scholarly journals Characterisation of Gel-Forming Mucins Produced In Vivo and In Ex Vivo Conjunctival Explant Cultures

2021 ◽  
Vol 22 (19) ◽  
pp. 10528
Author(s):  
Sara I. Van Acker ◽  
Bert Van den Bogerd ◽  
Zoë P. Van Acker ◽  
Agnė Vailionytė ◽  
Michel Haagdorens ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Ex Vivo ◽  
Tear Film ◽  
Goblet Cells ◽  
Film Stability ◽  
Explant Cultures ◽  
Current Paradigm ◽  
Conjunctival Goblet Cells

One key element to the health of the ocular surface encompasses the presence of gel-forming mucins in the pre-ocular tear film. Conjunctival goblet cells are specialized epithelial cells that secrete mucins necessary for tear film stability and general homeostasis. Their dysfunction can be linked to a range of ocular surface inflammation disorders and chronic injuries. To obtain new perspectives and angles to tackle mucin deficiency, the need for an accurate evaluation of their presence and corresponding mucin secretion in ex vivo conjunctival cultures has become a requisite. In vitro, goblet cells show a significant decrease in the production and secretion of gel-forming mucins, accompanied by signs of dedifferentiation or transdifferentiation. Explant cultures on laminin-treated CLP-PEG hydrogels can, however, support the production of gel-forming mucins. Together, we challenge the current paradigm to evaluate the presence of cultured goblet cells solely based on their general mucin (MUC) content through imaging analyses, showing the need for additional techniques to assess the functionality of goblet cells. In addition, we broadened the gel-forming mucin profile of in vivo goblet cells with MUC5B and MUC6, while MUC2 and MUC6 is added to the profile of cultured goblet cells.

scholarly journals Role of ion channels in the functional response of conjunctival goblet cells to dry eye

2018 ◽  
Vol 315 (2) ◽  
pp. C236-C246 ◽  
Author(s):  
Donald G. Puro
Keyword(s):  
Ion Channels ◽  
Dry Eye ◽  
Ocular Surface ◽  
Adaptive Response ◽  
Tear Film ◽  
Goblet Cells ◽  
Cation Conductance ◽  
Conjunctival Goblet Cells

Optimal vision requires an ocular surface with a stable tear film whose many critical tasks include providing >70% of the eye’s refractive power. However, for millions, tear film instability produces uncomfortable sight-impairing dry eye. Despite the multitude of etiologies for dry eye, a universal hallmark is hyperosmolarity of the tear film. Presently, knowledge of how the ocular surface responds to hyperosmolarity remains incomplete with little understood about the role of ion channels. This bioelectric analysis focused on conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to dry eye. In freshly excised rat conjunctiva, perforated-patch recordings demonstrated that a ≥10% rise in osmolarity triggers goblet cells to rapidly generate a ~15-mV hyperpolarization due to the oxidant-dependent activation of ATP-sensitive K+ (KATP) channels. High-resolution membrane capacitance measurements used to monitor exocytosis revealed that this hyperpolarization results in an approximately fourfold boost in exocytotic activity evoked by cholinergic input, which in vivo occurs via a neural reflex and depends chiefly on calcium influxing down its electro-gradient. We discovered that this adaptive response is transient. During 30–80 min of hyperosmolarity, development of a depolarizing nonspecific cation conductance fully counterbalances the KATP-driven hyperpolarization and thereby eliminates the exocytotic boost. We conclude that hyperosmotic-induced hyperpolarization is a previously unappreciated mechanism by which goblet cells respond to transient ocular dryness. Loss of this voltage increase during long-term dryness/hyperosmolarity may account for the clinical conundrum that goblet cells in chronically dry eyes can remain filled with mucin even though the tear film is hyperosmotic and mucin-deficient.

scholarly journals 11-oxygenated estrogens are a novel class of human estrogens but do not contribute to the circulating estrogen pool

Endocrinology ◽  
2020 ◽  
Author(s):  
Lise Barnard ◽  
Lina Schiffer ◽  
Renate Louw du-Toit ◽  
Jennifer A Tamblyn ◽  
Shiuan Chen ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Cancer Cell Line ◽  
Substantial Contribution ◽  
Cytochrome P450 Aromatase ◽  
Explant Cultures ◽  
Steroid Analysis ◽  
17Β Estradiol ◽  
Estrogen Biosynthesis

Abstract Androgens are the obligatory precursors of estrogens. In humans, classic androgen biosynthesis yields testosterone, thought to represent the predominant circulating active androgen in both men and women. However, recent work has shown that 11-ketotestosterone, derived from the newly described 11-oxygenated androgen biosynthesis pathway, makes a substantial contribution to the active androgen pool in women. Considering that classic androgens are the obligatory substrates for estrogen biosynthesis catalyzed by cytochrome P450 aromatase, we hypothesized that 11-oxygenated androgens are aromatizable. Here we utilize steroid analysis by tandem mass spectrometry to demonstrate that human aromatase generates 11-oxygenated estrogens from 11-oxygenated androgens in three different cell-based aromatase expression systems and in human ex vivo placenta explant cultures. We also show that 11-oxygenated estrogens are generated as a byproduct of the aromatization of classic androgens. We show that 11β-hydroxy-17β-estradiol binds and activates estrogen receptors α and β and that 11β-hydroxy-17β-estradiol and the classic androgen pathway-derived active estrogen, 17β-estradiol, are equipotent in stimulating breast cancer cell line proliferation and expression of estrogen-responsive genes. 11-oxygenated estrogens were, however, not detectable in serum from individuals with high aromatase levels (pregnant women) and elevated 11-oxygenated androgen levels (patients with congenital adrenal hyperplasia or adrenocortical carcinoma). Our data shows that while 11-oxygenated androgens are aromatizable in vitro and ex vivo, the resulting 11-oxygenated estrogens are not detectable in circulation, suggesting that 11-oxygenated androgens function primarily as androgens in vivo.

scholarly journals Contribution of Mucins towards the Physical Properties of the Tear Film: A Modern Update

2019 ◽  
Vol 20 (24) ◽  
pp. 6132 ◽  
Author(s):  
Georgi As. Georgiev ◽  
Petar Eftimov ◽  
Norihiko Yokoi
Keyword(s):  
Ocular Surface ◽  
Tear Film ◽  
The Novel ◽  
Mucin Secretion ◽  
Lipid Layer ◽  
The Impact ◽  
Ophthalmic Disease

Instability of the tear film (TF) protecting the ocular surface results in dry eye syndrome (DES), the most prevalent public health ophthalmic disease affecting the quality of life of 10 to 30% of the human population worldwide. Although the impact of the tear film lipid layer (TFLL) and of the aqueous tears (AT) to the TF stability is extensively studied, in contrast the contribution of the secretory mucins (SM) and of the membrane-associated mucins (MAM), i.e., one of the most abundant molecular classes in AT and in the corneal epithelium respectively, remains poorly defined. However, it is well known that in DES both types of mucins are quantitatively or qualitatively deficient. Numerous studies since the 1990s until now have proposed direct involvement of SM and MAM in the material properties (viscoelasticity, hydration, and protection of the ocular surface; synergistic cooperation with the rest of the TF layers; etc.) and stability of TF. These theories will be reviewed here in the context of the classical and modern in vitro and in vivo results that allow their reappraisal and in view of the novel mucin secretion enhancing pharmaceuticals, which have opened innovative routes for the therapy of DES.

scholarly journals Gut-Derived Butyrate Suppresses Ocular Surface Inflammation

2021 ◽  
Author(s):  
Laura Schaefer ◽  
Humberto Hernandez ◽  
Rosalind A. Coats ◽  
Zhiyuan Yu ◽  
Stephen C. Pflugfelder ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Tear Film ◽  
Intestinal Microbiome ◽  
Inflammatory Disorder ◽  
Corneal Epithelial ◽  
Film Instability ◽  
Epithelial Cultures ◽  
Pre Treatment

Abstract Dry eye is a common ocular inflammatory disorder characterized by tear film instability and reduced tear production. There is increasing evidence that homeostasis of the ocular surface is impacted by the intestinal microbiome. We are interested in investigating the potential role of microbially produced small molecules in mediating the interaction between the intestinal microbiota and the ocular surface. One such molecule is butyrate, a short-chain fatty acid (SCFA) produced by certain members of the gut microbiota through fermentation of dietary fiber. Here we show that SCFA transporter Slc5a8 is expressed in vivo in murine conjunctival and corneal epithelium. Pre-treatment of in vitro corneal epithelial cultures or bone marrow-derived dendritic cells (BMDCs) with phenylbutyrate (PBA) reduces lipopolysaccharide-induced pro-inflammatory Tnf expression. Corneal epithelial cultures and BMDCs isolated from Slc5a8 knockout mice are unable to respond to PBA pre-treatment, suggesting that Slc5a8 is required for the protective effect of PBA. Finally, the treatment of mice undergoing desiccating stress with oral tributyrin, a prodrug form of butyrate, reduces inflammation at the ocular surface in vivo, and this effect partially requires Slc5a8. Together these data support our hypothesis that SCFAs produced in the gut participate in the maintenance of ocular surface homeostasis.

scholarly journals Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Anne V. Lyngstadaas ◽  
Markus V. Olsen ◽  
Jeffrey A. Bair ◽  
Robin R. Hodges ◽  
Tor P. Utheim ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ocular Surface ◽  
Tear Film ◽  
Goblet Cells ◽  
Optimal Level ◽  
Annexin A1 ◽  
Mucin Secretion ◽  
Intracellular Ca ◽  
Dependent Protein ◽  
Conjunctival Goblet Cells

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.

scholarly journals In vivo fluorescence imaging of conjunctival goblet cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Seonghan Kim ◽  
Seunghun Lee ◽  
Hoonchul Chang ◽  
Moses Kim ◽  
Myoung Joon Kim ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Imaging ◽  
Ocular Surface ◽  
Periodic Acid ◽  
Ophthalmic Solution ◽  
Goblet Cells ◽  
Wide Field ◽  
Ocular Surface Diseases ◽  
Conjunctival Goblet Cells

Abstract Conjunctival goblet cells (GCs) are specialized epithelial cells that secrete mucins onto the ocular surface to maintain the wet environment. Assessment of GCs is important because various ocular surface diseases are associated with their loss. Although there are GC assessment methods available, the current methods are either invasive or difficult to use. In this report, we developed a simple and non-invasive GC assessment method based on fluorescence imaging. Moxifloxacin ophthalmic solution was used to label GCs via topical administration, and then various fluorescence microscopies could image GCs in high contrasts. Fluorescence imaging of GCs in the mouse conjunctiva was confirmed by both confocal reflection microscopy and histology with Periodic acid-Schiff (PAS) labeling. Real-time in-vivo conjunctival GC imaging was demonstrated in a rat model by using both confocal fluorescence microscopy and simple wide-field fluorescence microscopy. Different GC densities were observed in the forniceal and bulbar conjunctivas of the rat eye. Moxifloxacin based fluorescence imaging provides high-contrast images of conjunctival GCs non-invasively and could be useful for the study or diagnosis of GC related ocular surface diseases.

scholarly journals Evidence of Polyphenols Efficacy against Dry Eye Disease

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 190
Author(s):  
Gaia Favero ◽  
Enrico Moretti ◽  
Kristína Krajčíková ◽  
Vladimíra Tomečková ◽  
Rita Rezzani
Keyword(s):  
Dry Eye ◽  
Ocular Surface ◽  
Tear Film ◽  
Dry Eye Disease ◽  
Eye Disease ◽  
Beneficial Effects ◽  
Underlying Causes ◽  
Ophthalmic Drug

Dry eye disease is a multifactorial pathology compromising the quality of life of patients, resulting in significant damage of the ocular surface and discomfort. The current therapeutical strategies are not able to definitively resolve the underlying causes and stop the symptoms. Polyphenols are promising natural molecules that are receiving increasing attention for their activity/effects in counteracting the main pathologic mechanisms of dry eye disease and reducing its symptoms. In the present review, a deep literature search focusing on the main polyphenols tested against dry eye disease was conducted, analyzing related in vitro, in vivo, and clinical studies to provide a comprehensive and current review on the state of the art. Polyphenols present multiple effects against dry eye diseases-related ocular surface injury. In particular, the observed beneficial effects of polyphenols on corneal cells are the reduction of the pathological processes of inflammation, oxidative stress, and apoptosis and modulation of the tear film. Due to numerous studies reporting that polyphenols are effective and safe for treating the pathological mechanisms of this ocular surface disease, we believe that future studies should confirm and extend the evidence of polyphenols efficacy in clinical practice against dry eye disease and help to develop new ophthalmic drug(s).

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

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