Comparative influence of differentiation and proliferation on gene expression in human meibomian gland epithelial cells.

We recently discovered that by changing environmental signals, differentiated immortalized human meibomian gland epithelial cells (IHMGECs) de-differentiate into proliferating cells. We also discovered that following exposure to appropriate stimuli, these proliferative cells re-differentiate into differentiated IHMGECs. We hypothesize that this plasticity of differentiated and proliferative IHMGECs is paralleled by very significant alterations in cellular gene expression. To begin to test this hypothesis, we compared the gene expression patterns of IHMGECs during differentiation and proliferation. IHMGECs were cultured for four days in either differentiating or proliferating media. After four days of culture, cells were processed for the analysis of gene expression by using Illumina BeadChips and bioinformatic software. Our study identified significant differences in the expression of more than 9200 genes in differentiated and proliferative IHMGECs. Differentiation was associated with significant increases in the expression of specific genes (e.g. S100 calcium binding protein P; 7,194,386-fold upregulation) and numerous ontologies (e.g. 83 biological process [bp] ontologies with ≥100 genes were upregulated), such as those related to development, transport and lysosomes. Proliferation also led to a significant rise in specific gene expressions (e.g. cathelicidin antimicrobial peptide; 859,100-fold upregulation) and many ontologies (115 biological process [bp] ontologies with ≥100 genes were upregulated), with most of the highly significant ontologies related to cell cycle (z scores > 13.9). Our findings demonstrate that gene expression in differentiated and proliferative IHMGECs is extremely different. These results may have significant implications for the regeneration of HMGECs and the reversal of MG dropout in MG dysfunction.

Toxicity of the cosmetic preservatives parabens, phenoxyethanol and chlorphenesin on human meibomian gland epithelial cells.

Recently, we discovered that the cosmetic preservatives, benzalkonium chloride and formaldehyde, are especially toxic to human meibomian gland epithelial cells (HMGECs). Exposure to these agents, at concentrations approved for human use, leads within hours to cellular atrophy and death. We hypothesize that these effects are not unique, and that other cosmetic preservatives also exert adverse effects on HMGECs. Such compounds include parabens, phenoxyethanol and chlorphenesin, which have been reported to be toxic to corneal and conjunctival epithelial cells, the liver and kidney, as well as to irritate the eye. To test our hypothesis, we examined the influence of parabens, phenoxyethanol and chlorphenesin on the morphology, signaling, survival, proliferation and lipid expression of immortalized (I) HMGECs. These cells were cultured under proliferating or differentiating conditions with varying concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin for up to 5 days. We monitored the signaling ability, appearance, number and neutral lipid content of the IHMGECs, as well as their lysosome accumulation. Our findings show that a 30-min exposure of IHMGECs to these preservatives results in a significant reduction in the activity of the Akt pathway. This effect is dose-dependent and occurs at concentrations equal to (chlorphenesin) and less than (all others) those dosages approved for human use. Further, a 24-h treatment of the IHMGECs with concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin close to, or at, the approved human dose induces cellular atrophy and death. At all concentrations tested, no preservative stimulated IHMGEC proliferation. Of particular interest, it was not possible to evaluate the influence of these preservatives, at close to human approved dosages, on IHMGEC differentiation, because the cells did not survive the treatment. In summary, our results support our hypothesis and show that methylparaben, ethylparaben, phenoxyethanol and chlorphenesin are toxic to IHMGECs.

Effect of brimonidine, an α2 adrenergic agonist, on human meibomian gland epithelial cells.

We recently discovered that the anti-glaucoma pharmaceuticals timolol, a ß adrenergic antagonist, and pilocarpine, a cholinergic compound, negatively influence the morphology, proliferative capacity and survival of human meibomian gland epithelial cells (HMGECs). We hypothesize that another class of anti-glaucoma drugs, the α2 adrenergic agonists, also acts directly on HMGECs to affect their structure and function. We tested this hypothesis. Immortalized (i) HMGECs were cultured with brimonidine, as well as clonidine (α2 agonist), phenylephrine (α1 agonist), RX821002 (inverse α2 agonist) and MK912 (neutral α2 agonist) for up to 7 days. Cells were counted with a hemocytometer, and evaluated for morphology, signaling pathway activity, protein biomarker expression, and the accumulation of neutral lipids, phospholipids and lysosomes. Our findings demonstrate that brimondine treatment induces a dose-dependent decrease in Akt signaling and proliferation of iHMGECs. In contrast, brimonidine also promotes a dose-dependent differentiation of iHMGECs, including an increase in neutral lipid, phospholipid and lysosome levels. These effects were paralleled by an inhibition of p38 signaling, and duplicated by cellular exposure to clonidine, but not phenylephrine. Brimonidine also enhanced the cellular content of sterol regulatory binding protein-1, a master regulator of lipid synthesis. Of particular interest, the putative α2 antagonists, RX821002 and MK912, did not interfere with brimonidine action, but rather stimulated IHMGEC differentiation. Our results support our hypothesis and demonstrate that α2 adrenergic agonists act directly on iHMGECs. However, these compounds do not elicit an overall negative effect. Rather, the α2 agonists promote the differentiation of iHMGECs.

Toxicity of cosmetic preservatives on human ocular surface and adnexal cells.

Cosmetic products, such as mascara, eye shadow, eyeliner and eye makeup remover are used extensively to highlight the eyes or clean the eyelids, and typically contain preservatives to prevent microbial growth. These preservatives include benzalkonium chloride (BAK) and formaldehyde (FA)-releasing preservatives. We hypothesize that these preservatives, at concentrations (BAK = 1 mg/ml; FA = 0.74 mg/ml) approved for consumer use, are toxic to human ocular surface and adnexal cells. Accordingly, we tested the influence of BAK and FA on the morphology, survival, and proliferation and signaling ability of immortalized human meibomian gland (iHMGECs), corneal (iHCECs) and conjunctival (iHConjECs) epithelial cells. iHMGECs, iHCECs and iHConjECs were cultured with different concentrations of BAK (5 µg/ml to 0.005 µg/ml) or FA (1 mg/ml to 1 µg/ml) under basal, proliferating or differentiating conditions up to 7 days. We used low BAK levels, because we found that 0.5 mg/ml and 50 µg/ml BAK killed iHMGECs within 1 day after a 15 min exposure. Experimental procedures included analyses of cell appearance, cell number, and neutral lipid content (LipidTox), lysosome accumulation (LysoTracker) and AKT signaling in all 3 cell types. Our results demonstrate that BAK and FA cause dose-dependent changes in the morphology, survival, proliferation and AKT signaling of iHMGECs, iHCECs and iHConjECs. Many of the concentrations tested induced cell atrophy, poor adherence, decreased proliferation and death, after 5 days of exposure. Cellular signaling, as indicated by AKT phosphorylation after 15 (FA) or 30 (BAK) minutes of treatment, was also reduced in a dose-dependent fashion in all 3 cell types, irrespective of whether cells had been cultured under proliferating or differentiating conditions. Our results support our hypothesis and demonstrate that the cosmetic preservatives, BAK and FA, exert many toxic effects on cells of the ocular surface and adnexa.

Meibomian Gland Dysfunction in Primary and Secondary Sjögren Syndrome.

PURPOSE: We hypothesized that women with primary (pSS) and secondary Sjögren syndrome (sSS; with systemic lupus erythematosus [SLE] or rheumatoid arthritis [RA]) have meibomian gland dysfunction (MGD). We sought to test our hypothesis. METHODS: Subjects with pSS, sSS + SLE, sSS + RA, and non-SS-related MGD were recruited from the Sjögren's Syndrome Foundation or outpatient clinics at Tufts University School of Dental Medicine or Brigham and Women's Hospital. The control population was recruited from the Greater Boston area. After providing written informed consent, the subjects underwent an eye examination and/or completed two questionnaires that assess symptoms of dry eye disease (DED). RESULTS: Our results demonstrate that pSS and sSS patients have MGD. These subjects had meibomian gland orifice metaplasia, an increased number of occluded meibomian gland orifices, and a reduced quality of meibomian gland secretions. Further, patients with pSS, sSS + SLE, sSS + RA, and MGD had significant alterations in their tear film, lid margin, cornea, and conjunctiva. Symptoms of DED were increased ∼10-fold in all pSS, sSS, and MGD groups relative to controls. CONCLUSIONS: Our findings support our hypothesis and show that individuals with pSS, sSS + SLE, and sSS + RA have MGD. In addition, our study indicates that patients with pSS and sSS have both aqueous-deficient and evaporative DED.

Type I Interferon Signaling Is Required for Dacryoadenitis in the Nonobese Diabetic Mouse Model of Sjögren Syndrome.

Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.

Does estrogen deficiency cause lacrimal gland inflammation and aqueous-deficient dry eye in mice?

Researchers have proposed that estrogen deficiency will lead to a Sjögren's syndrome (SjS)-like lacrimal gland inflammation, aqueous tear deficiency and dry eye. The purpose of this study was to determine whether this proposal is correct. Lacrimal glands were obtained from adult, age-matched wild type (WT) and aromatase knockout (ArKO) mice, in which estrogen synthesis is completely eliminated. Tissues were also obtained from autoimmune MRL/Mp-lpr/lpr (MRL/lpr) mice as inflammation controls. Tear volumes in WT and ArKO mice were measured and glands were processed for molecular biological and histological evaluation. Our results demonstrate that estrogen absence does not lead to a SjS-like inflammation in lacrimal tissue or to an aqueous-deficient dry eye. There was no upregulation of genes associated with inflammatory pathways in lacrimal glands of male or female ArKO mice. Such inflammatory activity was prominent in autoimmune MRL/lpr tissues. We also found no evidence of inflammation in lacrimal gland tissue sections of estrogen-deficient mice, and tear volumes of ArKO males were actually increased as compared to those WT controls. Interestingly, our study did show that estrogen absence influences the expression of thousands of lacrimal gland genes, and that this impact is sex- and genotype-specific. Our findings demonstrate that estrogen absence is not a risk factor for the development of SjS-like lacrimal gland inflammation or for aqueous-deficient dry eye in mice.

Characterization of full-length recombinant human Proteoglycan 4 as an ocular surface boundary lubricant.

Proteoglycan 4 (PRG4, or lubricin) is a lubricating mucin-like glycoprotein recently discovered at the ocular surface, where it functions as a boundary lubricant and appears to play a protective role. Recent technological advances have enabled abundant expression of full-length recombinant human PRG4 (rhPRG4). The objectives of this study were to 1) biochemically characterize the gross structure and glycosylations of full-length rhPRG4, and 2) assess the ocular surface boundary lubricating ability of rhPRG4 at both human cornea-eyelid and human cornea-polydimethylsiloxane (PDMS) biointerfaces. rhPRG4 expressed by a Chinese hamster ovary cell line was characterized and compared to native bovine PRG4 by SDS-PAGE western blotting, and protein identity was assessed by tandem mass spectrometry (MS/MS). Human corneas were articulated against PDMS or human eyelids, at effective sliding velocities of 0.3-30 mm/s under physiological loads of ∼15 kPa, to assess and compare the ocular lubricating ability of rhPRG4 to PRG4. Samples were tested serially in PRG4, rhPRG4 (both 300 µg/ml), then saline. Western blotting indicated that rhPRG4 had immunoreactivity at the appropriate apparent molecular weight, and possessed O-linked glycosylation consistent with that of PRG4. rhPRG4 protein identity was confirmed by MS/MS. Both PRG4 and rhPRG4 significantly, and similarly, reduced friction compared to saline at both human cornea - PDMS and human cornea-eyelid biointerfaces. In conclusion, the rhPRG4 studied here demonstrated appropriate higher order structure, O-linked glycosylations, and ocular surface boundary lubricating. Purified rhPRG4 may have clinical utility as a topical treatment of dry eye disease or contact lens biomaterial coating to promote more comfortable wear.

Reduction of methyl isothiocyanate atmospheric emissions after application of metam sodium by shank injection.

Regulatory initiatives in the United States have created the impetus to reassess application methods for metam sodium (sodium -methyldithiocarbamate), a methyl isothiocyanate (MITC) generator, to reduce flux to the atmosphere. This paper compares flux rates in the years 1990 through 2002 with flux rates based on four studies conducted during the period 2008 through 2010 in California, Michigan, Wisconsin, and Washington using current shank-injection/compaction methods. Up to a 100-fold reduction in peak flux rates and total loss of MITC have been observed. A combination of the following factors led to these reductions in flux: soil moisture goals set at 70% of the field water holding capacity; improved design of shank-injection systems to break up the voids after injection; effective shank compaction to further reduce volatilization; and the use of water sealing, where applicable. These refinements in the application methods for metam sodium provide a means to merge environmental and agricultural goals in the United States and in other countries that use metam sodium. This paper documents the reduced atmospheric emissions of MITC under commercial production conditions when applied using good agricultural practices. This research also shows that MITC flux can be effectively managed without the use of high barrier tarp material.

Characterization of ocular gland morphology and tear composition of pinnipeds.

OBJECTIVE: The importance of tear film integrity to ocular health in terrestrial mammals is well established, however, in marine mammals, the role of the tear film in protection of the ocular surface is not known. In an effort to better understand the function of tears in maintaining health of the marine mammal eye surface, we examined ocular glands of the California sea lion and began to characterize the biochemical nature of the tear film of pinnipeds. PROCEDURES: Glands dissected from California sea lion eyelids and adnexa were examined for gross morphology, sectioned for microscopic analysis, and stained with hematoxylin and eosin. The tear film was examined using interferometry. Tears were collected from humans and pinnipeds for the analysis of protein and carbohydrate content. RESULTS: The sea lion has sebaceous glands in the lid, but these glands are different in size and orientation compared with typical meibomian glands of terrestrial mammals. Two other accessory ocular glands located dorsotemporally and medially appeared to be identical in morphology, with tubulo-acinar morphology. An outer lipid layer on the ocular surface of the sea lion was not detected using interferometry, consistent with the absence of typical meibomian glands. Similar to human tears, the tears of pinnipeds contain several proteins but the ratio of carbohydrate to protein was greater than that in human tears. CONCLUSIONS: Our findings indicate that the ocular gland architecture and biochemical nature of the tear film of pinnipeds have evolved to adapt to the challenges of an aquatic environment.

TFOS Lifestyle Report Executive Summary: A Lifestyle Epidemic - Ocular Surface Disease.

The Tear Film & Ocular Surface Society (TFOS) Workshop entitled 'A Lifestyle Epidemic: Ocular Surface Disease' was a global initiative undertaken to establish the direct and indirect impacts of everyday lifestyle choices and challenges on ocular surface health. This article presents an executive summary of the evidence-based conclusions and recommendations of the 10-part TFOS Lifestyle Workshop report. Lifestyle factors described within the report include contact lenses, cosmetics, digital environment, elective medications and procedures, environmental conditions, lifestyle challenges, nutrition, and societal challenges. For each topic area, the current literature was summarized and appraised in a narrative-style review and the answer to a key topic-specific question was sought using systematic review methodology. The TFOS Lifestyle Workshop report was published in its entirety in the April 2023 and July 2023 issues of The Ocular Surface journal. Links to downloadable versions of the document and supplementary material, including report translations, are available on the TFOS website: http://www.TearFilm.org.

TFOS Lifestyle: Impact of cosmetics on the ocular surface.

In this report the use of eye cosmetic products and procedures and how this represents a lifestyle challenge that may exacerbate or promote the development of ocular surface and adnexal disease is discussed. Multiple aspects of eye cosmetics are addressed, including their history and market value, psychological and social impacts, possible problems associated with cosmetic ingredients, products, and procedures, and regulations for eye cosmetic use. In addition, a systematic review that critically appraises randomized controlled trial evidence concerning the ocular effects of eyelash growth products is included. The findings of this systematic review highlight the evidence gaps and indicate future directions for research to focus on ocular surface outcomes associated with eyelash growth products.

Androgen regulation of gene expression in human meibomian gland and conjunctival epithelial cells.

PURPOSE: Androgens exert a significant influence on the structure, function and/or pathophysiology of the meibomian gland and conjunctiva. We sought to determine whether this hormone action involves the regulation of epithelial cell gene expression in these tissues. METHODS: Immortalized human meibomian gland and conjunctival epithelial cells were treated with placebo or dihydrotestosterone (DHT) and processed for molecular biologic procedures. Gene expression was evaluated with BeadChips and data were analyzed with bioinformatic and statistical software. RESULTS: Androgen treatment significantly influenced the expression of approximately 3,000 genes in immortalized human meibomian gland and conjunctival epithelial cells. The nature of DHT action on gene activity was predominantly cell-specific. Similarly, DHT exerted a significant, but primarily cell-specific, influence on many gene ontologies and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These included groups of genes related, for example, to lipid dynamics, innate immunity, cell cycle, Janus kinase (JAK)-signal transducer and activator of transcription (stat) cascades, oxidative phosphorylation, the proteasome, and mammalian target of rapamycin (mTOR), Wnt, and peroxisome proliferator-activated receptor (PPAR) signaling. CONCLUSIONS: Our findings support our hypothesis that androgens regulate gene expression in human meibomian gland and conjunctival epithelial cells. Our ongoing studies are designed to determine whether many of these genes are translated and play a role in the health and well being of the eye.

Report of the TFOS/ARVO Symposium on global treatments for dry eye disease: an unmet need.

In September 2010, a Symposium in Florence, Italy, was held to address the unmet need for global treatments for dry eye disease (DED). It was sponsored by The Tear Film & Ocular Surface Society (TFOS; www.TearFilm.org) and co-sponsored by the Association for Research in Vision & Ophthalmology (www.arvo.org). The Symposium objectives were two-fold: first, to discuss accepted and emerging clinical endpoints of DED with regulatory experts from around the world; and second, to consider how to improve clinical trials of treatments for DED. The Symposium focused on the personal and collective burden of DED, as well as the developmental and regulatory challenges associated with generating new DED therapeutics. This article provides a synopsis of many of the presentations, discussions and recommendations of this Symposium.

Dose-dependent and synergistic effects of proteoglycan 4 on boundary lubrication at a human cornea-polydimethylsiloxane biointerface.

OBJECTIVES: Proteoglycan 4 (PRG4), also known as lubricin, is a boundary lubricating mucin-like glycoprotein present on several tissue surfaces in the body. The objectives of this study were to (1) implement and characterize an in vitro boundary lubrication test at a human cornea-polydimethylsiloxane (PDMS) biointerface and (2) determine the dose-dependent and synergistic effects of PRG4, with hyaluronan (HA), on ocular surface boundary lubrication using this test. METHODS: Human corneas and model PDMS material were articulated against each other, at effective sliding velocities v(eff) between 0.3 and 30 mm/sec under physiologic loads of approximately 8 to 25 kPa. Samples were tested serially in (1) saline, PRG4 at 30, 100, 300 µg/mL resuspended in saline, then saline again or (2) saline, AQuify Comfort Eye Drops (containing 0.1% HA), 300 µg/mL PRG4 in saline, 300 µg/mL PRG4 in AQuify, then saline again. Both static and kinetic friction coefficients were calculated. RESULTS: PRG4 effectively lowered friction at the cornea-PDMS biointerface, both alone in a dose-dependent manner and in combination with HA. PRG4 reduced kinetic friction coefficients, <µ(kinetic, Neq)>, from approximately 0.30 in saline, to approximately 0.30, 0.24, and 0.17 in 30, 100, and 300 µg/mL PRG4, respectively. Values of <µ(kinetic, Neq)> in AQuify, approximately 0.32, were similar to those in saline; however, when combined with 300 µg/mL PRG4, values of <µ(kinetic, Neq)> were reduced to approximately 0.15. CONCLUSIONS: PRG4 functions as an effective ocular surface boundary lubricant, both alone in a dose-dependent manner and in combination with HA.

Soil fate of agricultural fumigants in raised-bed, plasticulture systems in the southeastern United States.

Soil concentrations and degradation rates of methyl isothio-cyanate (MITC), chloropicrin (CP), 1,3-dichloropropene (1,3-D), and dimethyl disulfide (DMDS) were determined under fumigant application scenarios representative of commercial raised bed, plastic mulched vegetable production systems. Five days after application, 1,3-D, MITC, and CP were detected at concentrations up to 3.52, 0.72, and 2.45 µg cm, respectively, in the soil atmosphere when applications were made in uniformly compacted soils with a water content >200% of field capacity and covered by a virtually impermeable or metalized film. By contrast, DMDS, MITC, and CP concentrations in the soil atmosphere were 0.81, 0.02, and 0.05 µg cm, respectively, 5 d after application in soil containing undecomposed plant residue, numerous large (>3 mm) clods, and water content below field capacity and covered by low-density polyethylene. Ranked in order of impact on the persistence of fumigants in soil were soil water content (moisture), soil tilth (the physical condition of soil as related to its fitness as a planting bed), the type of plastic film used to cover fumigated beds, and soil texture. Fumigants were readily detected 13 d after application when applied in uniformly compacted soils with water contents >200% of capacity and covered by a virtually impermeable or metalized film. By contrast, 1,3-D and MITC had dissipated 5 d after application in soils with numerous large (>3 mm) clods and water contents below field capacity that were covered by low-density polyethylene. Soil degradation of CP, DMDS, and MITC were primarily attributed to biological mechanisms, whereas degradation of 1,3-D was attributed principally to abiotic factors. This study demonstrates improved soil retention of agricultural fumigants in application scenarios representative of good agricultural practices.

The Carbonic Anhydrase Inhibitor Dorzolamide Stimulates the Differentiation of Human Meibomian Gland Epithelial Cells.

PURPOSE: Clinical studies have indicated that the long-term use of topical antiglaucoma drugs, such as carbonic anhydrase inhibitors (CAIs), may lead to meibomian gland dysfunction (MGD). We hypothesize that these adverse effects involve a direct influence on human MG epithelial cells (HMGECs). The purpose our present investigation was to test our hypothesis and determine whether exposure to dorzolamide, a CAI, impacts the proliferation, intracellular signaling and differentiation of HMGECs. MATERIALS AND METHODS: We cultured immortalized (i) HMGECs with vehicle or various concentrations of dorzolamide for 6 days. Cells were enumerated with a hemocytometer, and examined for their morphology, Akt signaling activity, accumulation of neutral lipids, phospholipids and lysosomes, and the expression of protein biomarkers for lipogenesis regulation, lysosomes and autophagosomes. RESULTS: Our results show that a high, 500 µg/ml concentration of dorzolamide causes a significant decrease in Akt signaling and the proliferation of iHMGECs. However, the high dose of dorzolamide also promotes the differentiation of iHMGECs. This response features increases in the number of lysosomes, the accumulation of phospholipids, and the expression of the light chain 3A biomarker for autophagosomes. In contrast, the therapeutic amount (50 µg/ml) of dorzolamide has no impact on the proliferative or differentiative abilities of iHMGECs. CONCLUSIONS: Our results support our hypothesis and demonstrate that the CAI dorzolamide does exert a direct influence on the proliferation and differentiation of iHMGECs. However, this effect is elicited only by a high, and not a therapeutic, amount of dorzolamide. Abbreviations: AKT: phosphoinositide 3-kinase-protein kinase B; BPE: bovine pituitary extract; CAD: cationic amphiphilic drug; DED: dry eye disease; DMEM/F12: 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12; EGF: epidermal growth factor; FBS: fetal bovine serum; iHMGECs: immortalized human meibomian gland epithelial cells; KSFM: keratinocyte serum-free medium; LAMP-1: lysosomal-associated membrane protein 1; LC3A: light chain 3A; MGD: meibomian gland dysfunction; SREBP-1: sterol regulatory element-binding protein 1.

The Role of Hypoxia-Inducible Factor 1α in the Regulation of Human Meibomian Gland Epithelial Cells.

Purpose: We recently discovered that a hypoxic environment is beneficial for meibomian gland (MG) function. The mechanisms underlying this effect are unknown, but we hypothesize that it is due to an increase in the levels of hypoxia-inducible factor 1α (HIF1α). In other tissues, HIF1α is the primary regulator of cellular responses to hypoxia, and HIF1α expression can be induced by multiple stimuli, including hypoxia and hypoxia-mimetic agents. The objective of this study was to test our hypothesis. Methods: Human eyelid tissues were stained for HIF1α. Immortalized human MG epithelial cells (IHMGECs) were cultured for varying time periods under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the hypoxia-mimetic agent roxadustat (Roxa). IHMGECs were then processed for the analysis of cell number, HIF1α expression, lipid-containing vesicles, neutral and polar lipid content, DNase II activity, and intracellular pH. Results: Our results show that HIF1α protein is present in human MG acinar epithelial cells in vivo. Our findings also demonstrate that exposure to 1% O2 or to Roxa increases the expression of HIF1α, the number of lipid-containing vesicles, the content of neutral lipids, and the activity of DNase II and decreases the pH in IHMGECs in vitro. Conclusions: Our data support our hypothesis that the beneficial effect of hypoxia on the MG is mediated through an increased expression of HIF1α.

Dihydrotestosterone suppression of proinflammatory gene expression in human meibomian gland epithelial cells.

PURPOSE: We discovered that dihydrotestosterone (DHT) decreases the ability of lipopolysaccharide, a bacterial toxin, to stimulate the secretion of leukotriene B4, a potent proinflammatory mediator, by immortalized human meibomian gland epithelial cells (IHMGECs). We hypothesize that this hormone action reflects an androgen suppression of proinflammatory gene activity in these cells. Our goal was to test this hypothesis. For comparison, we also examined whether DHT treatment elicits the same effect in immortalized human corneal (IHC) and conjunctival (IHConj) ECs. METHODS: Differentiated cells were cultured in media containing vehicle or 10 nM DHT. Cells (n = 3 wells/treatment group) were then processed for RNA isolation and the analysis of gene expression by using Illumina BeadChips, background subtraction, cubic spline normalization and Geospiza software. RESULTS: Our results demonstrate that DHT significantly suppressed the expression of numerous immune-related genes in HMGECs, such as those associated with antigen processing and presentation, innate and adaptive immune responses, chemotaxis, and cytokine production. DHT also enhanced the expression of genes for defensin ß1, IL-1 receptor antagonist, and the anti-inflammatory serine peptidase inhibitor, Kazal type 5. In contrast, DHT had no effect on proinflammatory gene expression in HCECs, and significantly increased 33 gene ontologies linked to the immune system in HConjECs. CONCLUSIONS: Our findings support our hypothesis that androgens suppress proinflammatory gene expression in IHMGECs. This hormone effect may contribute to the typical absence of inflammation within the human meibomian gland.

Expression of Lubricin in the Human Amniotic Membrane.

PURPOSE: Lubricin, a boundary lubricant, is the body's unique antiadhesive, antifibrotic, antifriction, and antiinflammatory glycoprotein. This amphiphile is produced by numerous tissues and acts to regulate a number of processes, such as homeostasis, shear stress, tissue development, innate immunity, inflammation, and wound healing. We hypothesize that lubricin is also synthesized and expressed by the amniotic membrane (AM), which also possesses antiadhesive, antifibrotic, and antiinflammatory properties. We also hypothesize that lubricin, at least in part, mediates these AM capabilities. Our goal was to test our hypothesis. METHODS: We obtained multiple samples of fresh, cryopreserved (CP), and freeze-dried (FD) human AMs, as well as fresh placental tissue as positive controls, and processed them for light microscopy, immunofluorescence, and western blot analyses. We also evaluated the ability of recombinant human lubricin to associate with FD-AMs. RESULTS: Our results demonstrate that all fresh placental, fresh AM, and CP-AM samples contained lubricin. Lubricin was expressed in placental chorionic villi, AM epithelial and stromal cells, and CP-AM epithelia. No lubricin could be detected in FD-AMs but could be restored in FD-AMs after overnight incubation with recombinant human lubricin. CONCLUSIONS: This study supports our hypothesis that lubricin is expressed in human AMs. In addition, our data show that preservation methods influence the extent of this expression. Indeed, the disappearance of lubricin in FD-AMs may explain why dried AM reportedly loses its antiinflammatory and antiscarring abilities. It is possible that lubricin may mediate, at least in part, many of the biological properties of AMs.