Meibomian Gland Cells Research Articles

Could Ocular Glands Be Infected by SARS-CoV-2?

The aim of the study was to investigate the expression levels of ACE2 in ocular glands and to investigate the effect of S protein on them. Male C57BL/6J mice were used for the experiments. The expression levels of ACE2 are highest in the Meibomian glands, followed by the conjunctiva, the cornea, and the lacrimal glands. Co-immunoprecipitation assays confirmed direct binding between ACE2 and S protein in ocular surface epithelia and Meibomian glands. CD45+ cell infiltration was found in the S protein treatment group, which was accompanied by upregulation of inflammation-related cytokines. There was also prominent cell apoptosis in the S protein treatment group. In conclusion, not only the cornea and the conjunctiva, but also the Meibomian glands express ACE2, and S protein could induce ocular surface epithelial cell and Meibomian gland cell inflammation and apoptosis.

The effect of airborne particulate matter 2.5 (PM2.5) on meibomian gland

Exposure to particulate matters in air pollution of 2.5 μm or less (PM2.5) was associated with loss of meibomian glands. The aim of this study was to verify that PM2.5 could directly impact meibomian gland epithelial cells and damage their function. To investigate the impact of PM2.5 on meibomian gland, immortalized human meibomian gland epithelial cells were treated with various concentrations of PM2.5in vitro. Meibomian gland cell microstructure, cell viability, expression of proliferating cell nuclear antigen and IL-1β, and intracellular accumulation of acidic vesicles were measured by transmission electron microscopy, cell counting, Western blot and LysoTracker staining, respectively. To further study the effect of PM2.5in vivo, male C57BL/6J mice were treated with 5 mg/ml PM2.5 or vehicle for 3 months. Corneal fluorescein staining and ocular examinations were done before and after the treatment. Eyelids tissues were processed for morphological studies, immunostaining and Oil Red O staining. Our data suggest that exposure to PM2.5 caused significant meibomian gland dropout, clogged gland orifice and increased corneal fluorescein staining that were consistent with the clinical presentations of meibomian gland dysfunction. Prominent changes in the morphology and ultrastructure of meibomian glands was observed with PM2.5 treatment. PM2.5 promoted ductal keratinization, inhibited cell proliferation, induced cell apoptosis and increased Interleukin-1β production in meibomian gland epithelial cells. This study may explain the association between PM2.5 exposure and meibomian gland dropout observed in clinic. PM2.5 resuspension instillation could be used to induce a meibomian gland dysfunction animal model.

Thermosensitive TRP Channels Are Functionally Expressed and Influence the Lipogenesis in Human Meibomian Gland Cells.

While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1, TRPV3, TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8's functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.

Isolation and Culture of Human Meibomian Gland Ductal Cells.

Hyperkeratinization of meibomian gland (MG) ducts is currently recognized as the primary pathologic mechanism of meibomian gland dysfunction (MGD). This research figured out a method to isolate the MG ducts and established a novel system to culture the human meibomian gland ductal cells (HMGDCs) for investigating the process of MGD. The MG ducts were obtained from the eyelids of recently deceased donors and subjected to enzymatic digestion. The acini were then removed to isolate independent ducts. These MG ducts were subsequently cultivated on Matrigel-coated wells and covered with a glass plate to obtain HMGDCs. The HMGDCs were further cultivated until passage 2, and when they reached 60% confluence, they were treated with IL-1β and rosiglitazone for a duration of 48 hours. Immunofluorescence staining and Western blot techniques were employed to identify ductal cells and analyze the effects of IL-1β on HMGDCs in an in vitro setting. Ophthalmic micro-forceps and insulin needles can be employed for the purpose of isolating ducts. Within this particular culture system, the rapid expansion of HMGDCs occurred in close proximity to the duct tissue. MG ducts specifically expressed keratin 6 (Krt6) and hardly synthesized lipids. Furthermore, the expression of Krt6 was significantly higher (P < 0.0001) in HMGDCs compared to human meibomian gland cells. Upon treatment with IL-1β, HMGDCs exhibited an overexpression of keratin 1, which was effectively blocked by the administration of rosiglitazone. The present study successfully isolated human MG ducts and cultured HMGDCs, providing a valuable in vitro model for investigating the mechanism of MGD. Additionally, the potential therapeutic efficacy of rosiglitazone in treating hyperkeratinization of ducts in patients with MGD was identified.

Potential benefits of Malva sylvestris in dry-eye disease pathology in vitro based on antioxidant, wound-healing and anti-inflammatory properties

Dry eye disease (DED) is a common chronic ocular surface disease. Available therapies are effective but often associated with side effects. This study investigates the potential of a Malva sylvestris L. flower extract and two defined preparations, a mucilage and a polyphenol rich fraction, on cells that are essential for the DED pathology. Furthermore, single compounds were isolated and characterised out of the polyphenol fraction. The M. sylvestris extract and its two fractions reduced reactive oxygen species (ROS) in an ultraviolet-induced model and promoted wound healing capacity of HCE-T cells, but only the polyphenol fraction and the flower extract exhibited significant radical scavenging activity. The flower extract and the polyphenol fraction inhibited cytokine secretion (IL-6, TNF-α, IL-8) from HCE-T cells and THP-1 cells. In contrast, the mucilage fraction led to an increase in mediator secretion. The NF-κB activity and calcium influx in THP-1 and Jurkat cells, respectively was decreased by treatment with the flower extract and the polyphenol fraction, whereas the mucilage fraction had no influence on these parameters. Moreover, the flower extract and the mucilage fraction at low concentration could stimulate meibomian gland cells’ lipid accumulation. The isolated single compounds showed no effect on analysed parameters, except a coumarin derivative and malvin which showed ROS inhibition effects.

Culture of primary human meibomian gland cells from surgically excised eyelid tissue

Meibomian gland dysfunction is one of the most common ocular diseases, with therapeutic treatment being primarily palliative due to our incomplete understanding of meibomian gland (MG) pathophysiology. To progress in vitro studies of human MG, this study describes a comprehensive protocol, with detailed troubleshooting, for the successful isolation, cultivation and cryopreservation of primary MG cells using biopsy-size segments of human eyelid tissue that would otherwise be discarded during surgery. MG acini were isolated and used to establish and propagate lipid-producing primary human MG cells. The primary cell viability during culture procedure was maintained through the application of Rho-associated coiled-coil containing protein kinase inhibitor (Y-27632, 10 μM) and collagen I from rat tails. Transcriptomic analysis of differentiated primary human MG cells confirmed cell origin and revealed high-level expression of many lipogenesis-related genes such as stearoyl-CoA desaturase (SCD), ELOVL Fatty Acid Elongase 1 (ELOVL1) and fatty acid synthase (FASN). Primary tarsal plate fibroblasts were also successfully isolated, cultured and cryopreserved. Established primary human MG cells and tarsal plate fibroblasts presented in this study have potential for applications in 3D models and bioengineered tissue that facilitate research in understanding of MG biology and pathophysiology.

Study of pathological processes of meibomian gland dysfunction by in vitro culture airlifting conditions

ABSTRACT Meibomian gland dysfunction (MGD) is a group of disorders linked by functional abnormalities of the meibomian glands. Current studies on MGD pathogenesis focus on meibomian gland cells, providing information on a single cell’s response to experimental manipulation, and do not maintain the architecture of an intact meibomian gland acinus and the acinar epithelial cells’ secretion state in vivo. In this study, rat meibomian gland explants were cultured by a Transwell chamber-assisted method under an air-liquid interface (airlift) in vitro for 96 h. Analyses for tissue viability, histology, biomarker expression, and lipid accumulation were performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and TUNEL assays, hematoxylin and eosin (H&E) staining, immunofluorescence, Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), transmission electron microscopy (TEM), and western blotting (WB). MTT, TUNEL, and H&E staining indicated better tissue viability and morphology than the submerged conditions used in previous studies. Levels of MGD biomarkers, including keratin 1 (KRT1) and 14 (KRT14) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), along with oxidative stress markers, including reactive oxygen species, malondialdehyde, and 4-hydroxy-2-nonenal, gradually increased over culture time. The MGD pathophysiological changes and biomarker expression of meibomian gland explants cultured under airlift conditions were similar to those reported by previous studies, indicating that abnormal acinar cell differentiation and glandular epithelial cell hyperkeratosis may contribute to obstructive MGD occurrence.

Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine–α1 Adrenergic Receptor–Mitochondrial Impairment Axis of Lacrimal Gland

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. In the present study, we found that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased c-FOS+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenoceptors (ARs), the α1 subtype played the predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with βAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

Establishment of a human meibomian gland carcinoma cell model and analysis of differently expressed genes

Meibomian gland carcinoma (MGC) is a malignant eyelid tumor with a high malignancy degree and poor prognosis. However, the lack of suitable cell and animal models has limited the study of MGC pathogenesis. In the present study, we established and identified one human MGC cell and one meibomian gland (MG) cell model by fresh surgical resection tissue block primary culture and differentially expressed gene assays. The outgrowth of MGC and MG cells was periodically observed after primary culture, and the first passage of MGC cells proceeded on the 14th day, whereas that for MG cells after three weeks. Cell ultrastructures were observed by transmission electron microscopy (TEM). Immunofluorescence staining showed that MGC and MG cells were both positive for cytokeratin (CK) and androgen receptor (AR). Orange granules were observed in the cytoplasm of MGC and MG cells using Oil red O staining, but they were stronger for MG cells than for MGC. CCK-8 detection demonstrated that the proliferation ability of MGC cells was stronger than that of MG cells. Moreover, during RNA sequence analyses, 3023 differential expressed genes were detected between MGC and MG cells. These genes were involved in biological processes such as cell division and positive regulation of cell migration; the signaling pathways mainly covered cell cycle and DNA replication. Further, the tumorigenic potential of MGC cells was examined by inoculating them subcutaneously into the right abdomen of three severely immunodeficient NOD -SCID mice. Transplanted tumors formed on day 11 after inoculation. The xenograft mouse tissues retained the same histological characteristics as the human MGC original tumor and MGC primary cells. Altogether, these results showed that the MGC and MG models were successfully cultured and established, and differentially expressed genes were successfully detected. We provided a useful model and molecular basis for studying the biological characteristics and pathogenesis of human MGC.

Periplocin ameliorates mouse age-related meibomian gland dysfunction through up-regulation of Na/K-ATPase via SRC pathway

Age-related meibomian gland dysfunction (MGD) is the main cause of evaporative dry eye disease in an aging population. Decreased meibocyte cell renewal and lipid synthesis are associated with age-related MGD. Here, we found an obvious decline of Ki67, ΔNp63, and Na+/K+ ATPase expression in aged meibomian glands. Potential Na+/K+ ATPase agonist periplocin, a naturally occurring compound extracted from the traditional herbal medicine cortex periplocae, could promote the proliferation and stem cell activity of meibocyte cells in vitro. Moreover, we observed that periplocin treatment effectively increased the expression of Na+ /K+ ATPase, accompanied with the enhanced expression of Ki67 and ΔNp63 in aged meibomian glands, indicating that periplocin may accelerate meibocyte cell renewal in aged mice. LipidTox staining showed increased lipid accumulation after periplocin treatment in cultured meibomian gland cells and aged meibomian glands. Furthermore, we demonstrated that the SRC pathway was inhibited in aged meibomian glands; however, it was activated by periplocin. Accordingly, the inhibition of the SRC signaling pathway by saracatinib blocked periplocin-induced proliferation and lipid accumulation in meibomian gland cells. In sum, we suggest periplocin-ameliorated meibocyte cell renewal and lipid synthesis in aged meibomian glands via the SRC pathway, which could be a promising candidate for age-related MGD.

Diquafosol tetrasodium elicits total cholesterol release from rabbit meibomian gland cells via P2Y2 purinergic receptor signalling

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears. Hence, DQS-containing eye drops have been approved as a therapeutic option for dry eye disease in some Asian countries, including Japan. Recent clinical reports state that instilling DQS-containing eye drops significantly increases the lipid layer thickness in tears. Therefore, we examined this compound’s direct actions on holocrine lipid-secreting meibomian gland cells and their function. Isolated meibomian gland cells (meibocytes) were procured from rabbits and cultivated in serum-free culture medium. Differentiated meibocytes with pioglitazone were used for the subsequent experiments. Intracellular Ca2+ signalling of the cells was dramatically elevated with DQS addition in a dose-dependent manner. This DQS-induced elevation was almost completely cancelled by the coexistence of the selective P2Y2 receptor antagonist AR-C118925XX. DQS treatment also facilitated total cholesterol (TC) release from cells into the medium. This effect of DQS on TC was suppressed significantly by the intracellular Ca2+ chelator BAPTA-AM as well as by AR-C118925XX. DNA fragmentation analysis revealed that DQS may have enhanced the apoptotic DNA fragmentation caused spontaneously by cells. Thus, DQS could stimulate meibocytes to release lipids through the P2Y2 receptor and possibly facilitate holocrine cell maturation.

The effect of tanshinone on meibomian gland cell proliferation, differentiation and lipid synthesis

Objective To investigate the effects of cryptotanshinone and tanshinone ⅡA, two major monomer components of tanshinone, on the proliferation, differentiation and lipid synthesis of rat meibomian gland epithelial cells in vitro. Methods The eyelid meibomian gland tissue was isolated from 2-month-old SD rats and co-cultured with 3T3 trophoblasts for 5 days.Cryptotanshinone and tanshinone ⅡA were prepared with DMSO into different concentrations.The cells were grouped to 0.125 μmol/L, 0.250 μmol/L, 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L drug groups and treated for 48 hours, respectively.Only dimethyl sulfoxide (DMSO) was added in medium in the DMSO control group.The expressions of keratin 14 (K14) and p63 in frozen sections of meibomian gland tissue and clones of meibomian gland cells were detected by immunofluorescence.Real-time fluorescence quantitative PCR was used to assay the relative expression of K16, cell proliferation related antigen 67 (Ki67) and CCAAT enhancer binding protein α (C/EBPα) in meibomian gland cell clones.Crystal violet staining and oil red staining were used to evaluate the colony formation rate and lipid synthesis of meibomian gland epithelial cells. Results Primary cultured meibomian gland cells were cloned on day 4-5 in vivo, and the cloning area was increased on day 7 after culture, p63 and K14 were positively expressed in clones.Compared with the DMSO control group, the relative expression levels of Ki67 mRNA were significantly elevated in the 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L cryptotanshinone groups (all at P 0.05). No lipid drop was found in the tarsal gland cell clones; however, the accumulation of lipid was seen in the cell clusters at the margin of the clones by oil red O staining.The average clone formation rate of tarsal gland cells in the 1.250 μmol/L cryptotanshinone group was (2.55±0.20)%, which was significantly higher than (2.05±0.13)% in the DMSO control group (t=4.379, P 0.05). Conclusions Cryptotanshinone and tanshinone ⅡA promote the proliferation of meibomian gland epithelia cells, but play less impacts to lipid synthesis of meibomian gland epithelia cells in vitro.cryptotanshinone promote the clone tormation of meibomian gland epithelia cells. Key words: Tanshinone; Meibomian gland epithelial cells; Proliferation; Differentiation; Lipid metabolism/drug effects; Cell culture

E-Cadherin Is Important for Meibomian Gland Function as Revealed by a New Human ex Vivo Slice Culture Model

Meibomian glands within the eyelid are important for the maintenance of the integrity and health of the ocular surface. Patients with the blistering skin disease pemphigus vulgaris (PV), which is caused by autoantibodies against desmosomal cadherins, often have dry eye disease. Therefore, we studied the regulation of cell cohesion in human meibomian gland epithelial cells (HMGECs). During serum-induced differentiation for 1 to 6 days, HMGECs drastically enhanced intercellular cohesion, whereas lipid production did not change. The expression profiles of the desmosomal PV antigens desmoglein (Dsg) 3and 1 but not of the adherens junction component E-cadherin (Ecad) was dependent on the presence of serum. Surprisingly, after 1 day but not after 6 days of serum-induced differentiation, an inhibitory antibody against Ecad drastically reduced intercellular cohesion and blocked lipid production ofHMGECs. In contrast, antibodies against desmosomal cadherins, including human and mouse pemphigus autoantibodies, had no effect on monolayer integrity and lipid production. Because lipid production was unaltered in meibomian glands from Dsg3-deficient mice, we established an exvivo slice culture model of human eyelids to allow studies in a more physiologic environment. Here, the inhibitory antibody against Ecad but not a Dsg3-specific PV antibody interfered with stimulated lipid production. Together, these data demonstrate that cell cohesion is maintained differently in meibomian glandcellsand indicate that Ecad is important for meibomian gland function.

Short Tandem Repeat (STR) Profiles of Commonly Used Human Ocular Surface Cell Lines

ABSTRACTPurpose: The purpose of this study is to establish the short tandem repeat (STR) profiles of several human cell lines commonly used in ocular surface research.Materials and Methods: Independently DNA was extracted from multiple passages of three human corneal epithelial cell lines, two human conjunctival epithelial cell lines and one meibomian gland cell line, from different laboratories actively involved in ocular surface research. The samples were then subjected to STR analysis on a fee-for-service basis in an academic setting and the data compared against that in available databases.Results: The STR profiles for the human corneal epithelial cells were different among the three cell lines studied and for each line the profiles were identical across the samples provided by three laboratories. Profiles for the human conjunctival epithelial cells were different among the two cell lines studied. Profiles for the meibomian gland cell line were identical across the samples provided by three laboratories. No samples were contaminated by elements of other cell lines such as HeLa.Conclusions: This comprehensive study provides verification of STR profiles for commonly used human ocular surface cell lines that can now be used as a reference by others in the field to authenticate the cell lines in use in their own laboratories.

Serotonin hormonally regulates lacrimal gland secretory function via the serotonin type 3a receptor

Tears are extracellular fluid secreted from the lacrimal gland (LG). Tears consist of a dynamic tri-layered film composed of secretions from the LG, Meibomian gland, and conjunctival goblet cells. The LG secretes the aqueous component of the tear, the Meibomian gland secretes the lipid component, and conjunctival goblet cells secrete mucin. The regulation of LG activity via the autonomic nervous system has been recognized as fundamental to maintaining aqueous tear flow. Here, we describe the role of a hormone, peripheral serotonin, in tear secretion. We found that blood serotonin concentration, changed by feeding a diet deprived of the serotonin precursor tryptophan, correlated with tear secretion, and that a sustained decrease in serotonin resulted in LG atrophy and autophagy. The combination of a decrease in serotonin with the interruption of autonomic neural stimuli to the LG preceded these alterations. Furthermore, we found that the serotonin type 3a receptor expressed in LG acinar cells is involved in tear secretion via intracellular calcium mobilization. Our findings demonstrate that hormonal regulation by serotonin, in cooperation with the autonomic nervous system, regulates tear secretion.

Limited Azithromycin Localization to Rabbit Meibomian Glands Revealed by LC-MS-Based Bioanalysis and DESI Imaging.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye, and although it affects approximately 4% of the population, treatment options remain limited. Topical azithromycin is one of the most promising pharmacological agents because of its multiple mechanisms of action and long sustainability. Azithromycin is frequently used as an off-label medication in the U.S. However, although azithromycin is presumed to act directly on meibomian gland cells, the mechanisms of action that contribute to its clinical efficacy remain unclear because no studies using a pharmacokinetic approach have been performed. Therefore, we aimed to clarify whether topical azithromycin reaches the meibomian glands sufficiently to generate a biological effect. We measured azithromycin concentrations in rabbit meibomian glands collected using a recently developed method. Moreover, we also visualized the azithromycin micro-distribution using desorption electrospray ionization (DESI) imaging. Azithromycin concentration in the meibomian glands reached only 0.8 µg/g tissue following a single application of a 1% azithromycin ophthalmic solution and was 1000-fold lower than the concentration in conjunctival epithelium. Similarly, no signal was observed in the meibomian glands on DESI images. Our results clearly demonstrated that topical azithromycin had limited access to the meibomian glands and was predominantly distributed in ocular surface tissues such as the palpebral conjunctiva and lid margins. These findings provide new insight into the clinical responses to topical azithromycin therapy and will aid in the further development of effective drugs with more suitable pharmacokinetic properties.

Meibomian gland cells display a differentiation-dependent composition of desmosomes.

Meibomian glands are a modified type of sebaceous glands within the eye lid, which produce an oily secretion important for the stabilization and the prevention of evaporation of the tear film. The holocrine secretory mode of Meibomian glands is characterized by the centripetal movement, the maturation and finally degeneration of the acinar epithelial cells. The process of maturation and degeneration is paralleled by altered expression pattern of certain proteins and the intracellular accumulation of Meibomian gland lipids. In this study, we investigated the correlation between the differentiation status of Meibomian acinus cells and the presence of adhesive junctions. By ultrastructural analyses, we showed for the first time that the frequency of desmosomes increased with the degree of differentiation. Importantly, we detected a differentiation-dependent distribution pattern of desmosomes within the Meibomian gland cells of the acinus, whereas molecules of other cell junctions, e.g., adherens junctions, are equally distributed. Together, these findings provide new insights into the processes of Meibomian gland secretion and may be important for the interpretation of Meibomian gland dysfunction causing diseases like the dry eye syndrome.

Characterization of Rat Meibomian Gland Ion and Fluid Transport.

PurposeWe establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG.MethodsFreshly excised rat MG tissues were characterized as follows: (1) mRNA expression of selected epithelial ion channels/transporters were measured by RT-PCR, (2) localization of epithelial sodium channel (ENaC) mRNAs was performed by in situ hybridization, and (3) protein expression and localization of βENaC, the Na+/K+/Cl− cotransporter (NKCC), and the Na+/K+ ATPase were evaluated by immunofluorescence. Primary isolated rat MG cells were cocultured with 3T3 feeder cells and a Rho-associated kinase (ROCK) inhibitor (Y-27632) for expansion. Passaged rat MG cells were cultured as planar sheets under air-liquid interface (ALI) conditions for gene expression and electrophysiologic studies. Passaged rat MG cells also were cultured in matrigel matrices to form spheroids, which were examined ultrastructurally by transmission electron microscopy (TEM) and functionally using swelling assays.ResultsExpression of multiple ion channel/transporter genes was detected in rat MG tissues. β-ENaC mRNA and protein were localized more to MG peripheral acinar cells than central acinar cells or ductular epithelial cells. Electrophysiologic studies of rat MG cell planar cultures demonstrated functional sodium, chloride, and potassium channels, and cotransporters activities. Transmission electron microscopic analyses of rat MG spheroids revealed highly differentiated MG cells with abundant lysosomal lamellar bodies. Rat MG spheroids culture-based measurements demonstrated active volume regulation by ion channels.ConclusionsThis study demonstrates the presence and function of ion channels and volume transport by rat MG. Two novel primary MG cell culture models that may be useful for MG research were established.

Can tetracycline antibiotics duplicate the ability of azithromycin to stimulate human meibomian gland epithelial cell differentiation?

Azithromycin and tetracyclines are commonly prescribed in the United States for the treatment of meibomian gland dysfunction (MGD). The efficacy of these antibiotics has been believed to be their antiinflammatory and antibacterial actions, which suppress MGD-associated posterior blepharitis and growth of lid bacteria. However, we recently discovered that azithromycin can act directly on human meibomian gland epithelial cells (HMGECs) to stimulate their function. In this study, we sought to determine whether tetracycline antibiotics can duplicate this azithromycin effect. Immortalized HMGEC were cultured in the presence of a vehicle, azithromycin, doxycycline, minocycline, or tetracycline for 5 days. Cells were evaluated for cholesterol and neutral lipid staining, and the lipid composition of cellular lysates was analyzed by high-performance thin-layer chromatography. Our results demonstrate that azithromycin's ability to stimulate the differentiation of human meibomian gland cells is unique, and is not duplicated by doxycycline, minocycline, or tetracycline. Azithromycin, but not the other antibiotics, significantly increased the cellular accumulation of cholesterol, cholesterol esters, phospholipids, and lysosomes. These differentiative actions of azithromycin were paralleled by an increased expression of sterol regulatory element-binding protein 1. Our findings show that the stimulatory effects of azithromycin on HMGEC function are unique and are not duplicated by the antibiotics doxycycline, minocycline, or tetracycline. Our results further suggest that this stimulatory influence of azithromycin may contribute to its beneficial effect in treating MGD and its associated evaporative dry eye disease.

A Role for ELOVL4 in the Mouse Meibomian Gland and Sebocyte Cell Biology

The meibum lipidome contains lipids with extremely long chain fatty acid (ELCFA) residues, longer than C28. Particular lipids based on extremely long chain (O-acyl)-ω-hydroxy fatty acids (OAHFA) are found in all mammal meibum and are proposed to stabilize the tear film by forming the interphase between its lipid and aqueous sublayers. The enzyme ELOVL4 is required for synthesis of ELCFA. We investigated whether Stgd3 mice, harboring mutations in ELOVL4 that have been shown to decrease the levels of its biosynthetic lipid products, would represent a model system in which to define the role of such lipids in meibum. Ocular phenotypes of wild-type mice were compared with those of Stgd3 mice. ELOVL4 expression in eyelid and back skin was characterized by immunohistochemical analysis. Anatomical changes within the eyelids of mutant mice were examined by hematoxylin and eosin staining of paraffin-embedded tissue. Mutant mice had increased eyelid blink rates, a reluctance to maintain their eyes fully open, protruding meibomian gland (MG) orifices, and anatomical changes within the MG. In wild-type mice, ELOVL4 was strongly expressed within the holocrine meibomian and sebaceous glands. The enzyme localized to structures encircling lipid deposits within cells in both the early and late stages of differentiation. No ELOVL4 was detected within the central meibomian duct. Stgd3 mice show changes that resemble clinical findings in patients with the evaporative type of dry eye disease, suggesting that further studies in this mouse model will provide a basis for better understanding of the causes of human dry eye.