell niche. HA is upregulated after injury participating in the formation of the provisional matrix, and has a key role in regulating the wound healing process.

Hyaluronan supports the limbal stem cell phenotype during ex vivo culture.

Background: Hyaluronan (HA) has previously been identified as an integral component of the limbal stem cell niche in vivo. In this study, we investigated whether a similar HA matrix is also expressed in vitro providing a niche supporting limbal epithelial stem cells (LESCs) during ex vivo expansion. We also investigated whether providing exogenous HA in vitro is beneficial to LESCs during ex vivo expansion.

Structural Determinants of Substrate Recognition and Catalysis by Heparan Sulfate Sulfotransferases.

Heparan sulfate (HS) and heparin contain imprinted "sulfation codes", which dictate their diverse physiological and pathological functions. A group of orchestrated biosynthetic enzymes cooperate in polymerizing and modifying HS chains. The biotechnological development of enzymes that can recreate this sulfation pattern on synthetic heparin is challenging, primarily due to the paucity of quantitative data for sulfotransferase enzymes.

Extracellular Matrix Deposition and Remodeling after Corneal Alkali Burn in Mice.

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury.

Steroid disulfates - Sulfation double trouble.

Sulfation pathways have recently come into the focus of biomedical research. For steroid hormones and related compounds, sulfation represents an additional layer of regulation as sulfated steroids are more water-soluble and tend to be biologically less active. For steroid diols, an additional sulfation is possible, carried out by the same sulfotransferases that catalyze the first sulfation step.

Distribution and Function of Glycosaminoglycans and Proteoglycans in the Development, Homeostasis and Pathology of the Ocular Surface.

The ocular surface, which forms the interface between the eye and the external environment, includes the cornea, corneoscleral limbus, the conjunctiva and the accessory glands that produce the tear film. Glycosaminoglycans (GAGs) and proteoglycans (PGs) have been shown to play important roles in the development, hemostasis and pathology of the ocular surface. Herein we review the current literature related to the distribution and function of GAGs and PGs within the ocular surface, with focus on the cornea.

Epithelial Cell Migration and Proliferation Patterns During Initial Wound Closure in Normal Mice and an Experimental Model of Limbal Stem Cell Deficiency.

Purpose: Establishing the dynamics of corneal wound healing is of vital importance to better understand corneal inflammation, pathology, and corneal regeneration. Numerous studies have made great strides in investigating multiple aspects of corneal wound healing; however, some aspects remain to be elucidated. This study worked toward establishing (1) if epithelial limbal stem cells (LSCs) are necessary for healing all corneal wounds, (2) the mechanism by which epithelial cells migrate toward the wound, and (3) if centrifugal epithelial cell movement exists.

Hyaluronan Derived From the Limbus is a Key Regulator of Corneal Lymphangiogenesis.

Purpose: We recently reported that the glycosaminoglycan hyaluronan (HA), which promotes inflammatory angiogenesis in other vascular beds, is an abundant component of the limbal extracellular matrix. Consequently, we have explored the possibility that HA contributes to lymphangiogenesis in the inflamed cornea.

Methods: To study the role of HA on lymphangiogenesis, we used mice lacking the hyaluronan synthases and injury models that induce lymphangiogenesis.

Structural basis of oligosaccharide processing by glycosaminoglycan sulfotransferases.

Heparan sulfate (HS) is a sulfated polysaccharide that plays a key role in morphogenesis, physiology and pathogenesis. The biosynthesis of HS takes place in the Golgi apparatus by a group of enzymes that polymerize, epimerize and sulfate the sugar chain. This biosynthetic process introduces varying degrees of sulfate substitution, which are tightly regulated and directly dictate binding specificity to different cytokines, morphogens and growth factors.

Human DHEA sulfation requires direct interaction between PAPS synthase 2 and DHEA sulfotransferase SULT2A1.

The high-energy sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS), generated by human PAPS synthase isoforms PAPSS1 and PAPSS2, is required for all human sulfation pathways. Sulfotransferase SULT2A1 uses PAPS for sulfation of the androgen precursor dehydroepiandrosterone (DHEA), thereby reducing downstream activation of DHEA to active androgens. Human mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation.

Hyaluronan Rich Microenvironment in the Limbal Stem Cell Niche Regulates Limbal Stem Cell Differentiation.

Purpose: Limbal epithelial stem cells (LSCs), located in the basal layer of the corneal epithelium in the corneal limbus, are vital for maintaining the corneal epithelium. LSCs have a high capacity of self-renewal with increased potential for error-free proliferation and poor differentiation. To date, limited research has focused on unveiling the composition of the limbal stem cell niche, and, more important, on the role the specific stem cell niche may have in LSC differentiation and function.