TGF-ß-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-ß), a versatile signaling molecule that coordinates various cell responses, has a central role in corneal wound healing. Upon corneal injury, TGF-ß is rapidly released into the extracellular environment, triggering cell migration and proliferation, the differentiation of keratocytes into myofibroblasts, and the initiation of the repair process. TGF-ß-mediated processes are essential for wound closure; however, excessive levels of TGF-ß can lead to fibrosis and scarring, causing impaired vision. Three primary isoforms of TGF-ß exist-TGF-ß1, TGF-ß2, and TGF-ß3. Although TGF-ß isoforms share many structural and functional similarities, they present distinct roles in corneal regeneration, which adds an additional layer of complexity to understand the role of TGF-ß in corneal wound healing. Further, aberrant TGF-ß activity has been linked to various corneal pathologies, such as scarring and Peter's Anomaly. Thus, understanding the molecular and cellular mechanisms by which TGF-ß1-3 regulate corneal wound healing will enable the development of potential therapeutic interventions targeting the key molecule in this process. Herein, we summarize the multifaceted roles of TGF-ß in corneal wound healing, dissecting its mechanisms of action and interactions with other molecules, and outline its role in corneal pathogenesis.

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. Mice were subjected to unilateral corneal debridement or alkali burn (AB) injuries and wound healing assessed over time using fluorescein stain, in vivo confocal microscopy and histology. RESULTS: TSG-6 is endogenously expressed in the human and mouse cornea and established corneal epithelial cell lines and is upregulated after injury. A loss of TSG-6 has no structural and functional effect in the cornea during homeostasis. No differences were noted in the rate of corneal epithelial wound closure between BALB/c, TSG-6+/- and TSG-6-/- mice. TSG-6-/- mice presented decreased inflammatory response within the first 24 h of injury and accelerated corneal wound healing following AB when compared to control mice. CONCLUSION: TSG-6 is endogenously expressed in the cornea and upregulated after injury where it propagates the inflammatory response following chemical injury.

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. Methods: The cornea and limbal rim were dissected from mouse corneas, subjected to mRNA extraction, and sequenced using a NextSeq 500 (Illumina) and data processed using CLC Genomics Workbench 20 (Qiagen) and the STRING database to identify key components of the LSCN. Their expression was confirmed by real-time PCR, Western blotting, and immunohistochemistry. Furthermore, the differential expression of key compounds in different corneal cell types were determined with single-cell RNA sequencing. Results: We identified that the hyaladherins inter-alpha-inhibitor (IαI), TSG-6 and versican are highly expressed in the limbus. Specifically, HA/HC complexes are present in the LSCN, in the stroma underlying the limbal epithelium, and surrounding the limbal vasculature. For IαI, heavy chains 5 and 2 (HC5 and HC2) were found to be the most highly expressed HCs in the mouse and human limbus and were associate with HA-forming HA/HC-specific matrices. Conclusions: The LSCN contains HA/HC complexes, which have been previously correlated with stem cell niches. The identification of HA/HC complexes in the LSCN could serve as a new therapeutic avenue for treating corneal pathology. Additionally, HA/HC complexes could be used as a substrate for culturing LESCs before LESC transplantation.

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. For this, high-molecular-weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl). ROS lead to the fragmentation of HA, with the oxHA products produced by HOCl exhibiting an altered chemical structure while those produced by H2O2 do not. HMWHA promotes the viability of human corneal epithelial cells (hTCEpi), while low MWHA (LMWHA), ultra-LMWHA (ULMWHA), and most forms of oxHA do not. HMWHA and LMWHA promote hTCEpi proliferation, while ULMWHA and all forms of oxHA do not. LMWHA and some forms of oxHA promote hTCEpi migration, while HMWHA does not. Finally, all native forms of HA and oxHA produced by HOCl promote in vivo corneal wound healing, while oxHA produced by H2O2 does not. Taken together, our results show that HA fragmentation by ROS can alter the physiological activity of HA by altering its MW and structure.

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. Hence, a thorough understanding of the timeline of MG development, maturation and aging is required for regenerative purposes along with signaling molecules & pathways controlling proper differentiation of MG lineage in mammalian eye. Understanding the factors that contribute to the development of MGs, developmental abnormalities of MGs, and changes in the quality & quantity of meibum with developing phases of MGs are essential for developing potential treatments for MGD. In this review, we compiled a timeline of events and the factors involved in the structural and functional development of MGs and the associated developmental defects of MGs during development, maturation and aging.

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. This study investigated whether changes in the distribution of HA before and after injury affects the biomechanical properties of the cornea in vivo. Methods: Corneas of wild-type (wt) mice and mice lacking enzymes involved in the biosynthesis of HA were analyzed before, immediately after, and 7 and 14 days after a corneal alkali burn (AB). The corneas were evaluated using both a ring light and fluorescein stain by in vivo confocal microscopy, optical coherence elastography (OCE), and immunostaining of corneal whole mounts. Results: Our results show that wt mice and mice lacking HA synthase (Has)1 and 3 present an increase in corneal stiffness 7 and 14 days after AB without a significant increase in HA expression and absence of scarring at 14 days after AB. In contrast, mice lacking Has2 present a significant decrease in corneal stiffness, with a significant increase in HA expression and scarring at 14 days after AB. Conclusions: Our findings show that the mechanical properties of the cornea are significantly modulated by changes in HA distribution following alkali burn.

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. METHOD: Human LESCs (hLESCs) were isolated from donor corneas and a mouse corneal epithelial progenitor cell line (TKE2) was obtained. The HA matrix was identified surrounding LESCs in vitro using immunocytochemistry, flow cytometry and red blood exclusion assay. Thereafter, LESCs were maintained on HA coated dishes or in the presence of HA supplemented in the media, and viability, proliferation, cell size, colony formation capabilities and expression of putative stem cell markers were compared with cells maintained on commonly used coated dishes. RESULTS: hLESCs and TKE2 cells express an HA-rich matrix in vitro, and this matrix is essential for maintaining LESCs. Further supplying exogenous HA, as a substrate and supplemented to the media, increases LESC proliferation, colony formation capabilities and the expression levels of putative limbal stem cell markers. CONCLUSION: Our data show that both exogenous and endogenous HA help to maintain the LESC phenotype. Exogenous HA provides improved culture conditions for LESC during ex vivo expansion. Thus, HA forms a favorable microenvironment for LESCs during ex vivo expansion and, therefore, could be considered as an easy and cost-effective substrate and/or supplement for culturing LESCs in the clinic.

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. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal 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. The steroid disulfates that are formed are the focus of this review. We discuss both their biochemical production as well as their putative biological function. Steroid disulfates have also been linked to various clinical conditions in numerous untargeted metabolomics studies. New analytical techniques exploring the biosynthetic routes of steroid disulfates have led to novel insights, changing our understanding of sulfation in human biology. They promise a bright future for research into sulfation pathways, hopefully too for the diagnosis and treatment of several associated diseases.

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. The unique organization of ECM components within the cornea is essential for the maintenance of corneal transparency and function. Many studies have described the importance of GAGs within the epithelial and stromal compartment, while very few studies have analyzed the ECM of the endothelial layer. Importantly, GAGs have been shown to be essential for maintaining corneal homeostasis, epithelial cell differentiation and wound healing, and, more recently, a role has been suggested for the ECM in regulating limbal stem cells, corneal innervation, corneal inflammation, corneal angiogenesis and lymphangiogenesis. Reports have also associated genetic defects of the ECM to corneal pathologies. Thus, we also highlight the role of different GAGs and PGs in ocular surface homeostasis, as well as in pathology.

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. Methods: To establish different aspects of corneal epithelial wound healing we subjected mice lacking hyaluronan synthase 2 (previously shown to lack LSCs) and wild-type mice to different corneal debridement injury models. Results: Our data show that both LSCs and corneal epithelial cells contribute toward closure of corneal wounds. In wild-type mice, removal of the limbal rim delayed closure of 1.5-mm wounds, and not of 0.75-mm wounds, indicating that smaller wounds do not rely on LSCs as do larger wounds. In mice shown to lack LSCs, removal of the limbal rim did not affect wound healing, irrespective of the wound size. Finally, transient amplifying cells and central epithelial cells move toward a central corneal wound in a centripetal manner, whereas central epithelial cells may move in a centrifugal manner to resurface peripheral corneal wounds. Conclusions: Our findings show the dimensions of the corneal wound dictate involvement of LSCs. Our data suggest that divergent findings by different groups on the dynamics of wound healing can be in part owing to differences in the wounding models used.

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. Results: Here we report that HA regulates corneal lymphangiogenesis, both during post-natal development and in response to adult corneal injury. Furthermore, we show that injury to the cornea by alkali burn upregulates both HA production and lymphangiogenesis and that these processes are ablated in HA synthase 2 deficient mice. Conclusion: These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis might prevent the corneal scarring and rejection that frequently results from corneal transplantation.

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. Here, we report the use of molecular dynamics simulations to investigate the dynamics of substrate recognition of two glycosaminoglycan (GAG) sulfotransferases, N-deacetylase-N-sulfotransferase and 2-O-sulfotransferase to the HS chain during the biosynthetic process. We performed multiple simulations of the binding of the sulfotransferase domains to both the HS oligosaccharide substrate and sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate. Analysis of extended simulations provide detailed and useful insights into the atomic interactions that are at work during oligosaccharide processing. The fast information matching method was used to detect the enzyme global dynamics and to predict the pairwise contact of residues responsible for GAG-enzyme binding and unbinding. The correlation between HS displacement and the location of the modified GAG chain were calculated, indicating a possible route for HS and heparin during sulfotransferase processing. Our data also show sulfotransferases contain a conserved interspaced positively charged amino acid residues that form a patch which controls the protein-GAG binding equilibrium. Together, our findings provide further understanding on the fine-tuned complex mechanism of GAG biosynthesis. Our findings can also be extrapolated to other systems for calculating rates of protein-GAG binding.

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 PAPSS2 mutations manifest with undetectable DHEA sulfate, androgen excess, and metabolic disease, suggesting that ubiquitous PAPSS1 cannot compensate for deficient PAPSS2 in supporting DHEA sulfation. In knockdown studies in human adrenocortical NCI-H295R1 cells, we found that PAPSS2, but not PAPSS1, is required for efficient DHEA sulfation. Specific APS kinase activity, the rate-limiting step in PAPS biosynthesis, did not differ between PAPSS1 and PAPSS2. Co-expression of cytoplasmic SULT2A1 with a cytoplasmic PAPSS2 variant supported DHEA sulfation more efficiently than co-expression with nuclear PAPSS2 or nuclear/cytosolic PAPSS1. Proximity ligation assays revealed protein-protein interactions between SULT2A1 and PAPSS2 and, to a lesser extent, PAPSS1. Molecular docking studies showed a putative binding site for SULT2A1 within the PAPSS2 APS kinase domain. Energy-dependent scoring of docking solutions identified the interaction as specific for the PAPSS2 and SULT2A1 isoforms. These findings elucidate the mechanistic basis for the selective requirement for PAPSS2 in human 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. Our work investigates the composition of the extracellular matrix in the LSC niche and how it regulates LSC differentiation and function. Methods: Hyaluronan (HA) is naturally synthesized by hyaluronan synthases (HASs), and vertebrates have the following three types: HAS1, HAS2, and HAS3. Wild-type and HAS and TSG-6 knockout mice-HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, TSG-6-/--were used to determine the importance of the HA niche in LSC differentiation and specification. Results: Our data demonstrate that the LSC niche is composed of a HA rich extracellular matrix. HAS1-/-;HAS3-/-, HAS2Δ/ΔCorEpi, and TSG-6-/- mice have delayed wound healing and increased inflammation after injury. Interestingly, upon insult the HAS knock-out mice up-regulate HA throughout the cornea through a compensatory mechanism, and in turn this alters LSC and epithelial cell specification. Conclusions: The LSC niche is composed of a specialized HA matrix that differs from that present in the rest of the corneal epithelium, and the disruption of this specific HA matrix within the LSC niche leads to compromised corneal epithelial regeneration. Finally, our findings suggest that HA has a major role in maintaining the LSC phenotype.

Extrinsic and Intrinsic Mechanisms by Which Mesenchymal Stem Cells Suppress the Immune System.

Mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells that have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Recent studies have demonstrated that MSCs possess a unique ability to exert suppressive and regulatory effects on both adaptive and innate immunity in an autologous and allogeneic manner. A vital step in stem cell transplantation is overcoming the potential graft-versus-host disease, which is a limiting factor to transplantation success. Given that MSCs attain powerful differentiation capabilities and also present immunosuppressive properties, which enable them to survive host immune rejection, MSCs are of great interest. Due to their ability to differentiate into different cell types and to suppress and modulate the immune system, MSCs are being developed for treating a plethora of diseases, including immune disorders. Moreover, in recent years, MSCs have been genetically engineered to treat and sometimes even cure some diseases, and the use of MSCs for cell therapy presents new perspectives for overcoming tissue rejection. In this review, we discuss the potential extrinsic and intrinsic mechanisms that underlie MSCs' unique ability to modulate inflammation, and both innate and adaptive immunity.

The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth.

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeletons. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

On the catalytic mechanism of polysaccharide lyases: evidence of His and Tyr involvement in heparin lysis by heparinase I and the role of Ca2+.

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca(2+)-heparin, in which α-L-iduronate-2-O-sulfate residues adopt (1)C4 conformation preferentially, is a substrate, while Na(+)-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates ß-elimination by abstracting the C5 proton of the α-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.