Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca2+ concentration ([Ca2+]i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca2+i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca2+]i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca2+ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10-6 M), which depletes ER Ca2+ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10-5 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca2+]i was significantly lower after chelating Ca2+i with 2 mM EGTA in Ca2+-free buffer. The peak [Ca2+]i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10-5 M and BzATP at 10-4 M. Incubating CGCs with dantrolene alone decreased [Ca2+]i and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca2+i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca2+]i. This study contributes to the understanding of diseases arising from defective Ca2+ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca2+]i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca2+]i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca2+]i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca2+]i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca2+i homeostasis.

Effect of chlorhexidine, povidone-iodine and betadine antiseptic eye drops on cultured human conjunctival goblet cell survival.

PURPOSE: To compare the effect of the ocular antiseptic treatments 0.05% chlorhexidine, 5% povidone-iodine (PI) and 5% betadine on cell viability and mucin secretion of primary cultured human goblet cells (GCs). METHOD: GC viability was analysed using lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Expression of mucin was visualised by immunohistochemical MUC5AC staining. RESULTS: PI and betadine significantly reduced GC survival compared to the control (mean cell survival 23 ± 6% and 23 ± 7%, respectively, p < 0.05), whereas chlorhexidine did not significantly affect GC viability (mean cell survival: 78 ± 17%), as measured by the LDH assay. Similar results were obtained from the MTT assay, where PI and betadine caused a significant loss of GCs (mean cell survival: 26 ± 12% and 26 ± 13%, respectively, p < 0.05). Chlorhexidine did not significantly alter GC survival compared to the control (mean cell survival: 79 ± 8%). PI and betadine caused a dispersion of mucin secretion, which chlorhexidine did not. CONCLUSION: The most used antiseptic treatments, PI and betadine, applied prior to ocular surgery are significantly more cytotoxic to conjunctival GCs than chlorhexidine treatment.

Purinergic 2X 4 (P2X4), but not P2X7, receptors increase cytosolic [Ca2+] and stimulate mucin secretion in rat conjunctival goblet cells to maintain ocular surface health.

Ionotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca2+, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca2+] ([Ca2+]i) measured. [Ca2+]i was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca2+]i was significantly increased when stimulated with ATP (10-7-10-4 M). Suramin, a non-selective P2XR antagonist at 10-4 M did not reduce ATP-induced peak [Ca2+]i. The potent P2X7 agonist, BzATP (10-7-10-4 M) increased the [Ca2+]i, although to a lesser extent than ATP. When measuring [Ca2+]i the effect of repeated applications of ATP at 10-5 or 10-6 M the response "desensitized" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca2+]i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca2+]i. ATP (10-5 M), BzATP (10-4 M) and 2MeSATP (10-5 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca2+]i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.

Anti-Inflammatory and Pro-Resolving Actions of the N-Terminal Peptides Ac2-26, Ac2-12, and Ac9-25 of Annexin A1 on Conjunctival Goblet Cell Function.

Annexin A1 (AnxA1) is the primary mediator of the anti-inflammatory actions of glucocorticoids. AnxA1 functions as a pro-resolving mediator in cultured rat conjunctival goblet cells to ensure tissue homeostasis through stimulation of intracellular [Ca2+] ([Ca2+]i) and mucin secretion. AnxA1 has several N-terminal peptides with anti-inflammatory properties of their own, including Ac2-26, Ac2-12, and Ac9-25. The increase in [Ca2+]i caused by AnxA1 and its N-terminal peptides in goblet cells was measured to determine the formyl peptide receptors used by the compounds and the action of the peptides on histamine stimulation. Changes in [Ca2+]i were determined by using a fluorescent Ca2+ indicator. AnxA1 and its peptides each activated formyl peptide receptors in goblet cells. AnxA1 and Ac2-26 at 10-12 mol/L and Ac2-12 at 10-9 mol/L inhibited the histamine-stimulated increase in [Ca2+]i, as did resolvin D1 and lipoxin A4 at 10-12 mol/L, whereas Ac9-25 did not. AnxA1 and Ac2-26 counter-regulated the H1 receptor through the p42/p44 mitogen-activated protein kinase/extracellular regulated kinase 1/2, ß-adrenergic receptor kinase, and protein kinase C pathways, whereas Ac2-12 counter-regulated only through ß-adrenergic receptor kinase. In conclusion, current data show that the N-terminal peptides Ac2-26 and Ac2-12, but not Ac9-25, share multiple functions with the full-length AnxA1 in goblet cells, including inhibition of histamine-stimulated increase in [Ca2+]i and counter-regulation of the H1 receptor. These actions suggest a potential pharmaceutical application of the AnxA1 N-terminal peptides Ac2-26 and Ac2-12 in homeostasis and ocular inflammatory diseases.

The First Transcriptomic Atlas of the Adult Lacrimal Gland Reveals Epithelial Complexity and Identifies Novel Progenitor Cells in Mice.

The lacrimal gland (LG) secretes aqueous tears. Previous studies have provided insights into the cell lineage relationships during tissue morphogenesis. However, little is known about the cell types composing the adult LG and their progenitors. Using scRNAseq, we established the first comprehensive cell atlas of the adult mouse LG to investigate the cell hierarchy, its secretory repertoire, and the sex differences. Our analysis uncovered the complexity of the stromal landscape. Epithelium subclustering revealed myoepithelial cells, acinar subsets, and two novel acinar subpopulations: Tfrchi and Car6hi cells. The ductal compartment contained Wfdc2+ multilayered ducts and an Ltf+ cluster formed by luminal and intercalated duct cells. Kit+ progenitors were identified as: Krt14+ basal ductal cells, Aldh1a1+ cells of Ltf+ ducts, and Sox10+ cells of the Car6hi acinar and Ltf+ epithelial clusters. Lineage tracing experiments revealed that the Sox10+ adult populations contribute to the myoepithelial, acinar, and ductal lineages. Using scRNAseq data, we found that the postnatally developing LG epithelium harbored key features of putative adult progenitors. Finally, we showed that acinar cells produce most of the sex-biased lipocalins and secretoglobins detected in mouse tears. Our study provides a wealth of new data on LG maintenance and identifies the cellular origin of sex-biased tear components.

Lacrimal Gland Epithelial Cells Shape Immune Responses through the Modulation of Inflammasomes and Lipid Metabolism.

Lacrimal gland inflammation triggers dry eye disease through impaired tear secretion by the epithelium. As aberrant inflammasome activation occurs in autoimmune disorders including Sjögren's syndrome, we analyzed the inflammasome pathway during acute and chronic inflammation and investigated its potential regulators. Bacterial infection was mimicked by the intraglandular injection of lipopolysaccharide (LPS) and nigericin, known to activate the NLRP3 inflammasome. Acute injury of the lacrimal gland was induced by interleukin (IL)-1α injection. Chronic inflammation was studied using two Sjögren's syndrome models: diseased NOD.H2b compared to healthy BALBc mice and Thrombospondin-1-null (TSP-1-/-) compared to TSP-1WTC57BL/6J mice. Inflammasome activation was investigated by immunostaining using the R26ASC-citrine reporter mouse, by Western blotting, and by RNAseq. LPS/Nigericin, IL-1α and chronic inflammation induced inflammasomes in lacrimal gland epithelial cells. Acute and chronic inflammation of the lacrimal gland upregulated multiple inflammasome sensors, caspases 1/4, and interleukins Il1b and Il18. We also found increased IL-1ß maturation in Sjögren's syndrome models compared with healthy control lacrimal glands. Using RNA-seq data of regenerating lacrimal glands, we found that lipogenic genes were upregulated during the resolution of inflammation following acute injury. In chronically inflamed NOD.H2b lacrimal glands, an altered lipid metabolism was associated with disease progression: genes for cholesterol metabolism were upregulated, while genes involved in mitochondrial metabolism and fatty acid synthesis were downregulated, including peroxisome proliferator-activated receptor alpha (PPARα)/sterol regulatory element-binding 1 (SREBP-1)-dependent signaling. We conclude that epithelial cells can promote immune responses by forming inflammasomes, and that sustained inflammasome activation, together with an altered lipid metabolism, are key players of Sjögren's syndrome-like pathogenesis in the NOD.H2b mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

The conjunctival extracellular matrix, related disorders and development of substrates for conjunctival restoration.

The conjunctiva can be damaged by numerous diseases with scarring, loss of tissue and dysfunction. Depending on extent of damage, restoration of function may require a conjunctival graft. A wide variety of biological and synthetic substrates have been tested in the search for optimal conditions for ex vivo culture of conjunctival epithelial cells as a route toward tissue grafts. Each substrate has specific advantages but also disadvantages related to their unique physical and biological characteristics, and identification and development of an improved substrate remains a priority. To achieve the goal of mimicking and restoring a biological material, requires information from the material. Specifically, extracellular matrix (ECM) derived from conjunctival tissue. Knowledge of the composition and structure of native ECM and identifying contributions of individual components to its function would enable using or mimicking those components to develop improved biological substrates. ECM is comprised of two components: basement membrane secreted predominantly by epithelial cells containing laminins and type IV collagens, which directly support epithelial and goblet cell adhesion differentiation and growth and, interstitial matrix secreted by fibroblasts in lamina propria, which provides mechanical and structural support. This review presents current knowledge on anatomy, composition of conjunctival ECM and related conjunctival disorders. Requirements of potential substrates for conjunctival tissue engineering and transplantation are discussed. Biological and synthetic substrates and their components are described in an accompanying review.

Spatial transcriptomics of the lacrimal gland features macrophage activity and epithelium metabolism as key alterations during chronic inflammation.

The lacrimal gland (LG) is an exocrine gland that produces the watery part of the tear film that lubricates the ocular surface. Chronic inflammation, such as Sjögren's syndrome (SS), is one of the leading causes of aqueous-deficiency dry eye (ADDE) disease worldwide. In this study we analyzed the chronic inflammation in the LGs of the NOD.B10Sn-H2b/J (NOD.H-2b) mice, a mouse model of SS, utilizing bulk RNAseq and Visium spatial gene expression. With Seurat we performed unsupervised clustering and analyzed the spatial cell distribution and gene expression changes in all cell clusters within the LG sections. Moreover, for the first time, we analyzed and validated specific pathways defined by bulk RNAseq using Visium technology to determine activation of these pathways within the LG sections. This analysis suggests that altered metabolism and the hallmarks of inflammatory responses from both epithelial and immune cells drive inflammation. The most significant pathway enriched in upregulated DEGs was the "TYROBP Causal Network", that has not been described previously in SS. We also noted a significant decrease in lipid metabolism in the LG of the NOD.H-2b mice. Our data suggests that modulation of these pathways can provide a therapeutic strategy to treat ADDE.

Resolvin D2 uses multiple Ca2+ -dependent signaling pathways to stimulate mucin secretion in rat and human conjunctival goblet cells.

The mucin layer of the tear film is produced by goblet cells in the conjunctiva to protect the ocular surface and maintain homeostasis. The pro-resolving lipid mediator resolvin D2 (RvD2) biosynthesized from an omega 3 fatty acid actively terminates inflammation and regulates mucin secretion from conjunctival goblet cells. Our objective was to determine which Ca2+ -dependent signaling pathways RvD2 uses to stimulate conjunctival goblet cell function (CGC). We hypothesize that RvD2 activates multiple intracellular Ca2+ signaling pathways to stimulate CGC secretion. Rat and human CGCs were cultured from conjunctival explants. The amount of RvD2 receptor GPR18/DRV2 message and protein were determined. The intracellular concentration of Ca2+ ([Ca2+ ]i ) was measured in CGCs using a fluorescent Ca2+ dye and mucin secretion was determined by measuring protein secretion enzymatically with a lectin. Goblet cells were incubated with signaling pathway inhibitors before stimulation with RvD2 and [Ca2+ ]i or secretion was measured. In rat and human CGCs RvD2 receptor and in rat CGCs IP3 (a molecule that releases Ca2+ from intracellular organelles) receptors 1-3 were detected. In both species of CGC RvD2 increased [Ca2+ ]i similarly to RvD1. In rat CGCs, the increase in [Ca2+ ]i and secretion stimulated by RvD2 was significantly blocked by inhibitors to phospholipase (PL-) C and IP3 -receptor, but not protein kinase C. Increase in [Ca2+ ]i was blocked by the PLD inhibitor, but not the PLA2 inhibitor. Secretion was blocked by PLA2 inhibitor, but not the PLD inhibitor. An inhibitor of the epidermal growth factor receptor blocked the increase in [Ca2+ ]i by RvD2 in both species of CGCs. In CGCs RvD2 activates multiple intracellular signaling pathways that are Ca2+ -dependent, along with one Ca2+ -independent and one cAMP/protein kinase A-dependent pathway. Activation of these pathways stimulate mucin secretion from rat and human CGCs into the tear film contributing to ocular surface homeostasis and health.

Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators.

Many conjunctival inflammatory diseases differ between the sexes and altered conjunctival goblet cells (CGCs) response is often involved. Inflammation is initiated by the release of pro-inflammatory mediators and terminated by the biosynthesis of specialized pro-resolution mediators (SPMs). Herein, we determined the sex-based difference in the responses of CGCs to inflammatory stimuli or pro-resolving lipid SPMs and their interaction with sex hormones. GCs were cultured from pieces of human conjunctiva in RPMI media. CGCs were transferred 24 h before the start of experiments to phenol red-free and FBS-free media to minimize exogenous hormones. RT-PCR, immunofluorescence microscopy (IF), and Western Blot (WB) were performed to determine the presence of sex hormone receptors. Cellular response to pro-inflammatory stimuli or SPMs was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) using fura 2/AM microscopy. Use of RT-PCR demonstrated estrogen receptor (ER) α in 4/5 males and 3/3 females; ERß in 2/4 males and 2/3 females; and androgen receptors (AR) in 3/3 male and 3/3 female CGCs. Positive immunoreactivity by IF and protein expression by WB was detected using antibodies for the ERα and ERß in 3/3 males and 3/3 females, while AR were only present in males. Significantly different Ca2+ responses between sexes were found with carbachol only at 10-3 M, but not with histamine or leukotriene (LT) B4 at any concentration used. Incubation with dihydrotestosterone (DHT), estrone (E1), or estradiol (E2) at 10-7 M for 30 min significantly inhibited the LTB4-stimulated [Ca2+]i increase in male and female CGCs. Incubation with DHT, E1, and E2 overnight significantly inhibited the LTB4 response in females, while DHT and E2 significantly inhibited the LTB4 response in males. The SPM lipoxin A4 (LXA4) (10-9-10-8 M), but not the resolvins D1 or D2, induced an [Ca2+]i increase that was significantly higher in males compared to females. We conclude that male and female CGCs showed differences in the expression of sex hormone receptors. Treatment with sex hormones altered pro-inflammatory mediator LTB4-induced response. Males compared to females have a higher response to the ω-6-fatty acid derived SPM LXA4, indicating males may terminate inflammation in conjunctival goblet cells faster than females.

Role of the Phospholipase C Pathway and Calcium Mobilization in Oxytocin-Induced Contraction of Lacrimal Gland Myoepithelial Cells.

Purpose: We reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP3) which increases intracellular calcium concentration ([Ca2+]i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC. Methods: Tear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca2+]i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. Results: OXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10-7 M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca2+]i with a maximum response at 10-6 M. Furthermore, the activation of the IP3 receptor to increase [Ca2+]i is crucial for OXT-induced MEC contraction since blocking the IP3 receptor with 2-APB completely abrogated this response. Conclusions: We conclude that OXT uses the PLC/Ca2+ pathway to stimulate MEC contraction and increase lacrimal gland secretion.

Biological tissues and components, and synthetic substrates for conjunctival cell transplantation.

The conjunctiva is the largest component of the ocular surface. It can be damaged by various pathological processes leading to scarring, loss of tissue and dysfunction. Depending on the amount of damage, restoration of function may require a conjunctival graft. Numerous studies have investigated biological and synthetic substrates in the search for optimal conditions for the ex vivo culture of conjunctival epithelial cells that can be used as tissue grafts for transplantation. These substrates have advantages and disadvantages that are specific to the characteristics of each material; the development of an improved material remains a priority. This review is the second of a two-part review in The Ocular Surface. In the first review, the structure and function of the conjunctiva was evaluated with a focus on the extracellular matrix and the basement membrane, and biological and mechanical characteristics of the ideal substrate with recommendations for further studies. In this review the types of biological and synthetic substrates used for conjunctival transplantation are discussed including substrates based on the extracellular matrix. .

Povidone iodine treatment is deleterious to human ocular surface conjunctival cells in culture.

OBJECTIVE: To determine the effect of povidone iodine (PI), an antiseptic commonly used prior to ocular surgery, on viability of mixed populations of conjunctival stratified squamous and goblet cells, purified conjunctival goblet cells and purified conjunctival stromal fibroblasts in primary culture. METHODS AND ANALYSIS: Mixed population of epithelial cells (stratified squamous and goblet cells), goblet cells and fibroblasts were grown in culture from pieces of human conjunctiva using either supplemented DMEM/F12 or RPMI. Cell type was evaluated by immunofluorescence microscopy. Cells were treated for 5 min with phosphate-buffered saline (PBS); 0.25%, 2.5%, 5% or 10% PI in PBS; or a positive control of 30% H2O2. Cell viability was determined using Alamar Blue fluorescence and a live/dead kit using calcein/AM and ethidium homodimer-1 (EH-1). RESULTS: Mixed populations of epithelial cells, goblet cells and fibroblasts were characterised by immunofluorescence microscopy. As determined with Alamar Blue fluorescence, all concentrations of PI significantly decreased the number of cells from all three preparation types compared with PBS. As determined by calcein/EH-1 viability test, mixed populations of cells and fibroblasts were less sensitive to PI treatment than goblet cells. All concentrations of PI, except for 0.25% used with goblet cells, substantially increased the number of dead cells for all cell populations. The H2O2 control also significantly decreased the number and viability of all three types of cells in both tests. CONCLUSION: We conclude that PI, which is commonly used prior to ocular surgeries, is detrimental to human conjunctival stratified squamous cells, goblet cells and fibroblasts in culture.

Lacrimal Gland Myoepithelial Cells Are Altered in a Mouse Model of Dry Eye Disease.

The purpose of this study was to determine the pathogenic changes that occur in myoepithelial cells (MECs) from lacrimal glands of a mouse model of Sjögren syndrome. MECs were cultured from lacrimal glands of C57BL/6J [wild type (WT)] and thrombospondin 1 null (TSP1-/-, alias Thbs1-/-) mice and from mice expressing α-smooth muscle actin-green fluorescent protein that labels MECs. MECs were stimulated with cholinergic and α1-adrenergic agonists, vasoactive intestinal peptide (VIP), and the purinergic agonists ATP and UTP. Then intracellular [Ca2+] was measured using fura-2, and contraction was observed using live cell imaging. Expression of purinergic receptors was determined by Western blot analysis, and mRNA expression was analyzed by microarray. The increase in intracellular [Ca2+]I with VIP and UTP was significantly smaller in MECs from TSP1-/- compared with WT mice. Cholinergic agonists, ATP, and UTP stimulated contraction in MECs, although contraction of MECs from TSP1-/- mice was reduced compared with WT mice. The amount of purinergic receptors P2Y1, P2Y11, and P2Y13 was significantly decreased in MECs from TSP1-/- compared with WT mice, whereas several extracellular matrix and inflammation genes were up-regulated in MECs from TSP1-/- mice. We conclude that lacrimal gland MEC function is altered by inflammation because the functions regulated by cholinergic agonists, VIP, and purinergic receptors are decreased in TSP1-/- compared with WT mice.

Simple limbal epithelial transplantation: Current status and future perspectives.

Damage to limbal stem cells as a result of injury or disease can lead to limbal stem cell deficiency (LSCD). This disease is characterized by decreased vision that is often painful and may progress to blindness. Clinical features include inflammation, neovascularization, and persistent cornea epithelial defects. Successful strategies for treatment involve transplantation of grafts harvested from the limbus of the alternate healthy eye, called conjunctival-limbal autograft (CLAU) and transplantation of limbal cell sheets cultured from limbal biopsies, termed cultured limbal epithelial transplantation (CLET). In 2012, Sangwan and colleagues presented simple limbal epithelial transplantation (SLET), a novel transplantation technique that combines the benefits of CLAU and CLET and avoids the challenges associated with both. In SLET a small biopsy from the limbus of the healthy eye is divided and distributed over human amniotic membrane, which is placed on the affected cornea. Outgrowth occurs from each small explant and a complete corneal epithelium is typically formed within 2 weeks. Advantages of SLET include reduced risk of iatrogenic LSCD occurring in the healthy cornea at harvest; direct transfer circumventing the need for cell culture; and the opportunity to perform biopsy harvest and transplantation in one operation. Success so far using SLET is comparable with CLAU and CLET. Of note, 336 of 404 (83%) operations using SLET resulted in restoration of the corneal epithelium, whereas visual acuity improved in 258 of the 373 (69%) reported cases. This review summarizes the results of 31 studies published on SLET since 2012. Progress, advantages, challenges, and suggestions for future studies are presented.

Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Under physiologic conditions, conjunctival goblet cells (CGCs) secrete mucins into the tear film to preserve ocular surface homeostasis. Specialized proresolving mediators (SPMs), like resolvins (Rvs), regulate secretion from CGCs and actively terminate inflammation. The purpose of this study was to determine if RvD2 stimulated mucin secretion and to investigate the cellular signaling components. Goblet cells were cultured from rat conjunctiva. Secretion was measured by an enzyme-linked lectin assay, change in intracellular [Ca2+] ([Ca2+]i) using Fura-2, and cellular cAMP levels by ELISA. RvD2 (10-11-10-8 M) stimulated secretion, increased cellular cAMP levels and the [Ca2+]i. RvD2-stimulated increase in [Ca2+]i and secretion was blocked by Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride but not by the cAMP exchange protein inhibitor α-[2-(3-chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile. Forskolin, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP (8-Br-cAMP) increased [Ca2+]i. Increasing cAMP with 8-Br-cAMP inhibited the increase in [Ca2+]i stimulated by the cAMP-independent agonist cholinergic agonist carbachol. In conclusion, RvD2 uses both cellular cAMP and [Ca2+]i to stimulate glycoconjugate secretion from CGCs, but the interaction of cAMP and [Ca2+]i is context dependent. Thus RvD2 likely assists in the maintenance of the mucous layer of the tear film to sustain ocular surface homeostasis and has potential as a novel treatment for dry eye disease.-Botten, N., Hodges, R. R., Li, D., Bair, J. A., Shatos, M. A., Utheim, T. P., Serhan, C. N., Dartt, D. A. Resolvin D2 elevates cAMP to increase intracellular [Ca2+] and stimulate secretion from conjunctival goblet cells.

Resolvin D1, but not resolvin E1, transactivates the epidermal growth factor receptor to increase intracellular calcium and glycoconjugate secretion in rat and human conjunctival goblet cells.

PURPOSE: To identify interactions of the epidermal growth factor receptor (EGFR) with the pro-resolving mediator receptors for RvD1 and RvE1 to stimulate an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion from cultured human and rat conjunctival goblet cells. METHODS: Goblet cells from human and rat conjunctiva were grown in culture using RPMI media. Cultured goblet cells were pre-incubated with inhibitors, and then stimulated with RvD1, RvE1, EGF or the cholinergic agonist carbachol (Cch). Increase in [Ca2+]i was measured using fura-2/AM. Goblet cell secretion was measured using an enzyme-linked lectin assay with UEA-1. Western blot analysis was performed with antibodies against AKT and ERK 1/2. RESULTS: In cultured human conjunctival goblet cells RvE1 -stimulated an increase in [Ca2+]i. RvD1-, but not the RvE1-, stimulated increase in [Ca2+]i and mucin secretion was blocked by the EGFR inhibitor AG1478 and siRNA for the EGFR. RvD1-, but not RvE1-stimulated an increase in [Ca2+]i that was also inhibited by TAPI-1, an inhibitor of the matrix metalloprotease ADAM 17. Inhibition of the EGFR also blocked RvD1-stimulated increase in AKT activity and both RvD1-and RvE1-stimulated increase in ERK 1/2 activity. Pretreatment with either RvD1 or RvE1 did not block the EGFR-stimulated increase in [Ca2+]i. CONCLUSIONS: We conclude that in cultured rat and human conjunctival goblet cells, RvD1 activates the EGFR, increases [Ca2+]i, activates AKT and ERK1/2 to stimulate mucin secretion. RvE1 does not transactivate the EGFR to increase [Ca2+]I and stimulate mucin secretion, but does interact with the receptor to increase ERK 1/2 activity.

Conjunctival Goblet Cells, the Overlooked Cells in Glaucoma Treatment.

Glaucoma is the leading cause of irreversible blindness worldwide. Although no definitive cure exists, lowering of the intraocular pressure decreases the rate of progression in the majority of patients with glaucoma. Antiglaucomatous treatment modalities consist predominantly of chronic use of eye drops. It has become increasingly evident that long-term exposure to eye drops has a significant impact on the ocular surface, and thereby on patient compliance and quality of life. Maintenance of the ocular surface is highly dependent on a stable tear film. Conjunctival goblet cells (GCs) of the ocular surface play an important role in providing the innermost mucin layer of the tear film and are essential for maintaining the ocular surface homeostasis. Recent studies have reported severe side effects of antiglaucomatous drops on GCs. In particular, a preservative containing antiglaucomatous drops have been shown to affect the viability and functions of the GCs. Furthermore, GC density has been suggested as a potential predictor of surgical outcome after filtration surgery. The present review provides an overview of the current literature on the impact of antiglaucomatous eye drops on GCs as well as the impact on the ocular surface. Moreover, the existing evidence of a possible association between GC density and glaucoma filtration surgery outcome is summarized. We conclude that prostaglandin analogs spare the conjunctival GCs more compared with other antiglaucomatous drops and that GCs may be a good predictor of surgical outcome after filtration surgery. Overall, given the multiple functions of GCs in the ocular surface homeostasis, dedicated strategies should be adopted to preserve this cell population during the course of glaucoma.

Thrombospondin-1 Is Necessary for the Development and Repair of Corneal Nerves.

Thrombospondin-1-deficient (TSP-1-/-) mice are used as an animal model of Sjögren's Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren's Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1-/- and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1-/- compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1-/- mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1-/- compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1-/- mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1-/- mice age.

Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study.

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-D-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.