Interaction of phospholipid transfer protein with human tear fluid mucins

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.     

In-depth analysis of the human tear proteome

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment.     

Peptidomic analysis of human reflex tear fluid

Tear fluid is a complex mixture of biological compounds, including carbohydrates, lipids, electrolytes, proteins, and peptides. Despite the physiological importance of tear fluid, little is known about the identity of its endogenous peptides. In this study, we analyzed and identified naturally occurring peptide molecules in human reflex tear fluid by means of LC-MALDI-TOF–TOF. Tandem MS analyses revealed 30 peptides, most of which have not been identified before. Twenty-six peptides are derived from the proline-rich protein 4 and 4 peptides are derived from the polymeric immunoglobulin receptor. Based on their structural characteristics, we suggest that the identified tear fluid peptides contribute to the protective environment of the ocular surface.     

Mucins and TFF peptides of the tear film and lacrimal apparatus

The three-dimensional organization of the tear film, which is produced and drained by the different structures of the ocular adnexa, is essential for maintainance and protection of the ocular surface. This is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins, the mucins, which are usually expressed in association with a class of peptides having a well-defined, structurally conserved trefoil domain, the mammalian trefoil factor family (TFF) peptides. In this review, the latest information regarding mucin and TFF peptide function and regulation in the human lacrimal system, the tear film and the ocular surface is summarized with regard to mucous epithelia integrity, rheological and antimicrobial properties of the tear film and tear outflow, age-related changes and certain disease states such as dry eye, dacryostenosis and dacryolith formation.     

Localization of TFF3 peptide to porcine conjunctival goblet cells

TFF-peptides (formerly P-domain peptides, trefoil factors) form a new family of mucin-associated peptides mainly in the gastrointestinal tract. TFF3 is a typical secretory product of intestinal goblet cells and occurs also in the respiratory tract. Here, polyclonal antisera specific for TFF3 were used in Western blot analysis and immunofluorescence to determine the presence and distribution of TFF3 in the porcine conjunctiva, which is the primary source for ocular mucins. Significant accumulation of TFF3 was detected in conjunctival goblet cells but not in the lacrimal glands. This peptide, together with ocular mucins, may play a role in the rheological function of the tear film.     

Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits

The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit (Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source. Lacrimal fluid was collected from NZW sexually mature female rabbits, and culture medium from rabbit lacrimal gland epithelial (acinar) and interstitial cells was isolated. Proteins from these fluids were separated by SDS-PAGE electrophoresis and analyzed by sequencing the intact proteins and sequencing or mass analysis of fragments derived by trypsin digestion. Proteins of approximately 85 and 67 kDa were identified as rabbit transferrin and serum albumin, respectively, while components of 17 and 7 kDa had N-terminal sequences identical to those of lipophilin CL and AL, respectively. BLAST searches of the nr database with the N-terminal sequence of a protein of 18 kDa did not identify any homologues. However, when used to scan the PROSITE database, it was found to contain a lipocalin signature sequence. It is closely related to two lipocalins previously isolated from rabbit saliva and nasal mucus. Further studies with the N-terminal and internal sequences confirmed that the lacrimal protein is a lipocalin that is truncated at the N-terminus as compared with other tear lipocalins and is more similar to odorant binding proteins from rodents.     

Role of lactoferrin in the tear film

The surface of the eye provides an inert barrier against infection. Through its unique combination of antimicrobial action and anti-inflammatory activities lactoferrin (Lf) in the tear film plays an important role in the maintenance of ocular health. In order to maintain clarity the eye must provide immunological defense without immunopathology. Along with physical barriers, soluble plasma factors and other proteins such as lysozyme, Lf produced by the acinar cells of the lacrimal gland serves a number of roles in defense for this purpose. Lf in tears provides antimicrobial efficacy by binding free iron thus reducing the availability of iron necessary for microbial growth and survival as well as pathogenesis. Lf has been shown to inhibit biofilm formation and thus may play a role in protecting contact lens surfaces from colonization. Virus particles' entry into epithelial cells is inhibited by Lf while an excess of Lf in tear film is thought to limit the opportunistic Lf-mediated bridging of adenovirus and host cell that occurs in other tissues. Lf dampens the classical complement activation pathway by binding to markers of inflammation and immune activation while pathogen-associated molecular patterns such as lipopolysaccharide (LPS) are targeted by Lf for removal through tears and hydrodynamic flushing. This review focuses on the role of Lf in human tear film and its contribution to ocular health during contact lens wear.     

Bevacizumab Eye Drop Treatment Stimulates Tear Secretion in Rats Through Changes in VEGF and NGF Lacrimal Gland Levels

VEGF and NGF are known to modulate corneal healing, neovascularisation and tear secretion. While a VEGF-NGF cross talk has been recently shown to modulate corneal healing in rats, it is not known whether it also plays a role in the regulation of lacrimal function. In this study we aim to investigate the effects of anti-VEGF eye drop treatment on lacrimal gland function and on the local expression of VEGF and NGF in rats. Tear function was measured in 3 months old rats by modified Schirmer test at baseline and after 3 weeks of topical anti-VEGF eye drop treatment. Whole lacrimal glands from rats were removed after treatment and analysed by ELISA for VEGF and NGF levels. To investigate if the effects of anti-VEGF were mediated by changes in the NGF-pathway, we repeated the experiments in RCS rats, a strain with NGF-pathway impairment associated with decreased tear flow. After topical treatment with anti-VEGF eye drops, an increase in tear secretion was observed in both wild-type and RCS rats. A significant decrease of VEGF levels was also observed in lacrimal glands of both RCS and SD rats, accompanied by a significant increase in NGF levels. Inhibition of VEGF at the ocular surface in rats results in changes of tear function and lacrimal gland levels of VEGF and NGF. Further studies on the VEGF/NGF cross-talk at the ocular surface may expand our knowledge on the pathogenesis of several diseases characterized by tear dysfunction.     

Tear proteins in health, disease, and contact lens wear

Although well known as manifestations of sorrow, emotions, frustration, and blackmail, tears have a more prosaic and important function as a lubricant and as a blood substitute for the cornea. Tears transport oxygen and carbon dioxide and play a central role in the cellular economy of the ocular surface and conjunctiva. In addition to proteins, tears contain lipids and glycoproteins, which increase the wetting effect of the aqueous component and delay evaporation. The total protein concentration of tears is about 10% of that of the plasma. About 80 proteins and polypeptide components have been detected by electrophoresis. Among 30 proteins identified in tears, about 50% are enzymes. Some of the tear enzymes are secreted by the lacrimal glands; others are produced by or released from epithelial cells of the cornea and the conjunctiva. Finally, a few enzymes originate from plasma and appear in tears only in cases with increased permeability of the conjunctival vessels. The aim of this review is to provide clinical and biochemical information about tear enzymes both for ophthalmologists and for biochemists interested in clinical and experimental tear enzymology. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 4, pp. 469–482.     

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.     

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.     

Mucin Deficiency Causes Functional and Structural Changes of the Ocular Surface

MUC5AC is the most abundant gel-forming mucin in the ocular system. However, the specific function is unknown. In the present study, a Muc5ac knockout (KO) mouse model was subject to various physiological measurements as compared to its wide-type (WT) control. Interestingly, when KO mice were compared to WT mice, the mean tear break up time (TBUT) values were significantly lower and corneal fluorescein staining scores were significantly higher. But the tear volume was not changed. Despite the lack of Muc5ac expression in the conjunctiva of KO mice, Muc5b expression was significantly increased in these mice. Corneal opacification, varying in location and severity, was found in a few KO mice but not in WT mice. The present results suggest a significant difference in the quality, but not the quantity, of tear fluid in the KO mice compared to WT mice. Dry eye disease is multifactorial and therefore further evaluation of the varying components of the tear film, lacrimal unit and corneal structure of these KO mice may help elucidate the role of mucins in dry eye disease. Because Muc5ac knockout mice have clinical features of dry eye, this mouse model will be extremely useful for further studies regarding the pathophysiology of the ocular surface in dry eye in humans.     

Identification of hemopexin in tear film

Human tear fluid is a complex mixture of aqueous lipids, proteins, enzymes, and other biochemical and cellular elements. By conventional comparative proteomic approaches, we investigated the proteome in human tear fluid and compared the tear protein profile of normal control subjects with that of patients suffering from the ocular inflammatory disease vernal keratoconjunctivitis (VKC). Collected tear samples were directed to two-dimensional polyacrylamide gel electrophoresis protein separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification. Six differentially expressed proteins—interleukin 4, phospholipase A2, albumin, lactoferrin, hemopexin, and lipocalin—were displayed. Hemopexin had not been reported previously in tear film. Enzyme-linked immunosorbent assay confirmed that hemopexin concentrations were significantly higher in VKC tear samples and increased with disease stages. The results implied clinical interest of hemopexin in the tear proteome and eye diseases.     

Diagnostic potential of tear proteomic patterns in Sjögren's syndrome

Histological and functional changes of the lacrimal gland might be reflected in proteomic patterns in tear fluids. In this study, we carried out a determination of the disease biomarkers in tear fluid for Sj?gren's syndrome (SS) and a performance of noninvasive diagnostic test based on the proteomic patterns. Thirty-one SS patients and 57 control subjects were enrolled to this study. Their details were 23 cases with primary SS, 8 with secondary SS, 14 with dry eyes, 22 with miscellaneous ocular diseases, and 21 of healthy volunteers. Protein profiling in tear fluids was identified by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Multiple protein changes were reproducibly detected in the primary SS group, including 10 potential novel biomarkers. Seven of the biomarkers (2094, 2743, 14191, 14702, 16429, 17453, 17792 m/z) were down-regulated and 3 biomarkers (3483, 4972, 10860 m/z) were up-regulated in primary SS group, comparing to the protein profiles of control subjects. When cutoff value of SS down-score was set less than 0.5, this result yielded 87% sensitivity and 100% specificity. The positive predictive value for this sample set was 100%. There was a significant inverse correlation between SS down-scores and epithelial damages of the ocular surface in primary SS patients. These findings support the potential of proteomic pattern technology in tear fluids as the noninvasive diagnostic test for primary SS.     

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

Background  

The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.     

Characterization of human reflex tear proteome reveals high expression of lacrimal proline‐rich protein 4 (PRR4)

In‐depth studies on the proteome of reflex tears are still inadequate. Hence, further studies on this subject will unravel the key proteins which are conjectured to possess vital functions in the protection of the ocular surface. Therefore, this study investigated the differences in the expression levels in proteome of reflex compared to basal tears. Basal (n = 10) and reflex (n = 10) tear samples from healthy subjects were collected employing the capillary method, subsequently pooled and the proteomes were characterized employing 1DE combined with LC‐ESI‐MS/MS strategy for label‐free quantitative (LFQ) analysis. The differentially expressed proteins were validated by 2DE combined with LC‐ESI‐MS/MS and targeted‐MS approach called accurate inclusion mass screening (AIMS) strategies. The analysis of the reflex tear proteome demonstrated increased abundance in proline‐rich protein 4 (PRR4) and zymogen granule protein 16 homolog B (ZG16B) for the first time. Other abundant lacrimal proteins, e.g. lactotransferrin and lysozyme remained constant. Predominantly, the lacrimal gland‐specific PRR4 represents the major increased protein in reflex tears in an attempt to wash out irritants that come into contact with the eye. Conversely, decreased abundance in Ig alpha‐1 chain C, polymeric immunoglobulin receptor, cystatin S/SN, clusterin and mammaglobin were observed. This study had further unraveled the intricate proteome regulation during reflex tearing, especially the potential role of PRR4, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface.     

Use of nucleofection to efficiently transfect primary rabbit lacrimal gland acinar cells

Lacrimal gland acinar cells are an important cell type to study due to their role in production and release of tear proteins, a function essential for ocular surface integrity and normal visual acuity. However, mechanistic studies are often limited by problems with transfection using either plasmid DNA or siRNA. Although various gene delivery methods are available, many have been unproductive due to consistently low transfection efficiencies. We have developed a method using nucleofection that can result in 50% transfection efficiency and 60% knockdown efficiency for plasmid DNA and siRNA, respectively. These results are vastly improved relative to previous studies, demonstrating that nucleofection offers an efficient transfection technique for primary lacrimal gland acinar cells.     

The lacrimal gland: development,wound repair and regeneration

The lacrimal gland (LG) is important as it has a significant role in maintaining the stability of the microenvironment of the ocular surface. When a loss of function occurs in the LG, a significant reduction in tear production and dry eye disease (DED) may occur. A mammalian LG is a secretory gland consisting of acini and ducts. The interaction between epithelial cells and mesenchymal cells plays a major role during development and the self-restoration process of the gland. Some factors, such as fibroblast growth factor 10 and bone morphogenetic protein 7, are associated with these processes. Though several strategies for LG regeneration have been established, there is still a long way to go before there is clarity about LG stem cells. In this review, current knowledge on LG development, LG self-repair, DED and correlative regeneration therapies are summarized.     

PRGL: A cell wall proline-rich protein containning GASA domain in Gerbera hybrida

PRPs (proline-rich proteins) are a group of cell wall proteins characterized by their proline and hy- droproline-rich repetitive peptides. The expression of PRPs in plants is stimulated by wounding and environmental stress. GASA (gibberellic acid stimulated in Arabidopsis) proteins are small peptides sharing a 60 amino acid conserved C-terminal domain containing twelve invariant cysteine residues. Most of GASAs reported are localized to apoplasm or cell wall and their expression was regulated by gibberellins (GAs). It has been reported that, in French bean, these two proteins encoding by two distinct genes formed a two-component chitin-receptor involved in plant-pathogen interactions when plant was infected. We cloned a full-length cDNA of PRGL (proline-rich GASA-like) gene which encodes a protein containing both PRP and GASA-like domains. It is demonstrated that PRGL is a new protein with characteristics of PRP and GASA by analyzing its protein structure and gene expression.     

Corneal cell proteins and ocular surface pathology

The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structure, the basement membrane, composed of a specific subset of collagen type IV and laminin isoforms in addition to ubiquitous extracellular matrix molecules. Matrix metalloprotein-ases have been identified in normal corneal tissue and cells and may play a role in the development of ulcerative corneal diseases. Changes in extracellular matrix molecule localization and synthesis have been noted in other types of corneal diseases as well, including bullous keratopathy and keratoconus.