Human Meibomian Gland Research Articles

Pharmacological in vitro profiling of Buddleja officinalis flower extracts in the context of dry eye disease

Dry eye disease (DED) is caused by inflammation on the ocular surface and insufficient quality or production of the tear film. Due to various harmful environmental conditions, a gradual increase of DED cases has been reported. This study aims for a comprehensive in vitro pharmacological and phytochemical profiling of two different Buddleja officinalis Maxim. extracts to assess their potential for the treatment of DED. A hydroethanolic (BO-HE) and a lead-like enhanced (BO-LLE) B. officinalis extract were phytochemically characterized by UHPLC-UV-MS and UHPLC-ELSD analyses. Afterwards, the effects of either BO-HE or BO-LLE on in vitro dry eye models, including human corneal epithelial cell-transformed (HCE-T) cells, immortalized human meibomian gland epithelial cells (IHMGECs) and human leukemia monocytic (THP-1) cells, and Jurkat cells, were investigated. Both extracts exhibited strong anti-inflammatory properties with free radical scavenging activities and reduction of intracellular reactive oxygen species (ROS) in UVB-exposed HCE-T cells. Treatment with BO-HE or BO-LLE showed wound healing capacities. Moreover, both extracts differentially modulate mediator secretion in UVB-exposed HCE-T cells. In IHMGECs, the size of secreted lipid droplets was larger in BO-LLE treated cells. As for immune cells, a significant reduction in levels of TNF-α and IL-6 secretion by lipopolysaccharide (LPS)-exposed THP-1 cells was observed. Additionally, BO-LLE effectively inhibited intracellular calcium influx in Jurkat cells even at low concentrations. The results of this study demonstrate pharmacological potential of B. officinalis flowers for the treatment of DED pathology with BO-LLE exerting a slightly more beneficial profile than BO-HE.

Activation of the mTOR pathway enhances PPARγ/SREBP-mediated lipid synthesis in human meibomian gland epithelial cells.

The involvement of the mechanistic targets of rapamycin (mTOR) pathway in lipid metabolism has been recently elucidated. However, its specific role in the Meibomian gland, where lipid metabolism is significant, remains not fully understood. We investigated the role of mTOR signaling system in the lipogenesis and differentiation of human meibomian gland epithelial cells (HMGECs). Treatment of HMGECs with rapamycin resulted in a reduction in lipid synthesis and the expression of PPARγ and SREBP-1, the major regulators of lipid synthesis. The phosphorylation of p70S6kinase and AKT, which are downstream signals of mTOR complexes 1 and 2, respectively, decreased following rapamycin treatment. In addition, when both mTOR complex 1 and 2 were suppressed using siRNA, there was a significant reduction in the expression of PPARγ and SREBP-1, along with a decrease in lipid synthesis in HMGECs. Our findings suggest that inhibiting the mTOR pathway diminishes the differentiation and adipogenesis of meibomian gland epithelial cells, and both mTOR complexes 1 and 2 appear to play a role in this activity.

13-cis Retinoic Acid-Mediated Modulation of Human Meibomian Gland Epithelial Cells Development: Implications for In Vitro Modeling of Meibomian Gland Dysfunction.

Purpose: This study aimed to investigate the effect of 13-cis retinoic acid (13-cis RA) on human meibomian gland epithelial cells (HMGECs) and explore the potential of using this experimental model as an in vitro approach for studying meibomian gland dysfunction (MGD). Methods: First, HMGECs were cultured with 13-cis RA at different doses and times, and cell viability and proliferation rates were assessed to determine the appropriate stimulation concentration and time. Subsequently, during the proliferation stage, the expression of proliferation, inflammation, and oxidative stress genes and their products were evaluated. The meibum synthesis capacity was determined during the differentiation stage. Additionally, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 was used as a control to assess the impact of 13-cis RA on PPARγ. Results: 13-cis RA significantly inhibited cell viability and proliferation in a time-dose response manner. Under the stimulation of 2 and 5 μM for 48 h during the proliferation stage, a significant decrease was observed in the expression of cell proliferation markers Ki67, antioxidant SOD-2, and Nrf-2. However, the expression of the pro-inflammatory factors IL-1β, IL-8, MMP9, and oxidative stress markers NOX-4 and reactive oxygen species increased. During the differentiation stage, it suppressed meibum synthesis and the expression of meibocyte differentiation-related proteins adipose differentiation-associated protein 4 (ADFP4), elongation of very long chain fatty acid protein 4 (ELOVL4), sterol regulatory element-binding protein 2 (SREBP-2), and PPARγ. Conclusion: 13-cis RA inhibited cell viability, promoted inflammation and oxidative stress, and suppressed meibum synthesis through the PPARγ pathway. Our study shed light on the effect of 13-cis RA on HMGECs and provided a promising direction for studying MGD in vitro.

Identification of Meibomian gland stem cell populations and mechanisms of aging.

Meibomian glands secrete lipid-rich meibum, which prevents tear evaporation. Aging-related Meibomian gland shrinkage may result in part from stem cell exhaustion and is associated with evaporative dry eye disease, a common condition lacking effective treatment. The identities and niche of Meibomian gland stem cells and the signals controlling their activity are poorly defined. Using snRNA-seq, in vivo lineage tracing, ex vivo live imaging, and genetic studies in mice, we identified markers for stem cell populations that maintain distinct regions of the gland and uncovered Hh signaling as a key regulator of stem cell proliferation. Consistent with this, human Meibomian gland carcinoma exhibited increased Hh signaling. Aged glands displayed decreased Hh and EGF signaling, deficient innervation, and loss of collagen I in niche fibroblasts, indicating that alterations in both glandular epithelial cells and their surrounding microenvironment contribute to age-related degeneration. These findings suggest new approaches to treat aging-associated Meibomian gland loss.

Meibomian Gland Shortening Is Associated With Altered Meibum Composition.

The purpose of this study was to investigate differences in the composition of meibum extracted from human meibomian glands displaying different morphological characteristics. Adult participants with evidence of meibomian gland shortening were enrolled. Glands were observed using infrared meibography, and meibum was collected from one short and one long gland from the lower eyelid of the same eye. Total protein concentration was evaluated using the detergent compatible (DC) protein assay and lipid analysis was performed using liquid chromatography mass spectrometry. Fifteen participants (8 women and 7 men) completed the study (mean age = 34.7 ± 7.7 years). The average volume of meibum collected from the short and long glands was 0.02 ± 0.01 and 0.05 ± 0.03µL, respectively (P=0.01). Average protein concentration in the short glands was significantly higher compared to the long glands (0.10 ± 0.03 vs. 0.08 ± 0.02 mg/mL, P=0.01). Among the non-polar lipids, the mole percent of cholesterol esters (CEs; P=0.02), triacylglycerols (TAGs; P=0.04), and ceramide (Cer; P=0.03) was significantly lower in short glands compared with long glands. Among the polar lipids, the mole percent of (O-acyl)-ω-hydroxy fatty acid (OAHFA; P=0.01) was significantly lower in short glands, whereas phosphatidylcholine (PC; P=0.02) and sphingomyelin (SM; P=0.01) were significantly higher in short glands than long glands. Meibum composition differed between short and long glands. The lower mole percent of CE, TAG, OAHFA, and Cer and a higher mole percent of PC and SM among short glands may indicate disease activity. This information may clarify the natural history of meibomian gland dysfunction and future targets for therapy.

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.

Molecular detection of lacrimal apparatus and ocular surface - related ABC transporter genes

The ocular system is in constant interaction with the environment and with numerous pathogens. The ATP-binding cassette (ABC) transporters represent one of the largest groups among the transmembrane proteins. Their relevance has been demonstrated for their defense function against biotic and abiotic stress factors, for metabolic processes in tumors and for their importance in the development of resistance to drugs. The aim of this study was to analyze which ABC transporters are expressed at the ocular surface and in the human lacrimal apparatus. Using RT-PCR, all ABC transporters known to date in humans were examined in tissue samples from human cornea, conjunctiva, meibomian glands and lacrimal glands. The RT-PCR analyses revealed the presence of all ABC transporters in the samples examined, although the results for some of the 48 transporters known in human and analyzed were different in the various tissues. The present results provide information on the expression of ABC transporters at the mRNA level on the ocular surface and in the lacrimal system. Their detection forms the basis for follow-up studies at the protein level, which will provide more information about their physiological significance at the ocular surface and in the lacrimal system and which may explain pathological effects such as drug resistance.

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.

Expression of ATP-Binding Cassette Transporter A1 (ABCA1) in Eyelid Tissues and Meibomian Gland Epithelial Cells.

To validate the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and distribution in human eyelid tissues and meibomian gland epithelial cells. Meibomian gland tissues from human eyelids were isolated by collagenase A digestion and cultured in defined keratinocyte serum-free medium (DKSFM). Infrared imaging was used to analyze the general morphology of meibomian glands. Hematoxylin and eosin (H&E) staining and Oil Red O staining were used to observe the morphological structure and lipid secretion in the human meibomian gland tissues. Quantitative real-time polymerase chain reaction, western blotting, and immunostaining were used to detect the mRNA and protein expression and cytolocalization of ABCA1 in the meibomian gland tissues and cultured cells. The degree of loss of human meibomian gland tissue was related to age. Meibomian gland lipid metabolism was also associated with age. Additionally, human meibomian gland tissues express ABCA1 mRNA and protein; glandular epithelial cells express more ABCA1 mRNA and protein than acinar cells, and their expression in acinar cells decreases with differentiation. Furthermore, the expression of ABCA1 was downregulated in abnormal meibomian gland tissues. ABCA1 was mainly localized on the cell membrane in primary human meibomian gland epithelial cells (pHMGECs), whereas it was localized in the cytoplasm of immortalized human meibomian gland epithelial cells (iHMGECs). The mRNA and protein levels of ABCA1 in pHMGECs were higher than those in iHMGECs. Meibomian gland tissues of the human eyelid degenerate with age. ABCA1 expression in acinar cells decreases after differentiation and plays an important role in meibomian gland metabolism.

Comparative Characterization of Human Meibomian Glands, Free Sebaceous Glands, and Hair-Associated Sebaceous Glands Based on Biomarkers, Analysis of Secretion Composition, and Gland Morphology.

Meibomian gland dysfunction (MGD) is one of the main causes of dry eye disease. To better understand the physiological functions of human meibomian glands (MGs), the present study compared MGs with free sebaceous glands (SGs) and hair-associated SGs of humans using morphological, immunohistochemical, and liquid chromatography-mass spectrometry (LCMS)-based lipidomic approaches. Eyelids with MGs, nostrils, lips, and external auditory canals with free SGs, and scalp with hair-associated SGs of body donors were probed with antibodies against cytokeratins (CK) 1, 8, 10, and 14, stem cell markers keratin 15 and N-cadherin, cell-cell contact markers desmoglein 1 (Dsg1), desmocollin 3 (Dsc3), desmoplakin (Dp), plakoglobin (Pg), and E-cadherin, and the tight junction protein claudin 5. In addition, Oil Red O staining (ORO) was performed in cryosections. Secretions of MGs as well as of SGs of nostrils, external auditory canals, and scalps were collected from healthy volunteers, analyzed by LCMS, and the data were processed using various multivariate statistical analysis approaches. Serial sections of MGs, free SGs, and hair-associated SGs were 3D reconstructed and compared. CK1 was expressed differently in hair-associated SGs than in MGs and other free SGs. The expression levels of CK8, CK10, and CK14 in MGs were different from those in hair-associated SGs and other free SGs. KRT15 was expressed differently in hair-associated SGs, whereas N-cadherin was expressed equally in all types of glands. The cell-cell contact markers Dsg1, Dp, Dsc3, Pg, and E-cadherin revealed no differences. ORO staining showed that lipids in MGs were more highly dispersed and had larger lipid droplets than lipids in other free SGs. Hair-associated SGs had a smaller number of lipid droplets. LCMS revealed that the lipid composition of meibum was distinctively different from that of the sebum of the nostrils, external auditory canals, and scalp. The 3D reconstructions of the different glands revealed different morphologies of the SGs compared with MGs which are by far the largest type of glands. In humans, MGs differ in their morphology and secretory composition and show major differences from free and hair-associated SGs. The composition of meibum differs significantly from that of sebum from free SGs and from hair-associated SGs. Therefore, the MG can be considered as a highly specialized type of holocrine gland that exhibits all the histological characteristics of SGs, but is significantly different from them in terms of morphology and lipid composition.

Oleic acid induces lipogenesis and NLRP3 inflammasome activation in organotypic mouse meibomian gland and human meibomian gland epithelial cells

The accumulation of oleic acid (OA) in the meibum from patients with meibomian gland dysfunction (MGD) suggests that it may contribute to meibomian gland (MG) functional disorder, as it is a potent stimulator of acne-related lipogenesis and inflammation in sebaceous gland. Therefore, we investigate whether OA induces lipogenesis and inflammasome activation in organotypic cultured mouse MG and human meibomian gland epithelial cells (HMGECs). Organotypic cultured mouse MG and HMGECs were exposed to OA or combinations with specific AMPK agonists 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Lipogenic status, ductal keratinization, squamous metaplasia, NLRP3/ASC/Caspase-1 inflammasome activation, proinflammatory cytokine IL-1β production, and AMPK pathway phosphorylation in MG were subsequently examined by lipid staining, immunofluorescence staining, immunohistochemical staining, ELISA assay, and Western blot analyses. We found that OA significantly induced lipid accumulation, ductal keratinization, and squamous metaplasia in organotypic cultured MG, as evidenced by increased lipids deposition within acini and duct, upregulated expression of lipogenic proteins (SREBP-1 and HMGCR), and elevation of K10/Sprr1b. Additionally, OA induced NLRP3/ASC/Caspase-1 inflammasome activation, cleavage of Caspase-1, and production of downstream proinflammatory cytokine IL-1β. The findings of lipogenesis and NLRP3-related proinflammatory response in OA-stimulated HMGECs were consistent with those in organotypic cultured MG. OA exposure downregulated phospho-AMPK in two models, while AICAR treatment alleviated lipogenesis by improving AMPK/ACC phosphorylation and SREBP-1/HMGCR expression. Furthermore, AMPK amelioration inhibited activation of the NLRP3/ASC/Caspase-1 axis and secretion of IL-1β, thereby relieving the OA-induced proinflammatory response. These results demonstrated that OA induced lipogenic disorder and NLRP3 inflammasome activation in organotypic cultured mouse MG and HMGECs by suppressing the AMPK signaling pathway, indicating OA may play an etiological role in MGD.

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.

Eyes on the Lid: The Impact of Fibroblast Growth Factor Receptor Targeting Drug on Human Meibomian Gland.

To determine whether fibroblast growth factor receptor (FGFR)-targeting drug could impact human meibomian gland. We followed up with three patients who were using pemigatinib for 4 to 10 weeks. The patients were evaluated for their ocular surface disease index, best-corrected visual acuity, Schirmer test, cornea staining, meibum expressibility score, tear meniscus height, noninvasive tear film breakup time, and meibomian gland area. The distribution of the FGFR family, FGF7, and FGF10 were evaluated by immunofluorescence staining and Western blot in fresh tarsal tissues from deidentified patients who underwent lid plastic surgeries. All patients developed apparent meibomian gland atrophy, shortening and narrowing of ducts, and significantly increased meibum expressibility and decreased noninvasive tear film breakup time within 5 to 8 weeks. Laboratory evaluations confirmed that human meibomian gland expresses abundant fibroblast growth factor receptors. These findings indicate that meibomian gland is a target tissue of FGFR inhibitors, and patients who use these drugs may develop meibomian gland dysfunction.

Comparison of different lipid staining methods in human meibomian gland epithelial cells

This study aimed to compare the utility of four different dyes for intracellular lipid detection in immortalized human meibomian gland epithelial cells (IHMGECs). IHMGECs were cultured in a serum-containing medium for 10 days in the presence or absence of Roxadustat (Roxa), a known inducer of IHMGEC differentiation. Cells were then fixed and stained with Oil Red O (ORO), Sudan III (SIII), LipidTOX green (LT), or Nile Red (NR). IHMGECs were evaluated for the number, size, and area of stained intracellular lipid vesicles or the intensity of staining using bright field (ORO, SIII) or fluorescence (LT, NR) microscopy. Data were captured with ImageJ and analyzed with Student's two-tailed t-test. Our findings demonstrate that different staining methods can yield significantly different patterns of intracellular lipid quantity and/or distribution in IHMGECs. ORO and SIII significantly increased the size and area of lipid-containing vesicles in Roxa-treated cells. Neither stain showed a change in the number of vesicles during IHMGEC differentiation. Vesicle size was significantly greater in cells stained with ORO, as compared to SIII. In addtion, LT, but not NR, showed a significant increase in intracellular lipid intensity in IHMGECs following Roxa -induced differentiation. Our results demonstrate significant differences in the distribution patterns and intensities of lipid-containing vesicles in IHMGECs after staining with ORO, SIII, LT, and NR. ORO, SIII, and LT, but not NR staining, are helpful methods to help identify and quantitate the extent of intracellular lipid accumulation during IHMGEC differentiation.

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.

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

Induction of meibocyte differentiation by three-dimensional, matrigel culture of immortalized human meibomian gland epithelial cells to form acinar organoids

PurposeRecent studies have shown that two-dimensional (2D) culture of primary rabbit and immortalized human meibomian gland epithelial cells (iHMGEC) do not recapitulate normal meibocyte differentiation and fail to express critical enzymes necessary for synthesis of meibum lipids. The purpose of this study was to test the hypothesis that 3D-spheroid culture of iHMGEC can facilitate meibocyte differentiation and induce the expression of acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), shown to be required for synthesis of meibum wax esters. MethodsiHMGEC were suspended in matrigel/basement membrane matrix and grown in proliferation media to form distinct cell clusters or spheroids. Cells were then treated with serum-free, differentiation media (advanced DMEM/F12) with and without FGF10 and synthetic agonists for the nuclear lipid receptor, peroxisome proliferator activator receptor gamma (PPARγ). Cells were then evaluated for differentiation markers using western blotting, immunocytochemistry (ICC) and real-time PCR. Control cells were grown in standard 2D culture systems. ResultsUnder proliferative conditions, 3D culture induced the formation of KRT5+ spheroids that contained a Ki67+/P63+ undifferentiated, basal cell population. When spheroids were switched to differentiation media containing PPARγ agonists, two different organoid populations were detected, a KRT6low population that was AWAT2+/PPARγ+ and a KRT6high population that was AWAT2-/PPARγ-, suggesting that iHMGEC exhibit a dual differentiation potential toward either a ductal or meibocyte organoid phenotype. ConclusionThe 3D culturing of iHMGEC can induce the formation of both meibocyte and ductal organoids and may thus serve as a better in vitro model system for studying the regulatory mechanisms controlling meibomian gland function.

Activation of ADRB2/PKA Signaling Pathway Facilitates Lipid Synthesis in Meibocytes, and Beta-Blocker Glaucoma Drug Impedes PKA-Induced Lipid Synthesis by Inhibiting ADRB2.

Meibomian gland dysfunction is one of the main causes of dry eye disease and has limited therapeutic options. In this study, we investigated the biological function of the beta 2-adrenergic receptor (ADRB2)/protein kinase A (PKA) pathway in lipid synthesis and its underlying mechanisms in human meibomian gland epithelial cells (HMGECs). HMGECs were cultured in differentiation media with or without forskolin (an activator of adenylate cyclase), salbutamol (an ADRB2 agonist), or timolol (an ADRB2 antagonist) for up to 4 days. The phosphorylation of the cAMP-response element-binding protein (CREB) and the expression of peroxisome proliferator activator receptor (PPAR)γ and sterol regulatory element-binding protein (SREBP)-1 were measured by immunoblotting and quantitative PCR. Lipid synthesis was examined by LipidTOX immunostaining, AdipoRed assay, and Oil Red O staining. PKA pathway activation enhanced PPARγ expression and lipid synthesis in differentiated HMGECs. When treated with agonists of ADBR2 (upstream of the PKA signaling system), PPARγ expression and lipid synthesis were enhanced in HMGECs. The ADRB2 antagonist timolol showed the opposite effect. The activation of the ADRB2/PKA signaling pathway enhances lipid synthesis in HMGECs. These results provide a potential mechanism and therapeutic target for meibomian gland dysfunction, particularly in cases induced by beta-blocker glaucoma drugs.

The morphogenesis of the rabbit meibomian gland in relation to sex hormones: Immunohistochemical and transmission electron microscopy studies

Rabbits have been proposed as a model for the human meibomian gland (MG), a large specific sebaceous gland in the eyelid that consists of secretory acini arranged laterally and related to the central duct via short ductules, with the central duct continuing as an excretory duct to open at the free margin of the lid. First detected at embryonic day 18 as an aggregation of mesenchymal cells in the tarsal plate, it completes its development approximately 2 weeks postnatal when the separation of the eyelids is completed. The Transmission electron microscopy supports the meibocytes’ gradient maturation to the meibum’s synthesis. While the differentiating cells, their cytoplasm, are well packed with lipid droplets, the basal cells are characterized by a high nuclear to cytoplasm ratio. The androgen and estrogen receptor proteins are expressed in the basal cell and the meibocytes, and increase in age increases in the expression of these proteins. Additionally, the cytokeratin (CK14) is expressed in the basal and differentiating cells of the acini and the ductal epithelium. Therefore, the duct cells of the MG are common in all stem cells. These data concluded that the MG plays a major role in maintaining the health of the ocular surface and preservation of visual acuity. Any abnormalities in the structure of the MG lead to its dysfunction and changes in lipid secretion.

Prolactin Inducible Protein, but Not Prolactin, Is Present in Human Tears, Is Involved in Tear Film Quality, and Influences Evaporative Dry Eye Disease.

PurposeDecreased production of the aqueous component of the tear film is an important cause of the development of dry eye disease (DED). Tear production is influenced by hormones and hormone-like factors. Prolactin (PLR), a multifunctional pituitary gland hormone, is regularly present in the lacrimal gland of rats and rabbits. In humans, serum PLR concentration correlates with tear quality. To gain deeper insights of possible effects of PRL, prolactin receptor (PRLR) and prolactin inducible protein (PIP), we analyzed the three proteins in the human lacrimal apparatus and in reflex tears of healthy volunteers as well as patients suffering from DED.MethodsGene expression of PRLR and PIP was analyzed by RT-PCR in cadaveric human lacrimal gland and ocular surface tissues, immortalized human corneal epithelial cells (HCE and hTEPI) and human Meibomian gland epithelial cells (HMGECs). At the protein level, the expression and localization of PRL, PRLR and PIP in formalin-fixed paraffin sections of the lacrimal apparatus were studied by immunohistochemistry. In addition, tear fluid from DED patients and healthy volunteers was analyzed by ELISA to determine the concentration of PRL and PIP.ResultsRT-PCR analyses revealed gene expression of PRLR and PIP in human tissue samples of cornea, lacrimal glands, and eyelids, whereas only PIP, but not PRLR, was detectable in immortalized corneal epithelial cells. Immunohistochemistry revealed for the first time the expression and localization of PRL, PRLR, and PIP in human tissues of the lacrimal apparatus and at the ocular surface. PRL and PRLR were detectable in corneal epithelium, lacrimal glands, and Meibomian glands. Reflex tears from DED patients revealed significantly increased PIP concentrations, whereas PRL was undetectable in tears of DED patients and healthy volunteers.ConclusionPRL, PRLR, and PIP are found in the lacrimal apparatus and on the ocular surface. PIP, but not PRL, is present in human tears and appears to be involved in the physiology of tear film quality. Our clinical data revealed that PIP may affect tear quality, but further functional analyses are needed to fully elucidate the effects of PRL and PIP-associated factors in tear secretion as well as in the connection of DED.