The Ocular Surface Glycocalyx and its Alteration in Dry Eye Disease: A Review

Abstract

Many studies have revealed that transmembrane mucins, large glycoproteins with heavily glycosylated glycans, are essential for maintaining ocular surface epithelium lubrication and wettability. Recent reports indicate that transmembrane mucins and galectin-3, a chimera type of galectin that binds β-galactoside in the glycan, play a crucial role in maintaining the epithelial glycocalyx barrier. This review summarizes current evidence regarding the role of galectin-3, the role of the three major transmembrane mucins (i.e., MUC1, MUC4, and MUC16), in the maintenance of ocular surface wettability and transcellular barrier. Pathological mechanisms of glycocalyx barrier disruption and epithelial surface wettability decreases in dry eye disease are also summarized. Lastly, new ophthalmic drugs that target transmembrane mucin are described.

Ocular Surface Lubrication, Wettability, and Barrier Function of Glycocalyx

The apical membrane of the ocular surface epithelium has microplicae, which act as a protrusion and increase the surface area of most superficial epithelial cells.¹ The glycocalyx on the microplicae are largely glycoprotein that cover the surface of epithelial and other cells.² The carbohydrate layer can be visualized using various stains, as well as by its affinity for lectins and carbohydrate-binding proteins, which can be labeled with a fluorescent dye or other visible marker. Most animal epithelial cells have a “fuzzy” coating (glycocalyx) on the apical surface of their plasma membranes. This glycocalyx is the boundary between the epithelium and the tears,³ and consists of several trans-membrane glycoproteins, which serve as the backbone of glycocalyx.^4,5 The glycocalyx can extend up to 500 nm from the plasma membrane at the ocular surface, and is mainly composed of membrane-tethered mucins that play a role in epithelial surface lubrication, hydration, and barrier function.^4–6 

The huge glycocalyx glycoproteins generally help to protect cells against mechanical and chemical damage, prevent pathogen penetration into the eye, reduce friction during blinking, and maintain the hydrophilicity of the ocular surface.² Both the corneal and conjunctival surface epithelium have a transcellular barrier in the form of epithelial glycocalyx made up of membrane-associated mucin and galectin-3,^7,8 and a paracellular barrier in the form of a tight junction made up of occludin, claudin, and junctional adhesion molecules.⁹ Although surface epithelial cell tight junctions create a barrier to ion and hydrophilic molecule passage, apical plasma membranes and their associated glycocalyx are readily permeable to lipophilic molecules. Furthermore, conjunctival epithelium tight junctions are more leaky than corneal epithelium tight junctions.¹⁰ 

Transmembrane Mucins in Glycocalyx

Mucins are the largest, most highly glycosylated glycoproteins and consist of at least 20 different glycoproteins with two characteristic features.^11,12 First, the mucin extracellular domain is made up of multiple tandem repeats of amino acids rich in serine and threonine, which provide sites for N-acetyl-galactosamine (GalNAc) linkage to various galactose attachments via O-glycosylation.^4,5 Second, extensive O-glycosylation, which accounts for 50% to 80% of molecule's mass, which contributes to a mucin's negative hydrophilic charge.¹³ 

Stratified corneal and conjunctival epithelia express the following four transmembrane-associated mucins: MUC1,⁴ MUC4,¹⁴ MUC16,⁵ and MUC20.¹⁵ Cell surface mucins, especially MUC1 and MUC16, are concentrated at microplicae tips and form a dense glycocalyx at the epithelial-tear film interface.^4,5,16 MUC4 mRNA and protein are predominant in the conjunctival epithelium, with an apparent diminution of mRNA toward the central corneal epithelium, and little if any in the central corneal epithelium.¹⁴ Glycogene expression microarrays have indicated that MUC20, a transmembrane mucin, is highly expressed in the human corneal and conjunctival epithelium,¹⁷ but its function is not known on ocular surface. The three main functions of ocular surface transmembrane mucins are (1) surface protection against frictional stress (boundary lubrication), (2) apical cell surface barrier formation against allergens, pathogens, and extracellular molecules (glycocalyx barrier), and (3) improve epithelium wettability by converting the hydrophobic plasma membrane to a hydrophilic one via extensive O-glycosylation.^4,5 A dense polymer mucin network also has a high hydration (i.e., water holding) capacity. Therefore, mucin may also suppress tear evaporation.¹⁸ 

The three main ocular surface membrane mucins are MUC1 (120–300 kDa), MUC4 (900 kDa), and MUC16 (20 MDa).¹⁹ Mucin molecular weight varies between individuals because of genetic polymorphisms in the number of tandem repeats in the protein backbone. MUC1 was first isolated from the surface of breast carcinoma cells and was the first mucin to be cloned and analyzed.²⁰ However, there is very little direct information on MUC1 function on the ocular surface. In vitro studies have shown that MUC1 serves as an antiadhesive molecule,²¹ a signaling molecule,²² and a pathogen barrier.^23–25 In general, MUC4 is involved in proliferation signaling via tyrosine kinase ErbB2 receptor activation in the MUC4-β epidermal growth factor (EGF) domains.²⁶ However, there are no data on the function of MUC4 at the human ocular surface. With 22,152 amino acids in its protein sequence and a fully glycosylated molecular weight of approximately 20 MDa,²⁷ MUC16 is the largest membrane-associated mucin.²⁸ On the ocular surface, MUC16 is a glycocalyx component that forms a protective covering, thereby contributing to pathogen and molecule barrier formation. Rose bengal dye staining is used to evaluate ocular surface damage in patients with dry eye disease (DED) because it stains cells with a disrupted glycocalyx barrier function. Interestingly, cultured stratified human corneal epithelial cells form islands that prevent rose bengal dye penetration.²⁹ The direct role of MUC16 in preventing rose bengal staining was shown using an small interfering RNA (siRNA) MUC16 knockdown, which led to an increase in rose bengal staining in cultured corneal epithelial cells.³⁰ A more recent study compared the roles of MUC1 and MUC16 in the barrier function. Corneal epithelial cells with knocked down MUC16 had a decrease in all barrier functions, including protection against dye penetration, protection against bacterial adherence and invasion, trans-epithelial resistance, and tight junction formation. In contrast, cells with knocked down MUC1 had a significantly increased barrier to dye penetration and bacterial invasion (Fig. 1).⁶ Therefore, MUC16 likely plays a larger role in glycocalyx barrier formation than other membrane-anchored ocular surface mucins. 

Figure 1

 

Comparison of mucins MUC1 and MUC16 in epithelial barrier function. Rose bengal stains conjunctival epithelial cells that lack an apical surface barrier. Islands of unstained cells were observed in all cells examined, but human corneal limbal epithelia MUC16 knockdown cells (D) had smaller unstained islands than both MUC1 knockdown (B) and control cell lines (A, C, E). Scale bar: 50 μm. Reprinted from Gipson IK, Spurr-Michaud S, Tisdale A, Menon BB. Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS One. 2014;9(6):e100393. © 2014 Gipson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License.

The Role of Galectin-3 in the Glycocalyx Barrier

Heavy O-glycosylation of MUC16 is essential for maintaining the ocular surface glycocalyx barrier. However, it was not known whether or not transmembrane mucins were the only molecules able to form an epithelial glycocalyx barrier. Galectins are a family of soluble lectins that have a conserved carbohydrate recognition domain (CRD) and bind β-galactoside-containing glycans.^31,32 A total of 15 galectins have been identified in mammals and are widely distributed among various types of cells and tissues.³³ Galectins are thought to mediate diverse biological processes and are involved in the regulation of cell activation, cell adhesion, differentiation, cytokine secretion, inflammation, wound healing, and apoptosis.^34–36 

A recent study investigated the interaction between ocular surface transmembrane mucins and galectin-3, which is the most highly expressed carbohydrate-binding protein in normal human conjunctiva.¹⁷ Galectin-3 is a 35 kDa protein originally identified as only a chimera type galectin. The galectin-3 N-terminus enables oligomer through pentamer formation, which contributes to the variable geometries of crosslinked galectin-3 lattices.^37,38 These crosslinked molecules included EGF receptors and α5β1 integrin, both of which help generate molecular lattices.^39,40 In the apical glycocalyx, galectin-3 colocalizes membrane-associated MUC1 and MUC16, with both mucins binding to galectin-3 affinity columns in a galactose dependent manner.⁸ Disrupting mucin-galectin interactions resulted in decreased galectin-3 protein levels on the cell surface with concomitant barrier function loss, as indicated by increased permeability to rose bengal dye. Furthermore, significant loss of corneal glycocalyx barrier function was observed in galectin-3 null mice and in stratified human corneal epithelial cells when galectin-3 biosynthesis was interrupted using siRNA.⁴¹ Together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function via carbohydrate-dependent interactions with transmembrane mucins on the apical surface of ocular surface epithelial cells. 

Mucin Expression and Glycosylation in Patients With DED

Eyes with DED have altered expression and glycosylation of transmembrane mucin, as revealed by an immunohistochemical study that examined the H185 antibody. This antibody recognizes the MUC16 carbohydrate epitope (but not that of MUC1).⁵ Conjunctival cells have an H185 antibody “mosaic” binding pattern, with cells having light, medium, or intense binding. In eyes with non-Sjögren's syndrome DED, the H185 mosaic binding pattern was lost and was replaced by a “starry sky” pattern” (lack of apical cell binding was the “dark sky,” increase in goblet cell binding was the “stars in the sky”; Fig. 2).⁴² 

Figure 2

 

MUC16 glycan expression pattern, as illustrated by immunofluorescence micrographs showing binding of the H185 antibody to filter paper-stripped conjunctival samples from normal and dry eye subjects. Normal subjects had a mosaic binding pattern of various levels of MUC16 glycans differentiation. In contrast, dry eye subjects had a starry sky pattern, indicating large areas with no H185 antibody binding and occasional areas with very intense H185 antibody binding. Reprinted with permission from Gipson IK, Argüeso P. Role of mucins in the function of the corneal and conjunctival epithelia. Int Rev Cytol. 2003;231:1–49. Copyright © 2003 Elsevier Inc. Please refer to Danjo et al.⁴² for details on the “stars in the sky.” Scale bar: 100 μm.

Mucin glycosylation is also altered in eyes with diseases of the ocular surface. Contact lens users, DED patients, and glaucoma patients treated with β-blockers have a decrease in conjunctival sialylated mucin chains, as evaluated using impression cytology. Glycosyltransferases are essential enzymes that catalyze glycol-chain initiation and elongation by transferring sugar residue onto core proteins. In mucins, the posttranslational addition of GalNAc to serine or threonine residues of the protein backbone is the initial transfer step for producing O-glycans via GalNAc transferases (GalNAc-Ts). Patients with early-stage ocular cicatricial pemphigoid (OCP) have an increased distribution of GalNAc-Ts.⁴³ This suggests that ocular surface epithelial cells compensate by synthesizing mucin O-glycans to maintain a wet surface at the apical epithelium. However, in patients with late-stage OCP and a keratinized epithelium, GalNAc-Ts were not detected on the epithelial surface via immunohistochemical analysis.⁴³ Therefore, evidence suggests that transmembrane mucin expression and mucin glycosylation are both altered in eyes with DED. 

Conjunctival mucosal epithelial membrane mucin expression and MUC1 immunoreactivity were decreased in patients with Sjögren's syndrome.^44,45 Additionally, non-Sjögren's syndrome dry eye patients have significantly lower MUC1, MUC2, MUC4, MUC5AC, and MUC7 gene expression than healthy subjects, as revealed with conjunctival impression cytology.⁴⁶ Furthermore, conjunctival MUC5AC and MUC16 mRNA expression was decreased in patients with an unstable tear film, caused by aqueous deficient or short breakup time (BUT) DED, compared to normal subjects.⁴⁷ In contrast, an increase in MUC1 sialylation has been observed in eyes with mild-to-moderate DED, but a decrease was observed in eyes with severe DED.⁴⁸ In yet another study, patients with Sjögren's syndrome DED had significantly higher mRNA expression and MUC16⁴⁹ and MUC1⁵⁰ levels compared to normal subjects. Unfortunately, this conflicting evidence has not yet been explained and is not well understood. However, postmenopausal women with non-Sjögren's mild or moderate DED had significantly higher than normal levels of MUC1 and MUC16 mRNA and/or protein, likely in response to ocular surface irritation and inflammation associated with early stages of DED.⁵¹ 

New Evaluation for Decreased Wettability and Glycocalyx Barrier Disruption on the Ocular Surface

A large amount of membrane-associated mucin glycans trap the CRD of galectin-3 released by epithelial cells. Galectin-3 oligomers then form bridges between transmembrane mucins, creating a galectin-lattice formation.⁸ This glycocalyx barrier protects the cell surface by creating a tremendously thick, insulating layer that shields the ocular surface from harmful environmental conditions (Fig. 3). 

Figure 3

 

Illustration of a normal and disrupted glycocalyx barrier. Under normal conditions, bonding between transmembrane mucin glycans and the galectin-3 CRD forms pentamers that are crucial to maintaining glycocalyx barrier function. In eyes with dry eye disease, mucin glycans are shorter and fewer in number. Galectin-3 is subsequently not held in place by mucin glycans and is released into the tear film on the ocular surface. Modified from Argüeso P. Glycobiology of the ocular surface: mucins and lectins. Jpn J Ophthalmol. 2013;57:150–155. © Japanese Ophthalmological Society 2013.

DED alters transmembrane mucin glycosylation^2,42 and downregulates glucosyltransferase protein expression, subsequently decreasing glycans elongation.⁴³ These pathological conditions result in a decrease in the quality and quantity of membrane tethered mucins and glycans are not able trap all of the galectin-3 that is released by ocular surface superficial epithelial cells. Therefore, in eyes with DED, galectin-3 is not retained in the glycocalyx and is secreted into the tear film on the ocular surface (Fig. 3).⁵² A nonquantitative immunoblotting study with a limited cohort design found that galectin-3 was not present in the tears of normal subjects (n = 4 subjects), but that it was present in the tears of subjects with various ocular surface abnormalities (n = 9 subjects), including bullous keratopathy, sarcoidosis, chronic blepharitis, toxic conjunctivitis, adenoviral conjunctivitis, and alkali burn.⁵³ Additionally, our group's previous study found that galectin-3 concentration was higher in the tears of 16 DED subjects (including 15 aqueous deficient DED, 1 Sjögren's syndrome, 2 graft-vs-host disease (GVHD), and 1 aqueous sufficient DED patients) than in 11 normal subjects.⁵² This study showed that tear galectin-3 concentration is negatively correlated with tear BUT, likely due to ocular surface glycocalyx disruption. 

Glycans polymer formation by transmembrane mucins is essential for maintaining ocular surface wettability. DED inhibits this process, resulting in a glycocalyx with a decreased capacity for holding water, which subsequently shortens tear BUT. Not surprisingly, glycocalyx disruption reduces ocular surface wettability, increasing the amount of galectin-3 that is released into the tear film from glycocalyx components. Furthermore, galectin-3 N-terminal extension proteolysis by metalloproteinases results in formation of galectin-3 monomers, which interfere with galectin-3 dependent oligomerization.^38,54 Previous studies have shown that ocular surface matrix metalloproteinase (MMP) 9 activity is significantly higher than normal in eyes with DED.^55,56 Furthermore, our group previously showed that the galectin-3C to galectin-3 ratio in tears increases in DED patients. Endogenous galectin-3 cleavage in tear samples was also impaired by a broad-spectrum proteinase inhibitor cocktail, but not by a pan-specific MMP inhibitor. This result suggests that proteases other than MMPs promote galectin-3 degradation in the tears of DED patients.⁵² Recent reports demonstrate neutrophil elastase in tears of patients with severe ocular surface disease. Neutrophil elastase is a potent sheddase of mucins.^57,58 Further, data from these studies indicate that galectin-3 could potentially be used as a biomarker for the evaluation and management of patients with ocular surface pathology. 

New and Existing DED Therapies

Several pharmaceutical agents specifically target mucin deficiency in DED by increasing ocular surface mucin expression and secretion. Diquafosol sodium (Diquas 3% ophthalmic solution; Santen Pharmaceutical Co., Ltd, Osaka, Japan) and rebamipide (Mucosta ophthalmic suspension UD2%; Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan) became available in Japan in December 2010 and January 2012, respectively. 

Diquafosol Sodium Ophthalmic Solution

Diquafosol is a purinergic P2Y2 receptor agonist that stimulates water secretion from conjunctival epithelial cells and promotes mucin secretion from conjunctival goblet cells.⁵⁹ Fluorescein penetration into the central cornea, measured using slit-lamp fluorophotometry, was significantly higher in rats with experimental DED than in normal rats. However, corneal permeability significantly decreased after 1 week of 3% diquafosol treatment.⁶⁰ These results indicate that diquafosol improves tear secretion volume and restores corneal epithelial barrier function. Additionally, exposing SV40 immortalized human corneal epithelial cells to diquafosol tetrasodium for 3 hours led to an increase in membrane-associated mucin (MUC1, MUC4, and MUC16) gene expression in a dose-dependent manner.⁶¹ Positive results were also seen in the clinical setting. A phase III study was conducted in which DED subjects were treated with either 3% diquafosol ophthalmic solution or 0.1% sodium hyaluronate ophthalmic solution (control) for 4 weeks. Subjects who received diquafosol had significant improvements over control subjects in mid-cornea staining scores (fluorescein and rose bengal).⁶² It is most important for the mid-cornea ocular surface to have a smooth, sufficient tear film because this region most heavily influences refractive power in eyes without irregular astigmatism. 

Rebamipide Ophthalmic Suspension

Rebamipide, marketed as Mucosta ophthalmic suspension, is an amino acid derivative of 2-(1H)-quinolinone that also targets mucin-deficient DED. The drug was originally used in Japan as a mucosal protective agent in patients healing from gastroduodenal ulcers and gastritis. Rebamipide enhances mucus glycoprotein synthesis, prostaglandin stimulation, and reactive oxygen species inhibition. It also temporarily activates genes that encode cyclooxygenase-2 and growth factor expression, including EGF and its receptor.⁶³ Studies show that non-steroidal anti-inflammatory drugs (NSAIDs) stimulate inflammatory cell infiltration of gastric ulcer scar tissue because of prostaglandin deficiencies that underlie poor ulcer healing and increase ulcer recurrence incidence.^63–65 A randomized, placebo-controlled, clinical trial in Japan showed that rebamipide can prevent NSAID-induced gastric mucosal damage in healthy volunteers.⁶⁶ Another randomized trial showed that the combined use of rebamipide ophthalmic solution and a topical NSAID following cataract surgery can be beneficial, particularly for alleviating postoperative dry eye in eyes with decreased conjunctival goblet cells.⁶⁷ Additionally, a phase III clinical trial compared 2% rebamipide ophthalmic suspension with 0.1% sodium hyaluronate ophthalmic solution for treating DED. The rebamipide group had significantly smaller changes in lissamine green conjunctival staining score, but significantly larger gains in tear BUT as ocular surface conditions and wettability improved.⁶⁸ Subjects in the rebamipide group also had significant improvements in foreign body sensation and eye pain, DED symptoms associated with glycocalyx barrier disruption. 

Laboratory studies on cultivated human corneal epithelial cell monolayers have shown that rebamipide increases in vitro MUC1, MUC4, and MUC16 gene expression for 2 hours after treatment and MUC1, MUC4, and MUC16 protein expression for 24 hours after treatment via EGF receptor activation.⁶⁹ Furthermore, rebamipide increased MUC16 protein expression level, but not MUC1, MUC4, and MUC20, in stratified cultivated human corneal epithelial cells,⁷⁰ which mimic the in vivo human corneal epithelium. This finding indicates that rebamipide may repair glycocalyx barrier disruption in eyes with DED through MUC16 protein expression normalization. 

Future Directions for DED Research and Treatment

Excellent research has shown that both transmembrane mucins and galectin-3 are prominent components in the ocular surface glycocalyx barrier. Very large glycans in transmembrane mucins are essential to maintaining ocular surface wettability because of their very large water holding capacity. The MUC16 protein is the largest glycoprotein in the human body. MUC16 is the most important glycoprotein for transcellular barrier function because of its major role in galectin-lattice formation due to the MUC16 glycans binding CRD of oligomerized galectin-3. 

Diquafosol and rebamipide are both effective in restoring glycocalyx barrier homeostasis in eyes with DED. Diquafosol is approved for clinical use in many Asian countries, including Japan, South Korea, Vietnam, Thailand, and China. However, rebamipide is only commercially available in Japan. These DED therapies offer some insight into how DED disrupts the glycocalyx, but these mechanisms are not well understood. Future studies that examine changes in ocular surface mucin quality and quantity that occur with DED are needed to better understand glycocalyx barrier disruption and recovery. Advanced techniques to examine mucins and the glycocalyx, including proteome analysis and genetics, are needed. Furthermore, previous studies about the actions of diquafosol and rebamipide on mucin expression and secretion were done in vitro. There are no data from humans on mucin expression pre- and postdrug therapy; therefore, this research is necessary to answer the remaining questions of the action of the drugs. 

Acknowledgments

Funding of the publication fee and administration was provided by the Dry Eye Society, Tokyo, Japan. The Dry Eye Society had no role in the contents or writing of the manuscript. 

Disclosure: Y. Uchino, None